folding problem kinetics: Topics by Science.gov

Sample records for folding problem kinetics

RNA folding: structure prediction, folding kinetics and ion electrostatics.

PubMed

Tan, Zhijie; Zhang, Wenbing; Shi, Yazhou; Wang, Fenghua

2015-01-01

Beyond the "traditional" functions such as gene storage, transport and protein synthesis, recent discoveries reveal that RNAs have important "new" biological functions including the RNA silence and gene regulation of riboswitch. Such functions of noncoding RNAs are strongly coupled to the RNA structures and proper structure change, which naturally leads to the RNA folding problem including structure prediction and folding kinetics. Due to the polyanionic nature of RNAs, RNA folding structure, stability and kinetics are strongly coupled to the ion condition of solution. The main focus of this chapter is to review the recent progress in the three major aspects in RNA folding problem: structure prediction, folding kinetics and ion electrostatics. This chapter will introduce both the recent experimental and theoretical progress, while emphasize the theoretical modelling on the three aspects in RNA folding.
Dynamics of protein folding: probing the kinetic network of folding-unfolding transitions with experiment and theory.

PubMed

Buchner, Ginka S; Murphy, Ronan D; Buchete, Nicolae-Viorel; Kubelka, Jan

2011-08-01

The problem of spontaneous folding of amino acid chains into highly organized, biologically functional three-dimensional protein structures continues to challenge the modern science. Understanding how proteins fold requires characterization of the underlying energy landscapes as well as the dynamics of the polypeptide chains in all stages of the folding process. In recent years, important advances toward these goals have been achieved owing to the rapidly growing interdisciplinary interest and significant progress in both experimental techniques and theoretical methods. Improvements in the experimental time resolution led to determination of the timescales of the important elementary events in folding, such as formation of secondary structure and tertiary contacts. Sensitive single molecule methods made possible probing the distributions of the unfolded and folded states and following the folding reaction of individual protein molecules. Discovery of proteins that fold in microseconds opened the possibility of atomic-level theoretical simulations of folding and their direct comparisons with experimental data, as well as of direct experimental observation of the barrier-less folding transition. The ultra-fast folding also brought new questions, concerning the intrinsic limits of the folding rates and experimental signatures of barrier-less "downhill" folding. These problems will require novel approaches for even more detailed experimental investigations of the folding dynamics as well as for the analysis of the folding kinetic data. For theoretical simulations of folding, a main challenge is how to extract the relevant information from overwhelmingly detailed atomistic trajectories. New theoretical methods have been devised to allow a systematic approach towards a quantitative analysis of the kinetic network of folding-unfolding transitions between various configuration states of a protein, revealing the transition states and the associated folding pathways at multiple levels, from atomistic to coarse-grained representations. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches. Copyright © 2010 Elsevier B.V. All rights reserved.
Identification of a key structural element for protein folding within beta-hairpin turns.

PubMed

Kim, Jaewon; Brych, Stephen R; Lee, Jihun; Logan, Timothy M; Blaber, Michael

2003-05-09

Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.
Exploration of the folding dynamics of human telomeric G-quadruplex with a hybrid atomistic structure-based model

NASA Astrophysics Data System (ADS)

Bian, Yunqiang; Ren, Weitong; Song, Feng; Yu, Jiafeng; Wang, Jihua

2018-05-01

Structure-based models or Gō-like models, which are built from one or multiple particular experimental structures, have been successfully applied to the folding of proteins and RNAs. Recently, a variant termed the hybrid atomistic model advances the description of backbone and side chain interactions of traditional structure-based models, by borrowing the description of local interactions from classical force fields. In this study, we assessed the validity of this model in the folding problem of human telomeric DNA G-quadruplex, where local dihedral terms play important roles. A two-state model was developed and a set of molecular dynamics simulations was conducted to study the folding dynamics of sequence Htel24, which was experimentally validated to adopt two different (3 + 1) hybrid G-quadruplex topologies in K+ solution. Consistent with the experimental observations, the hybrid-1 conformation was found to be more stable and the hybrid-2 conformation was kinetically more favored. The simulations revealed that the hybrid-2 conformation folded in a higher cooperative manner, which may be the reason why it was kinetically more accessible. Moreover, by building a Markov state model, a two-quartet G-quadruplex state and a misfolded state were identified as competing states to complicate the folding process of Htel24. Besides, the simulations also showed that the transition between hybrid-1 and hybrid-2 conformations may proceed an ensemble of hairpin structures. The hybrid atomistic structure-based model reproduced the kinetic partitioning folding dynamics of Htel24 between two different folds, and thus can be used to study the complex folding processes of other G-quadruplex structures.
High Performance Biocomputation

DTIC Science & Technology

2005-03-01

in some other fields (e.g. computational hydrodynamics, lattice quantum chroniodynamics, etc.) but appears wholly inappropriate here as pointed out...restrict the overall conformational space by putting the system on a lattice . These have been used to great effect to study folding kinetics. These...many important problems to be worked on, not a single unique challenge (contrast this to QCD , for example). " almost all problems require significant
How Kinetics within the Unfolded State Affects Protein Folding: an Analysis Based on Markov State Models and an Ultra-Long MD Trajectory

PubMed Central

Deng, Nan-jie; Dai, Wei

2013-01-01

Understanding how kinetics in the unfolded state affects protein folding is a fundamentally important yet less well-understood issue. Here we employ three different models to analyze the unfolded landscape and folding kinetics of the miniprotein Trp-cage. The first is a 208 μs explicit solvent molecular dynamics (MD) simulation from D. E. Shaw Research containing tens of folding events. The second is a Markov state model (MSM-MD) constructed from the same ultra-long MD simulation; MSM-MD can be used to generate thousands of folding events. The third is a Markov state model built from temperature replica exchange MD simulations in implicit solvent (MSM-REMD). All the models exhibit multiple folding pathways, and there is a good correspondence between the folding pathways from direct MD and those computed from the MSMs. The unfolded populations interconvert rapidly between extended and collapsed conformations on time scales ≤ 40 ns, compared with the folding time of ≈ 5 μs. The folding rates are independent of where the folding is initiated from within the unfolded ensemble. About 90 % of the unfolded states are sampled within the first 40 μs of the ultra-long MD trajectory, which on average explores ~27 % of the unfolded state ensemble between consecutive folding events. We clustered the folding pathways according to structural similarity into “tubes”, and kinetically partitioned the unfolded state into populations that fold along different tubes. From our analysis of the simulations and a simple kinetic model, we find that when the mixing within the unfolded state is comparable to or faster than folding, the folding waiting times for all the folding tubes are similar and the folding kinetics is essentially single exponential despite the presence of heterogeneous folding paths with non-uniform barriers. When the mixing is much slower than folding, different unfolded populations fold independently leading to non-exponential kinetics. A kinetic partition of the Trp-cage unfolded state is constructed which reveals that different unfolded populations have almost the same probability to fold along any of the multiple folding paths. We are investigating whether the results for the kinetics in the unfolded state of the twenty-residue Trp-cage is representative of larger single domain proteins. PMID:23705683
Predicting repeat protein folding kinetics from an experimentally determined folding energy landscape

PubMed Central

Street, Timothy O; Barrick, Doug

2009-01-01

The Notch ankyrin domain is a repeat protein whose folding has been characterized through equilibrium and kinetic measurements. In previous work, equilibrium folding free energies of truncated constructs were used to generate an experimentally determined folding energy landscape (Mello and Barrick, Proc Natl Acad Sci USA 2004;101:14102–14107). Here, this folding energy landscape is used to parameterize a kinetic model in which local transition probabilities between partly folded states are based on energy values from the landscape. The landscape-based model correctly predicts highly diverse experimentally determined folding kinetics of the Notch ankyrin domain and sequence variants. These predictions include monophasic folding and biphasic unfolding, curvature in the unfolding limb of the chevron plot, population of a transient unfolding intermediate, relative folding rates of 19 variants spanning three orders of magnitude, and a change in the folding pathway that results from C-terminal stabilization. These findings indicate that the folding pathway(s) of the Notch ankyrin domain are thermodynamically selected: the primary determinants of kinetic behavior can be simply deduced from the local stability of individual repeats. PMID:19177351
Protein folding simulations: from coarse-grained model to all-atom model.

PubMed

Zhang, Jian; Li, Wenfei; Wang, Jun; Qin, Meng; Wu, Lei; Yan, Zhiqiang; Xu, Weixin; Zuo, Guanghong; Wang, Wei

2009-06-01

Protein folding is an important and challenging problem in molecular biology. During the last two decades, molecular dynamics (MD) simulation has proved to be a paramount tool and was widely used to study protein structures, folding kinetics and thermodynamics, and structure-stability-function relationship. It was also used to help engineering and designing new proteins, and to answer even more general questions such as the minimal number of amino acid or the evolution principle of protein families. Nowadays, the MD simulation is still undergoing rapid developments. The first trend is to toward developing new coarse-grained models and studying larger and more complex molecular systems such as protein-protein complex and their assembling process, amyloid related aggregations, and structure and motion of chaperons, motors, channels and virus capsides; the second trend is toward building high resolution models and explore more detailed and accurate pictures of protein folding and the associated processes, such as the coordination bond or disulfide bond involved folding, the polarization, charge transfer and protonate/deprotonate process involved in metal coupled folding, and the ion permeation and its coupling with the kinetics of channels. On these new territories, MD simulations have given many promising results and will continue to offer exciting views. Here, we review several new subjects investigated by using MD simulations as well as the corresponding developments of appropriate protein models. These include but are not limited to the attempt to go beyond the topology based Gō-like model and characterize the energetic factors in protein structures and dynamics, the study of the thermodynamics and kinetics of disulfide bond involved protein folding, the modeling of the interactions between chaperonin and the encapsulated protein and the protein folding under this circumstance, the effort to clarify the important yet still elusive folding mechanism of protein BBL, the development of discrete MD and its application in studying the alpha-beta conformational conversion and oligomer assembling process, and the modeling of metal ion involved protein folding. (c) 2009 IUBMB.
Energy landscapes, folding mechanisms, and kinetics of RNA tetraloop hairpins.

PubMed

Chakraborty, Debayan; Collepardo-Guevara, Rosana; Wales, David J

2014-12-31

RNA hairpins play a pivotal role in a diverse range of cellular functions, and are integral components of ribozymes, mRNA, and riboswitches. However, the mechanistic and kinetic details of RNA hairpin folding, which are key determinants of most of its biological functions, are poorly understood. In this work, we use the discrete path sampling (DPS) approach to explore the energy landscapes of two RNA tetraloop hairpins, and provide insights into their folding mechanisms and kinetics in atomistic detail. Our results show that the potential energy landscapes have a distinct funnel-like bias toward the folded hairpin state, consistent with efficient structure-seeking properties. Mechanistic and kinetic information is analyzed in terms of kinetic transition networks. We find microsecond folding times, consistent with temperature jump experiments, for hairpin folding initiated from relatively compact unfolded states. This process is essentially driven by an initial collapse, followed by rapid zippering of the helix stem in the final phase. Much lower folding rates are predicted when the folding is initiated from extended chains, which undergo longer excursions on the energy landscape before nucleation events can occur. Our work therefore explains recent experiments and coarse-grained simulations, where the folding kinetics exhibit precisely this dependency on the initial conditions.
A new model for approximating RNA folding trajectories and population kinetics

NASA Astrophysics Data System (ADS)

Kirkpatrick, Bonnie; Hajiaghayi, Monir; Condon, Anne

2013-01-01

RNA participates both in functional aspects of the cell and in gene regulation. The interactions of these molecules are mediated by their secondary structure which can be viewed as a planar circle graph with arcs for all the chemical bonds between pairs of bases in the RNA sequence. The problem of predicting RNA secondary structure, specifically the chemically most probable structure, has many useful and efficient algorithms. This leaves RNA folding, the problem of predicting the dynamic behavior of RNA structure over time, as the main open problem. RNA folding is important for functional understanding because some RNA molecules change secondary structure in response to interactions with the environment. The full RNA folding model on at most O(3n) secondary structures is the gold standard. We present a new subset approximation model for the full model, give methods to analyze its accuracy and discuss the relative merits of our model as compared with a pre-existing subset approximation. The main advantage of our model is that it generates Monte Carlo folding pathways with the same probabilities with which they are generated under the full model. The pre-existing subset approximation does not have this property.
Fragility of Liquids, Polyamorphism, Nucleation, and Folding Directions, in the Landscape Paradigm

NASA Astrophysics Data System (ADS)

Angell, C. A.

1998-03-01

Combination of the observations that cold glasses are rigid over eons, warm glasses (near their glass transitions) can densify, and most cooling liquids would, by extrapolation of observed entropy-temperature functions, achieve negative entropies well above OK if not salvaged by kinetics leads to the notion that substances which cannot find deep energy minima by 3 dimensional ordering of their particles must ``live" on a landscape of innumerable closely spaced and interconnected energy ``basins of probability" , the configuration space of amorphous substances. This energy hypersurface, which current studies suggest has a comparable multiplicity per heavy-atom particle regardless of how the particles are connected, provides the source of both excess liquid entropy and low temperature glass rigidity. If the energy distribution is narrow, the total entropy can be excited over a narrow temperature interval above T_g. The liquid then exhibits a high excess heat capacity and, directionally bonded cases aside, so called ``fragile" kinetic characteristics. A dynamic crossover to a liquid with different behavior occurs when the landscape entropy is almost fully excited. In extreme (highly cooperative) cases the crossover can occur via a first order transition in which much of the landscape entropy is excited isothermally. While this is rare in liquids, and in any case is difficult to observe because of promoted crystallization, it is quite common in mesoscopic systems which lack ordered packing possibilities but do have low energy amorphous configurations available. This is the case with proteins which often have ``all or nothing" transitions between folded and unfolded states, and behave like ``glassy" systems in their native (folded) states. Since phase transitions in many particle systems require nucleation, the identification of the nucleation step and its kinetic distinction from overall folding kinetics should be an important part of the understanding of the protein folding problem. The possibility exists that in certain cases an aberrant step in the nucleation event, facilitated by mutant nucleotide sequences or by third agents (heterogeneous nucleating agents), will trigger folding down an alternative and pathogenic route to a second stable state. This possibility should be evaluated, using nucleation kinetics analysis techniques, as an approach to understanding the initiation of ``mad cow" disease cerebral pathology.
Kinetic evidence for folding and unfolding intermediates in staphylococcal nuclease.

PubMed

Walkenhorst, W F; Green, S M; Roder, H

1997-05-13

The complex kinetic behavior commonly observed in protein folding studies suggests that a heterogeneous population of molecules exists in solution and that a number of discrete steps are involved in the conversion of unfolded molecules to the fully native form. A central issue in protein folding is whether any of these kinetic events represent conformational steps important for efficient folding rather than side reactions caused by slow steps such as proline isomerization or misfolding of the polypeptide chain. In order to address this question, we used stopped-flow fluorescence techniques to characterize the kinetic mechanism of folding and unfolding for a Pro- variant of SNase in which all six proline residues were replaced by glycines or alanines. Compared to the wild-type protein, which exhibits a series of proline-dependent slow folding phases, the folding kinetics of Pro- SNase were much simpler, which made quantitative kinetic analysis possible. Despite the absence of prolines or other complicating factors, the folding kinetics still contain several phases and exhibit a complex denaturant dependence. The GuHCl dependence of the major observable folding phase and a distinct lag in the appearance of the native state provide clear evidence for an early folding intermediate. The fluorescence of Trp140 in the alpha-helical domain is insensitive to the formation of this early intermediate, which is consistent with a partially folded state with a stable beta-domain and a largely disordered alpha-helical region. A second intermediate is required to model the kinetics of unfolding for the Pro- variant, which shows evidence for a denaturant-induced change in the rate-limiting unfolding step. With the inclusion of these two intermediates, we are able to completely model the major phase(s) in both folding and unfolding across a wide range of denaturant concentrations using a sequential four-state folding mechanism. In order to model the minor slow phase observed for the Pro- mutant, a six-state scheme containing a parallel pathway originating from a distinct unfolded state was required. The properties of this alternate unfolded conformation are consistent with those expected due to the presence of a non-prolyl cis peptide bond. To test the kinetic model, we used simulations based on the six-state scheme and were able to completely reproduce the folding kinetics for Pro- SNase across a range of denaturant concentrations.
Calculation of Rate Spectra from Noisy Time Series Data

PubMed Central

Voelz, Vincent A.; Pande, Vijay S.

2011-01-01

As the resolution of experiments to measure folding kinetics continues to improve, it has become imperative to avoid bias that may come with fitting data to a predetermined mechanistic model. Towards this end, we present a rate spectrum approach to analyze timescales present in kinetic data. Computing rate spectra of noisy time series data via numerical discrete inverse Laplace transform is an ill-conditioned inverse problem, so a regularization procedure must be used to perform the calculation. Here, we show the results of different regularization procedures applied to noisy multi-exponential and stretched exponential time series, as well as data from time-resolved folding kinetics experiments. In each case, the rate spectrum method recapitulates the relevant distribution of timescales present in the data, with different priors on the rate amplitudes naturally corresponding to common biases toward simple phenomenological models. These results suggest an attractive alternative to the “Occam’s razor” philosophy of simply choosing models with the fewest number of relaxation rates. PMID:22095854
Practical Approaches to Protein Folding and Assembly

PubMed Central

Walters, Jad; Milam, Sara L.; Clark, A. Clay

2009-01-01

We describe here the use of several spectroscopies, such as fluorescence emission, circular dichroism, and differential quenching by acrylamide, in examining the equilibrium and kinetic folding of proteins. The first section regarding equilibrium techniques provides practical information for determining the conformational stability of a protein. In addition, several equilibrium-folding models are discussed, from two-state monomer to four-state homodimer, providing a comprehensive protocol for interpretation of folding curves. The second section focuses on the experimental design and interpretation of kinetic data, such as burst-phase analysis and exponential fits, used in elucidating kinetic folding pathways. In addition, simulation programs are used routinely to support folding models generated by kinetic experiments, and the fundamentals of simulations are covered. PMID:19289201
Free Energy Landscape and Multiple Folding Pathways of an H-Type RNA Pseudoknot

PubMed Central

Bian, Yunqiang; Zhang, Jian; Wang, Jun; Wang, Jihua; Wang, Wei

2015-01-01

How RNA sequences fold to specific tertiary structures is one of the key problems for understanding their dynamics and functions. Here, we study the folding process of an H-type RNA pseudoknot by performing a large-scale all-atom MD simulation and bias-exchange metadynamics. The folding free energy landscapes are obtained and several folding intermediates are identified. It is suggested that the folding occurs via multiple mechanisms, including a step-wise mechanism starting either from the first helix or the second, and a cooperative mechanism with both helices forming simultaneously. Despite of the multiple mechanism nature, the ensemble folding kinetics estimated from a Markov state model is single-exponential. It is also found that the correlation between folding and binding of metal ions is significant, and the bound ions mediate long-range interactions in the intermediate structures. Non-native interactions are found to be dominant in the unfolded state and also present in some intermediates, possibly hinder the folding process of the RNA. PMID:26030098
Development and Application of a High Throughput Protein Unfolding Kinetic Assay

PubMed Central

Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

2016-01-01

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729
Sampling the multiple folding mechanisms of Trp-cage in explicit solvent

PubMed Central

Juraszek, J.; Bolhuis, P. G.

2006-01-01

We investigate the kinetic pathways of folding and unfolding of the designed miniprotein Trp- cage in explicit solvent. Straightforward molecular dynamics and replica exchange methods both have severe convergence problems, whereas transition path sampling allows us to sample unbiased dynamical pathways between folded and unfolded states and leads to deeper understanding of the mechanisms of (un)folding. In contrast to previous predictions employing an implicit solvent, we find that Trp-cage folds primarily (80% of the paths) via a pathway forming the tertiary contacts and the salt bridge, before helix formation. The remaining 20% of the paths occur in the opposite order, by first forming the helix. The transition states of the rate-limiting steps are solvated native-like structures. Water expulsion is found to be the last step upon folding for each route. Committor analysis suggests that the dynamics of the solvent is not part of the reaction coordinate. Nevertheless, during the transition, specific water molecules are strongly bound and can play a structural role in the folding. PMID:17035504
Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.

PubMed

Meuzelaar, Heleen; Tros, Martijn; Huerta-Viga, Adriana; van Dijk, Chris N; Vreede, Jocelyne; Woutersen, Sander

2014-03-06

Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu - and Arg + are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.
Co-Translational Folding Trajectory of the HemK Helical Domain.

PubMed

Mercier, Evan; Rodnina, Marina V

2018-06-26

Protein folding begins co-translationally within the restricted space of the peptide exit tunnel of the ribosome. We have already shown that the N-terminal α-helical domain of the universally conserved N 5 -glutamine methyltransferase HemK is compacted within the exit tunnel and rearranges into the native fold upon emerging from the ribosome. However, the exact folding pathway of the domain remained unclear. Here we analyzed the rapid kinetics of translation and folding monitored by fluorescence resonance energy transfer and photoinduced electron transfer using global fitting to a model for synthesis of the 112-amino acid HemK fragment. Our results suggest that the co-translational folding trajectory of HemK starts within the tunnel and passes through four kinetically distinct folding intermediates that may represent sequential docking of helices to a growing compact core. The kinetics of the process is defined entirely by translation. The results show how analysis of ensemble kinetic data can be used to dissect complex trajectories of rapid conformational rearrangements in multicomponent systems.
A hybrid MD-kMC algorithm for folding proteins in explicit solvent.

PubMed

Peter, Emanuel Karl; Shea, Joan-Emma

2014-04-14

We present a novel hybrid MD-kMC algorithm that is capable of efficiently folding proteins in explicit solvent. We apply this algorithm to the folding of a small protein, Trp-Cage. Different kMC move sets that capture different possible rate limiting steps are implemented. The first uses secondary structure formation as a relevant rate event (a combination of dihedral rotations and hydrogen-bonding formation and breakage). The second uses tertiary structure formation events through formation of contacts via translational moves. Both methods fold the protein, but via different mechanisms and with different folding kinetics. The first method leads to folding via a structured helical state, with kinetics fit by a single exponential. The second method leads to folding via a collapsed loop, with kinetics poorly fit by single or double exponentials. In both cases, folding times are faster than experimentally reported values, The secondary and tertiary move sets are integrated in a third MD-kMC implementation, which now leads to folding of the protein via both pathways, with single and double-exponential fits to the rates, and to folding rates in good agreement with experimental values. The competition between secondary and tertiary structure leads to a longer search for the helix-rich intermediate in the case of the first pathway, and to the emergence of a kinetically trapped long-lived molten-globule collapsed state in the case of the second pathway. The algorithm presented not only captures experimentally observed folding intermediates and kinetics, but yields insights into the relative roles of local and global interactions in determining folding mechanisms and rates.

Kinetic Folding Mechanism of Erythropoietin

PubMed Central

Banks, Douglas D.; Scavezze, Joanna L.; Siska, Christine C.

2009-01-01

This report describes what to our knowledge is the first kinetic folding studies of erythropoietin, a glycosylated four-helical bundle cytokine responsible for the regulation of red blood cell production. Kinetic responses for folding and unfolding reactions initiated by manual mixing were monitored by far-ultraviolet circular dichroism and fluorescence spectroscopy, and folding reactions initiated by stopped-flow mixing were monitored by fluorescence. The urea concentration dependence of the observed kinetics were best described by a three-state model with a transiently populated intermediate species that is on-pathway and obligatory. This folding scheme was further supported by the excellent agreement between the free energy of unfolding and m-value calculated from the microscopic rate constants derived from this model and these parameters determined from separate equilibrium unfolding experiments. Compared to the kinetics of other members of the four-helical bundle cytokine family, erythropoietin folding and unfolding reactions were slower and less susceptible to aggregation. We tentatively attribute these slower rates and protection from association events to the large amount of carbohydrate attached to erythropoietin at four sites. PMID:19450492
Understanding the kinetic mechanism of RNA single base pair formation

PubMed Central

Xu, Xiaojun; Yu, Tao; Chen, Shi-Jie

2016-01-01

RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model. PMID:26699466
Probing the kinetic stabilities of Friedreich's ataxia clinical variants using a solid phase GroEL chaperonin capture platform.

PubMed

Correia, Ana R; Naik, Subhashchandra; Fisher, Mark T; Gomes, Cláudio M

2014-10-20

Numerous human diseases are caused by protein folding defects where the protein may become more susceptible to degradation or aggregation. Aberrant protein folding can affect the kinetic stability of the proteins even if these proteins appear to be soluble in vivo. Experimental discrimination between functional properly folded and misfolded nonfunctional conformers is not always straightforward at near physiological conditions. The differences in the kinetic behavior of two initially folded frataxin clinical variants were examined using a high affinity chaperonin kinetic trap approach at 25 °C. The kinetically stable wild type frataxin (FXN) shows no visible partitioning onto the chaperonin. In contrast, the clinical variants FXN-p.Asp122Tyr and FXN-p.Ile154Phe kinetically populate partial folded forms that tightly bind the GroEL chaperonin platform. The initially soluble FXN-p.Ile154Phe variant partitions onto GroEL more rapidly and is more kinetically liable. These differences in kinetic stability were confirmed using differential scanning fluorimetry. The kinetic and aggregation stability differences of these variants may lead to the distinct functional impairments described in Friedreich's ataxia, the neurodegenerative disease associated to frataxin functional deficiency. This chaperonin platform approach may be useful for identifying small molecule stabilizers since stabilizing ligands to frataxin variants should lead to a concomitant decrease in chaperonin binding.
Guiding the folding pathway of DNA origami

NASA Astrophysics Data System (ADS)

Dunn, Katherine E.; Dannenberg, Frits; Ouldridge, Thomas E.; Kwiatkowska, Marta; Turberfield, Andrew J.; Bath, Jonathan

2015-09-01

DNA origami is a robust assembly technique that folds a single-stranded DNA template into a target structure by annealing it with hundreds of short `staple' strands. Its guiding design principle is that the target structure is the single most stable configuration. The folding transition is cooperative and, as in the case of proteins, is governed by information encoded in the polymer sequence. A typical origami folds primarily into the desired shape, but misfolded structures can kinetically trap the system and reduce the yield. Although adjusting assembly conditions or following empirical design rules can improve yield, well-folded origami often need to be separated from misfolded structures. The problem could in principle be avoided if assembly pathway and kinetics were fully understood and then rationally optimized. To this end, here we present a DNA origami system with the unusual property of being able to form a small set of distinguishable and well-folded shapes that represent discrete and approximately degenerate energy minima in a vast folding landscape, thus allowing us to probe the assembly process. The obtained high yield of well-folded origami structures confirms the existence of efficient folding pathways, while the shape distribution provides information about individual trajectories through the folding landscape. We find that, similarly to protein folding, the assembly of DNA origami is highly cooperative; that reversible bond formation is important in recovering from transient misfoldings; and that the early formation of long-range connections can very effectively enforce particular folds. We use these insights to inform the design of the system so as to steer assembly towards desired structures. Expanding the rational design process to include the assembly pathway should thus enable more reproducible synthesis, particularly when targeting more complex structures. We anticipate that this expansion will be essential if DNA origami is to continue its rapid development and become a reliable manufacturing technology.
Guiding the folding pathway of DNA origami.

PubMed

Dunn, Katherine E; Dannenberg, Frits; Ouldridge, Thomas E; Kwiatkowska, Marta; Turberfield, Andrew J; Bath, Jonathan

2015-09-03

DNA origami is a robust assembly technique that folds a single-stranded DNA template into a target structure by annealing it with hundreds of short 'staple' strands. Its guiding design principle is that the target structure is the single most stable configuration. The folding transition is cooperative and, as in the case of proteins, is governed by information encoded in the polymer sequence. A typical origami folds primarily into the desired shape, but misfolded structures can kinetically trap the system and reduce the yield. Although adjusting assembly conditions or following empirical design rules can improve yield, well-folded origami often need to be separated from misfolded structures. The problem could in principle be avoided if assembly pathway and kinetics were fully understood and then rationally optimized. To this end, here we present a DNA origami system with the unusual property of being able to form a small set of distinguishable and well-folded shapes that represent discrete and approximately degenerate energy minima in a vast folding landscape, thus allowing us to probe the assembly process. The obtained high yield of well-folded origami structures confirms the existence of efficient folding pathways, while the shape distribution provides information about individual trajectories through the folding landscape. We find that, similarly to protein folding, the assembly of DNA origami is highly cooperative; that reversible bond formation is important in recovering from transient misfoldings; and that the early formation of long-range connections can very effectively enforce particular folds. We use these insights to inform the design of the system so as to steer assembly towards desired structures. Expanding the rational design process to include the assembly pathway should thus enable more reproducible synthesis, particularly when targeting more complex structures. We anticipate that this expansion will be essential if DNA origami is to continue its rapid development and become a reliable manufacturing technology.
Single molecule RNA folding studied with optical trapping

NASA Astrophysics Data System (ADS)

Vieregg, Jeffrey Robert

The RNA folding problem (predicting the equilibrium structure and folding pathway of an RNA molecule from its sequence) is one of the classic problems of biophysics. Recent discoveries of many new functions for RNA have increased its importance, and new instrumental techniques have provided new ways to characterize molecular behavior. In particular, optical trapping (optical tweezers) allows controlled mechanical force to be applied to single RNA molecules while their end-to-end extension is monitored in real time. This enables characterization of RNA folding dynamics at a level unreachable by traditional bulk methods. Furthermore, recent advances in statistical mechanics make it possible to recover equilibrium quantities such as free energy from reactions which occur away from equilibrium. This dissertation describes the application of optical trapping and non-equilibrium statistical mechanics to quantitatively characterize folding of RNA secondary structures. By measuring the folding free energy of several specially designed hairpins in solutions containing various amounts of sodium and potassium, we were able to determine that RNA secondary structure thermodynamics depends not only on monovalent cation concentration but also surprisingly, on species. We also investigated the temperature dependence of hairpin folding thermodynamics and kinetics, which provided a direct measurement of enthalpy and entropy for RNA folding at physiological temperatures. We found that the folding pathway was quite sensitive to both salt and temperature, as measured by the folding success rate of a biologically important hairpin from the HIV-1 viral genome. Finally, I discuss modeling of force-induced RNA folding and unfolding, as well as a series of efforts which have dramatically improved the performance of our optical trapping instrument.
Does proline isomerization shape the folding funnel of the wild type and mutant staphylococcal nuclease?

NASA Astrophysics Data System (ADS)

Tsong, Tian Yow; Su, Zheng-Ding

1999-10-01

Cis/trans isomerization of proline residues is known to exhibit high activation energies. These kinetic barriers often dominate the energy landscape of protein folding. There are 6 proline residues (at positions 11, 31, 42, 47, 56 and 117) in staphylococcal nuclease (SNase) [EC 3.1.31.1]. Stopped-flow CD222nm measuring the evolution of the secondary structure of protein has detected 5 kinetic barriers in SNase folding (ΔG≠ for τfr<15, τf1 16.9, τf2 18.5, τf3 19.5, and τfs 21.8 kcal/mol) and 3 kinetic barriers in unfolding (ΔG≠ for τur<15, τu1 17.4, τus 21.6 kcal/mol). To investigate systematically how individual proline residues and 6 proline residues in toto can shape the folding funnel we have expediently constructed 7 proline mutants for study. They are 6 single-proline-substituted mutants (P11A, P31A, P42A, P47A, P56A and P117A) and 1 proline-free mutant (PallA). Study of equilibrium folding/unfolding and stopped-flow kinetics of the wildtype and the 7 mutants of SNase have allowed us to identify sources of 3 main kinetic barriers in the SNase folding. The highest barrier (ΔG≠=21.8 kcal) belongs to the cis/trans isomerization of Pro117. The next barrier (ΔG≠=19.5 kcal) involves synergetic effects of proline residues which limits the rate of folding of the oligonucleotide binding (OB) domain in all 7 proline-containing SNase. For the proline-free mutant (PallA) the OB domain folds rapidly. Furthermore, we have found that the equilibrium folding/unfolding properties of these proline mutants are remarkably similar to that of the wildtype despite their startlingly different folding/unfolding kinetics. These results lead us to conclude that while free energy of folding (ΔGF=-4.5 kcal/mol) provides the driving force, it is the activation energy that forms a conduit or shapes a kinetic funnel for SNase folding. The landscape for SNase folding is extremely rugged. Data support our previously proposed Least Activation Path (LAP) model for protein folding [Su, Z.D. et al. Proc. Natl. Acad. Sci. USA 93, 2539-2544 (1996)]. The LAP concept depicts protein folding as the movement of the unfolded population of protein along deep valleys of the energy landscape to reach the free energy minimum of the native state. An analogy for the LAP model would be flow of water from a highland over the rugged surface of a landscape to reach the lowest point of the ground. The fine features of the landscape will dictate kinetics and pathways of the flow.
Folding kinetics of WW domains with the united residue force field for bridging microscopic motions and experimental measurements

PubMed Central

Zhou, Rui; Maisuradze, Gia G.; Suñol, David; Todorovski, Toni; Macias, Maria J.; Xiao, Yi; Scheraga, Harold A.; Czaplewski, Cezary; Liwo, Adam

2014-01-01

To demonstrate the utility of the coarse-grained united-residue (UNRES) force field to compare experimental and computed kinetic data for folding proteins, we have performed long-time millisecond-timescale canonical Langevin molecular dynamics simulations of the triple β-strand from the Formin binding protein 28 WW domain and six nonnatural variants, using UNRES. The results have been compared with available experimental data in both a qualitative and a quantitative manner. Complexities of the folding pathways, which cannot be determined experimentally, were revealed. The folding mechanisms obtained from the simulated folding kinetics are in agreement with experimental results, with a few discrepancies for which we have accounted. The origins of single- and double-exponential kinetics and their correlations with two- and three-state folding scenarios are shown to be related to the relative barrier heights between the various states. The rate constants obtained from time profiles of the fractions of the native, intermediate, and unfolded structures, and the kinetic equations fitted to them, correlate with the experimental values; however, they are about three orders of magnitude larger than the experimental ones for most of the systems. These differences are in agreement with the timescale extension derived by scaling down the friction of water and averaging out the fast degrees of freedom when passing from all-atom to a coarse-grained representation. Our results indicate that the UNRES force field can provide accurate predictions of folding kinetics of these WW domains, often used as models for the study of the mechanisms of proein folding. PMID:25489078
Folding kinetics of WW domains with the united residue force field for bridging microscopic motions and experimental measurements.

PubMed

Zhou, Rui; Maisuradze, Gia G; Suñol, David; Todorovski, Toni; Macias, Maria J; Xiao, Yi; Scheraga, Harold A; Czaplewski, Cezary; Liwo, Adam

2014-12-23

To demonstrate the utility of the coarse-grained united-residue (UNRES) force field to compare experimental and computed kinetic data for folding proteins, we have performed long-time millisecond-timescale canonical Langevin molecular dynamics simulations of the triple β-strand from the Formin binding protein 28 WW domain and six nonnatural variants, using UNRES. The results have been compared with available experimental data in both a qualitative and a quantitative manner. Complexities of the folding pathways, which cannot be determined experimentally, were revealed. The folding mechanisms obtained from the simulated folding kinetics are in agreement with experimental results, with a few discrepancies for which we have accounted. The origins of single- and double-exponential kinetics and their correlations with two- and three-state folding scenarios are shown to be related to the relative barrier heights between the various states. The rate constants obtained from time profiles of the fractions of the native, intermediate, and unfolded structures, and the kinetic equations fitted to them, correlate with the experimental values; however, they are about three orders of magnitude larger than the experimental ones for most of the systems. These differences are in agreement with the timescale extension derived by scaling down the friction of water and averaging out the fast degrees of freedom when passing from all-atom to a coarse-grained representation. Our results indicate that the UNRES force field can provide accurate predictions of folding kinetics of these WW domains, often used as models for the study of the mechanisms of proein folding.
RNA folding kinetics using Monte Carlo and Gillespie algorithms.

PubMed

Clote, Peter; Bayegan, Amir H

2018-04-01

RNA secondary structure folding kinetics is known to be important for the biological function of certain processes, such as the hok/sok system in E. coli. Although linear algebra provides an exact computational solution of secondary structure folding kinetics with respect to the Turner energy model for tiny ([Formula: see text]20 nt) RNA sequences, the folding kinetics for larger sequences can only be approximated by binning structures into macrostates in a coarse-grained model, or by repeatedly simulating secondary structure folding with either the Monte Carlo algorithm or the Gillespie algorithm. Here we investigate the relation between the Monte Carlo algorithm and the Gillespie algorithm. We prove that asymptotically, the expected time for a K-step trajectory of the Monte Carlo algorithm is equal to [Formula: see text] times that of the Gillespie algorithm, where [Formula: see text] denotes the Boltzmann expected network degree. If the network is regular (i.e. every node has the same degree), then the mean first passage time (MFPT) computed by the Monte Carlo algorithm is equal to MFPT computed by the Gillespie algorithm multiplied by [Formula: see text]; however, this is not true for non-regular networks. In particular, RNA secondary structure folding kinetics, as computed by the Monte Carlo algorithm, is not equal to the folding kinetics, as computed by the Gillespie algorithm, although the mean first passage times are roughly correlated. Simulation software for RNA secondary structure folding according to the Monte Carlo and Gillespie algorithms is publicly available, as is our software to compute the expected degree of the network of secondary structures of a given RNA sequence-see http://bioinformatics.bc.edu/clote/RNAexpNumNbors .
Effects of mutation, truncation, and temperature on the folding kinetics of a WW domain.

PubMed

Maisuradze, Gia G; Zhou, Rui; Liwo, Adam; Xiao, Yi; Scheraga, Harold A

2012-07-20

The purpose of this work is to show how mutation, truncation, and change of temperature can influence the folding kinetics of a protein. This is accomplished by principal component analysis of molecular-dynamics-generated folding trajectories of the triple β-strand WW domain from formin binding protein 28 (FBP28) (Protein Data Bank ID: 1E0L) and its full-size, and singly- and doubly-truncated mutants at temperatures below and very close to the melting point. The reasons for biphasic folding kinetics [i.e., coexistence of slow (three-state) and fast (two-state) phases], including the involvement of a solvent-exposed hydrophobic cluster and another delocalized hydrophobic core in the folding kinetics, are discussed. New folding pathways are identified in free-energy landscapes determined in terms of principal components for full-size mutants. Three-state folding is found to be a main mechanism for folding the FBP28 WW domain and most of the full-size and truncated mutants. The results from the theoretical analysis are compared to those from experiment. Agreements and discrepancies between the theoretical and experimental results are discussed. Because of its importance in understanding protein kinetics and function, the diffusive mechanism by which the FBP28 WW domain and its full-size and truncated mutants explore their conformational space is examined in terms of the mean-square displacement and principal component analysis eigenvalue spectrum analyses. Subdiffusive behavior is observed for all studied systems. Copyright © 2012. Published by Elsevier Ltd.
Effects of mutation, truncation and temperature on the folding kinetics of a WW domain

PubMed Central

Maisuradze, Gia G.; Zhou, Rui; Liwo, Adam; Xiao, Yi; Scheraga, Harold A.

2013-01-01

The purpose of this work is to show how mutation, truncation and change of temperature can influence the folding kinetics of a protein. This is accomplished by principal component analysis (PCA) of molecular dynamics (MD)-generated folding trajectories of the triple β-strand WW domain from the Formin binding protein 28 (FBP) [PDB: 1E0L] and its full-size, and singly- and doubly-truncated mutants at temperatures below and very close to the melting point. The reasons for biphasic folding kinetics [i.e., coexistence of slow (three-state) and fast (two-state) phases], including the involvement of a solvent-exposed hydrophobic cluster and another delocalized hydrophobic core in the folding kinetics, are discussed. New folding pathways are identified in free-energy landscapes determined in terms of principal components for full-size mutants. Three-state folding is found to be a main mechanism for folding FBP28 WW domain and most of the full-size and truncated mutants. The results from the theoretical analysis are compared to those from experiment. Agreements and discrepancies between the theoretical and experimental results are discussed. Because of its importance in understanding protein kinetics and function, the diffusive mechanism by which FBP28 WW domain and its full-size and truncated mutants explore their conformational space is examined in terms of the mean-square displacement, (MSD), and PCA eigenvalue spectrum analyses. Subdiffusive behavior is observed for all studied systems. PMID:22560992
Principal component analysis for protein folding dynamics.

PubMed

Maisuradze, Gia G; Liwo, Adam; Scheraga, Harold A

2009-01-09

Protein folding is considered here by studying the dynamics of the folding of the triple beta-strand WW domain from the Formin-binding protein 28. Starting from the unfolded state and ending either in the native or nonnative conformational states, trajectories are generated with the coarse-grained united residue (UNRES) force field. The effectiveness of principal components analysis (PCA), an already established mathematical technique for finding global, correlated motions in atomic simulations of proteins, is evaluated here for coarse-grained trajectories. The problems related to PCA and their solutions are discussed. The folding and nonfolding of proteins are examined with free-energy landscapes. Detailed analyses of many folding and nonfolding trajectories at different temperatures show that PCA is very efficient for characterizing the general folding and nonfolding features of proteins. It is shown that the first principal component captures and describes in detail the dynamics of a system. Anomalous diffusion in the folding/nonfolding dynamics is examined by the mean-square displacement (MSD) and the fractional diffusion and fractional kinetic equations. The collisionless (or ballistic) behavior of a polypeptide undergoing Brownian motion along the first few principal components is accounted for.
Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways.

PubMed

Aghera, Nilesh; Udgaonkar, Jayant B

2012-07-13

Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased. Copyright © 2012 Elsevier Ltd. All rights reserved.
Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments.

PubMed

Mizukami, Takuya; Abe, Yukiko; Maki, Kosuke

2015-01-01

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0-2.2 M) than the formation of the native state (0-1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7-2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Wołek, Karol; Cieplak, Marek, E-mail: mc@ifpan.edu.pl

In structure-based models of proteins, one often assumes that folding is accomplished when all contacts are established. This assumption may frequently lead to a conceptual problem that folding takes place in a temperature region of very low thermodynamic stability, especially when the contact map used is too sparse. We consider six different structure-based models and show that allowing for a small, but model-dependent, percentage of the native contacts not being established boosts the folding temperature substantially while affecting the time scales of folding only in a minor way. We also compare other properties of the six models. We show thatmore » the choice of the description of the backbone stiffness has a substantial effect on the values of characteristic temperatures that relate both to equilibrium and kinetic properties. Models without any backbone stiffness (like the self-organized polymer) are found to perform similar to those with the stiffness, including in the studies of stretching.« less
Transiently disordered tails accelerate folding of globular proteins.

PubMed

Mallik, Saurav; Ray, Tanaya; Kundu, Sudip

2017-07-01

Numerous biological proteins exhibit intrinsic disorder at their termini, which are associated with multifarious functional roles. Here, we show the surprising result that an increased percentage of terminal short transiently disordered regions with enhanced flexibility (TstDREF) is associated with accelerated folding rates of globular proteins. Evolutionary conservation of predicted disorder at TstDREFs and drastic alteration of folding rates upon point-mutations suggest critical regulatory role(s) of TstDREFs in shaping the folding kinetics. TstDREFs are associated with long-range intramolecular interactions and the percentage of native secondary structural elements physically contacted by TstDREFs exhibit another surprising positive correlation with folding kinetics. These results allow us to infer probable molecular mechanisms behind the TstDREF-mediated regulation of folding kinetics that challenge protein biochemists to assess by direct experimental testing. © 2017 Federation of European Biochemical Societies.
Protein functional landscapes, dynamics, allostery: a tortuous path towards a universal theoretical framework.

PubMed

Zhuravlev, Pavel I; Papoian, Garegin A

2010-08-01

Energy landscape theories have provided a common ground for understanding the protein folding problem, which once seemed to be overwhelmingly complicated. At the same time, the native state was found to be an ensemble of interconverting states with frustration playing a more important role compared to the folding problem. The landscape of the folded protein - the native landscape - is glassier than the folding landscape; hence, a general description analogous to the folding theories is difficult to achieve. On the other hand, the native basin phase volume is much smaller, allowing a protein to fully sample its native energy landscape on the biological timescales. Current computational resources may also be used to perform this sampling for smaller proteins, to build a 'topographical map' of the native landscape that can be used for subsequent analysis. Several major approaches to representing this topographical map are highlighted in this review, including the construction of kinetic networks, hierarchical trees and free energy surfaces with subsequent structural and kinetic analyses. In this review, we extensively discuss the important question of choosing proper collective coordinates characterizing functional motions. In many cases, the substates on the native energy landscape, which represent different functional states, can be used to obtain variables that are well suited for building free energy surfaces and analyzing the protein's functional dynamics. Normal mode analysis can provide such variables in cases where functional motions are dictated by the molecule's architecture. Principal component analysis is a more expensive way of inferring the essential variables from the protein's motions, one that requires a long molecular dynamics simulation. Finally, the two popular models for the allosteric switching mechanism, 'preexisting equilibrium' and 'induced fit', are interpreted within the energy landscape paradigm as extreme points of a continuum of transition mechanisms. Some experimental evidence illustrating each of these two models, as well as intermediate mechanisms, is presented and discussed.
Collapse kinetics and chevron plots from simulations of denaturant-dependent folding of globular proteins

PubMed Central

Liu, Zhenxing; Reddy, Govardhan; O’Brien, Edward P.; Thirumalai, D.

2011-01-01

Quantitative description of how proteins fold under experimental conditions remains a challenging problem. Experiments often use urea and guanidinium chloride to study folding whereas the natural variable in simulations is temperature. To bridge the gap, we use the molecular transfer model that combines measured denaturant-dependent transfer free energies for the peptide group and amino acid residues, and a coarse-grained Cα-side chain model for polypeptide chains to simulate the folding of src SH3 domain. Stability of the native state decreases linearly as [C] (the concentration of guanidinium chloride) increases with the slope, m, that is in excellent agreement with experiments. Remarkably, the calculated folding rate at [C] = 0 is only 16-fold larger than the measured value. Most importantly ln kobs (kobs is the sum of folding and unfolding rates) as a function of [C] has the characteristic V (chevron) shape. In every folding trajectory, the times for reaching the native state, interactions stabilizing all the substructures, and global collapse coincide. The value of (mf is the slope of the folding arm of the chevron plot) is identical to the fraction of buried solvent accessible surface area in the structures of the transition state ensemble. In the dominant transition state, which does not vary significantly at low [C], the core of the protein and certain loops are structured. Besides solving the long-standing problem of computing the chevron plot, our work lays the foundation for incorporating denaturant effects in a physically transparent manner either in all-atom or coarse-grained simulations. PMID:21512127
Collapse kinetics and chevron plots from simulations of denaturant-dependent folding of globular proteins.

PubMed

Liu, Zhenxing; Reddy, Govardhan; O'Brien, Edward P; Thirumalai, D

2011-05-10

Quantitative description of how proteins fold under experimental conditions remains a challenging problem. Experiments often use urea and guanidinium chloride to study folding whereas the natural variable in simulations is temperature. To bridge the gap, we use the molecular transfer model that combines measured denaturant-dependent transfer free energies for the peptide group and amino acid residues, and a coarse-grained C(α)-side chain model for polypeptide chains to simulate the folding of src SH(3) domain. Stability of the native state decreases linearly as [C] (the concentration of guanidinium chloride) increases with the slope, m, that is in excellent agreement with experiments. Remarkably, the calculated folding rate at [C] = 0 is only 16-fold larger than the measured value. Most importantly ln k(obs) (k(obs) is the sum of folding and unfolding rates) as a function of [C] has the characteristic V (chevron) shape. In every folding trajectory, the times for reaching the native state, interactions stabilizing all the substructures, and global collapse coincide. The value of (m(f) is the slope of the folding arm of the chevron plot) is identical to the fraction of buried solvent accessible surface area in the structures of the transition state ensemble. In the dominant transition state, which does not vary significantly at low [C], the core of the protein and certain loops are structured. Besides solving the long-standing problem of computing the chevron plot, our work lays the foundation for incorporating denaturant effects in a physically transparent manner either in all-atom or coarse-grained simulations.

Identification of kinetically hot residues in proteins.

PubMed Central

Demirel, M. C.; Atilgan, A. R.; Jernigan, R. L.; Erman, B.; Bahar, I.

1998-01-01

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed. PMID:9865946
Influence of the native topology on the folding barrier for small proteins

NASA Astrophysics Data System (ADS)

Prieto, Lidia; Rey, Antonio

2007-11-01

The possibility of downhill instead of two-state folding for proteins has been a very controversial topic which arose from recent experimental studies. From the theoretical side, this question has also been accomplished in different ways. Given the experimental observation that a relationship exists between the native structure topology of a protein and the kinetic and thermodynamic properties of its folding process, Gō-type potentials are an appropriate way to approach this problem. In this work, we employ an interaction potential from this family to get a better insight on the topological characteristics of the native state that may somehow determine the presence of a thermodynamic barrier in the folding pathway. The results presented here show that, indeed, the native topology of a small protein has a great influence on its folding behavior, mostly depending on the proportion of local and long range contacts the protein has in its native structure. Furthermore, when all the interactions present contribute in a balanced way, the transition results to be cooperative. Otherwise, the tendency to a downhill folding behavior increases.
Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

PubMed Central

Lim, Shion A.; Hart, Kathryn M.; Marqusee, Susan

2016-01-01

Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein’s folding process, which is described by its energy landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein’s energy landscape is maintained or altered throughout evolution is unclear. To study how a protein’s energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We found that despite large sequence divergence, the overall folding pathway is conserved over billions of years of evolution. There are robust trends in the rates of protein folding and unfolding; both modern RNases H evolved to be more kinetically stable than their most recent common ancestor. Finally, our study demonstrates how a partially folded intermediate provides a readily adaptable folding landscape by allowing the independent tuning of kinetics and thermodynamics. PMID:27799545
Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate

PubMed Central

Stump, Matthew R.; Gloss, Lisa M.

2008-01-01

Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central α-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with ΔG°(H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1. PMID:18096639
Chaperonin-based biolayer interferometry to assess the kinetic stability of metastable, aggregation-prone proteins

PubMed Central

Lea, Wendy A.; Naik, Subhashchandra; Chaudhri, Tapan; Machen, Alexandra J.; O’Neil, Pierce T.; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T.; Burns, Joshua R.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.

2017-01-01

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules/solution conditions that can stabilize or destabilize thermally stable proteins, multi-domain proteins, oligomeric proteins, and most importantly, aggregation prone metastable proteins. PMID:27505032
Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

PubMed Central

Mizukami, Takuya; Abe, Yukiko; Maki, Kosuke

2015-01-01

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin. PMID:26244984
Learning To Fold Proteins Using Energy Landscape Theory

PubMed Central

Schafer, N.P.; Kim, B.L.; Zheng, W.; Wolynes, P.G.

2014-01-01

This review is a tutorial for scientists interested in the problem of protein structure prediction, particularly those interested in using coarse-grained molecular dynamics models that are optimized using lessons learned from the energy landscape theory of protein folding. We also present a review of the results of the AMH/AMC/AMW/AWSEM family of coarse-grained molecular dynamics protein folding models to illustrate the points covered in the first part of the article. Accurate coarse-grained structure prediction models can be used to investigate a wide range of conceptual and mechanistic issues outside of protein structure prediction; specifically, the paper concludes by reviewing how AWSEM has in recent years been able to elucidate questions related to the unusual kinetic behavior of artificially designed proteins, multidomain protein misfolding, and the initial stages of protein aggregation. PMID:25308991
Ligand-promoted protein folding by biased kinetic partitioning.

PubMed

Hingorani, Karan S; Metcalf, Matthew C; Deming, Derrick T; Garman, Scott C; Powers, Evan T; Gierasch, Lila M

2017-04-01

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.
Ligand-Promoted Protein Folding by Biased Kinetic Partitioning

PubMed Central

Hingorani, Karan S.; Metcalf, Matthew C.; Deming, Derrick T.; Garman, Scott C.; Powers, Evan T.; Gierasch, Lila M.

2017-01-01

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems. PMID:28218913
Highly Anomalous Energetics of Protein Cold Denaturation Linked to Folding-Unfolding Kinetics

PubMed Central

Romero-Romero, M. Luisa; Inglés-Prieto, Alvaro; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

2011-01-01

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a “mirror image” of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions. PMID:21829584
Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.

PubMed

Bingaman, Jamie L; Messina, Kyle J; Bevilacqua, Philip C

2017-05-01

Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions. Copyright © 2017 Elsevier Inc. All rights reserved.
Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics

PubMed Central

Bingaman, Jamie L.; Messina, Kyle J.; Bevilacqua, Philip C.

2017-01-01

Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand-mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions. PMID:28315484
Comparative analysis of the folding dynamics and kinetics of an engineered knotted protein and its variants derived from HP0242 of Helicobacter pylori

NASA Astrophysics Data System (ADS)

Wang, Liang-Wei; Liu, Yu-Nan; Lyu, Ping-Chiang; Jackson, Sophie E.; Hsu, Shang-Te Danny

2015-09-01

Understanding the mechanism by which a polypeptide chain thread itself spontaneously to attain a knotted conformation has been a major challenge in the field of protein folding. HP0242 is a homodimeric protein from Helicobacter pylori with intertwined helices to form a unique pseudo-knotted folding topology. A tandem HP0242 repeat has been constructed to become the first engineered trefoil-knotted protein. Its small size renders it a model system for computational analyses to examine its folding and knotting pathways. Here we report a multi-parametric study on the folding stability and kinetics of a library of HP0242 variants, including the trefoil-knotted tandem HP0242 repeat, using far-UV circular dichroism and fluorescence spectroscopy. Equilibrium chemical denaturation of HP0242 variants shows the presence of highly populated dimeric and structurally heterogeneous folding intermediates. Such equilibrium folding intermediates retain significant amount of helical structures except those at the N- and C-terminal regions in the native structure. Stopped-flow fluorescence measurements of HP0242 variants show that spontaneous refolding into knotted structures can be achieved within seconds, which is several orders of magnitude faster than previously observed for other knotted proteins. Nevertheless, the complex chevron plots indicate that HP0242 variants are prone to misfold into kinetic traps, leading to severely rolled-over refolding arms. The experimental observations are in general agreement with the previously reported molecular dynamics simulations. Based on our results, kinetic folding pathways are proposed to qualitatively describe the complex folding processes of HP0242 variants.
The equilibrium properties and folding kinetics of an all-atom Go model of the Trp-cage.

PubMed

Linhananta, Apichart; Boer, Jesse; MacKay, Ian

2005-03-15

The ultrafast-folding 20-residue Trp-cage protein is quickly becoming a new benchmark for molecular dynamics studies. Already several all-atom simulations have probed its equilibrium and kinetic properties. In this work an all-atom Go model is used to accurately represent the side-chain packing and native atomic contacts of the Trp-cage. The model reproduces the hallmark thermodynamics cooperativity of small proteins. Folding simulations observe that in the fast-folding dominant pathway, partial alpha-helical structure forms before hydrophobic core collapse. In the slow-folding secondary pathway, partial core collapse occurs before helical structure. The slow-folding rate of the secondary pathway is attributed to the loss of side-chain rotational freedom, due to the early core collapse, which impedes the helix formation. A major finding is the observation of a low-temperature kinetic intermediate stabilized by a salt bridge between residues Asp-9 and Arg-16. Similar observations [R. Zhou, Proc. Natl. Acad. Sci. U.S.A. 100, 13280 (2003)] were reported in a recent study using an all-atom model of the Trp-cage in explicit water, in which the salt-bridge stabilized intermediate was hypothesized to be the origin of the ultrafast-folding mechanism. A theoretical mutation that eliminates the Asp-9-Arg-16 salt bridge, but leaves the residues intact, is performed. Folding simulations of the mutant Trp-cage observe a two-state free-energy landscape with no kinetic intermediate and a significant decrease in the folding rate, in support of the hypothesis.
The equilibrium properties and folding kinetics of an all-atom Go xAF model of the Trp-cage

NASA Astrophysics Data System (ADS)

Linhananta, Apichart; Boer, Jesse; MacKay, Ian

2005-03-01

The ultrafast-folding 20-residue Trp-cage protein is quickly becoming a new benchmark for molecular dynamics studies. Already several all-atom simulations have probed its equilibrium and kinetic properties. In this work an all-atom Go ¯ model is used to accurately represent the side-chain packing and native atomic contacts of the Trp-cage. The model reproduces the hallmark thermodynamics cooperativity of small proteins. Folding simulations observe that in the fast-folding dominant pathway, partial α-helical structure forms before hydrophobic core collapse. In the slow-folding secondary pathway, partial core collapse occurs before helical structure. The slow-folding rate of the secondary pathway is attributed to the loss of side-chain rotational freedom, due to the early core collapse, which impedes the helix formation. A major finding is the observation of a low-temperature kinetic intermediate stabilized by a salt bridge between residues Asp-9 and Arg-16. Similar observations [R. Zhou, Proc. Natl. Acad. Sci. U.S.A. 100, 13280 (2003)] were reported in a recent study using an all-atom model of the Trp-cage in explicit water, in which the salt-bridge stabilized intermediate was hypothesized to be the origin of the ultrafast-folding mechanism. A theoretical mutation that eliminates the Asp-9-Arg-16 salt bridge, but leaves the residues intact, is performed. Folding simulations of the mutant Trp-cage observe a two-state free-energy landscape with no kinetic intermediate and a significant decrease in the folding rate, in support of the hypothesis.
SeqRate: sequence-based protein folding type classification and rates prediction

PubMed Central

2010-01-01

Background Protein folding rate is an important property of a protein. Predicting protein folding rate is useful for understanding protein folding process and guiding protein design. Most previous methods of predicting protein folding rate require the tertiary structure of a protein as an input. And most methods do not distinguish the different kinetic nature (two-state folding or multi-state folding) of the proteins. Here we developed a method, SeqRate, to predict both protein folding kinetic type (two-state versus multi-state) and real-value folding rate using sequence length, amino acid composition, contact order, contact number, and secondary structure information predicted from only protein sequence with support vector machines. Results We systematically studied the contributions of individual features to folding rate prediction. On a standard benchmark dataset, the accuracy of folding kinetic type classification is 80%. The Pearson correlation coefficient and the mean absolute difference between predicted and experimental folding rates (sec-1) in the base-10 logarithmic scale are 0.81 and 0.79 for two-state protein folders, and 0.80 and 0.68 for three-state protein folders. SeqRate is the first sequence-based method for protein folding type classification and its accuracy of fold rate prediction is improved over previous sequence-based methods. Its performance can be further enhanced with additional information, such as structure-based geometric contacts, as inputs. Conclusions Both the web server and software of predicting folding rate are publicly available at http://casp.rnet.missouri.edu/fold_rate/index.html. PMID:20438647
General mechanism of two-state protein folding kinetics.

PubMed

Rollins, Geoffrey C; Dill, Ken A

2014-08-13

We describe here a general model of the kinetic mechanism of protein folding. In the Foldon Funnel Model, proteins fold in units of secondary structures, which form sequentially along the folding pathway, stabilized by tertiary interactions. The model predicts that the free energy landscape has a volcano shape, rather than a simple funnel, that folding is two-state (single-exponential) when secondary structures are intrinsically unstable, and that each structure along the folding path is a transition state for the previous structure. It shows how sequential pathways are consistent with multiple stochastic routes on funnel landscapes, and it gives good agreement with the 9 order of magnitude dependence of folding rates on protein size for a set of 93 proteins, at the same time it is consistent with the near independence of folding equilibrium constant on size. This model gives estimates of folding rates of proteomes, leading to a median folding time in Escherichia coli of about 5 s.
Detection-dependent kinetics as a probe of folding landscape microstructure.

PubMed

Yang, Wei Yuan; Gruebele, Martin

2004-06-30

The folding landscapes of polypeptides and proteins exhibit a hierarchy of local minima. The causes range from proline isomerization all the way down to microstructure in the free energy caused by residual frustration inherent in even the best 20 amino acid design. The corresponding time scales range from hours to submicroseconds. The smallest microstructures are difficult to detect. We have measured the folding/unfolding kinetics of the engineered trpzip2 peptide at different tryptophan fluorescence wavelengths, each yielding a different rate. Wavelength-dependent folding kinetics on 0.1-2 mus time scales show that different microstructures with a range of solvent exposure and local dynamics are populated. We estimate a lower limit for the roughness of the free energy surface based on the range of rates observed.
Spontaneous Unfolding-Refolding of Fibronectin Type III Domains Assayed by Thiol Exchange

PubMed Central

Shah, Riddhi; Ohashi, Tomoo; Erickson, Harold P.; Oas, Terrence G.

2017-01-01

Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand β-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, ∼0.1–1 s−1, for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils. PMID:27909052
Proline Can Have Opposite Effects on Fast and Slow Protein Folding Phases

PubMed Central

Osváth, Szabolcs; Gruebele, Martin

2003-01-01

Proline isomerization is well known to cause additional slow phases during protein refolding. We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10–100 min) and very fast (4 μs–2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation. Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines. Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle. The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature. Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule. The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales. PMID:12885665

Periodic and stochastic thermal modulation of protein folding kinetics.

PubMed

Platkov, Max; Gruebele, Martin

2014-07-21

Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.
How the folding rates of two- and multistate proteins depend on the amino acid properties.

PubMed

Huang, Jitao T; Huang, Wei; Huang, Shanran R; Li, Xin

2014-10-01

Proteins fold by either two-state or multistate kinetic mechanism. We observe that amino acids play different roles in different mechanism. Many residues that are easy to form regular secondary structures (α helices, β sheets and turns) can promote the two-state folding reactions of small proteins. Most of hydrophilic residues can speed up the multistate folding reactions of large proteins. Folding rates of large proteins are equally responsive to the flexibility of partial amino acids. Other properties of amino acids (including volume, polarity, accessible surface, exposure degree, isoelectric point, and phase transfer energy) have contributed little to folding kinetics of the proteins. Cysteine is a special residue, it triggers two-state folding reaction and but inhibits multistate folding reaction. These findings not only provide a new insight into protein structure prediction, but also could be used to direct the point mutations that can change folding rate. © 2014 Wiley Periodicals, Inc.
Folding of human telomerase RNA pseudoknot using ion-jump and temperature-quench simulations.

PubMed

Biyun, Shi; Cho, Samuel S; Thirumalai, D

2011-12-21

Globally RNA folding occurs in multiple stages involving chain compaction and subsequent rearrangement by a number of parallel routes to the folded state. However, the sequence-dependent details of the folding pathways and the link between collapse and folding are poorly understood. To obtain a comprehensive picture of the thermodynamics and folding kinetics we used molecular simulations of coarse-grained model of a pseudoknot found in the conserved core domain of the human telomerase (hTR) by varying both temperature (T) and ion concentration (C). The phase diagram in the [T,C] plane shows that the boundary separating the folded and unfolded state for the finite 47-nucleotide system is relatively sharp, implying that from a thermodynamic perspective hTR behaves as an apparent two-state system. However, the folding kinetics following single C-jump or T-quench is complicated, involving multiple channels to the native state. Although globally folding kinetics triggered by T-quench and C-jump are similar, the kinetics of chain compaction are vastly different, which reflects the role of initial conditions in directing folding and collapse. Remarkably, even after substantial reduction in the overall size of hTR, the ensemble of compact conformations are far from being nativelike, suggesting that the search for the folded state occurs among the ensemble of low-energy fluidlike globules. The rate of unfolding, which occurs in a single step, is faster upon C-decrease compared to a jump in temperature. To identify "hidden" states that are visited during the folding process we performed simulations by periodically interrupting the approach to the folded state by lowering C. These simulations show that hTR reaches the folded state through a small number of connected clusters that are repeatedly visited during the pulse sequence in which the folding or unfolding is interrupted. The results from interrupted folding simulations, which are in accord with non-equilibrium single-molecule folding of a large ribozyme, show that multiple probes are needed to reveal the invisible states that are sampled by RNA as it folds. Although we have illustrated the complexity of RNA folding using hTR as a case study, general arguments and qualitative comparisons to time-resolved scattering experiments on Azoarcus group I ribozyme and single-molecule non-equilibrium periodic ion-jump experiments establish the generality of our findings. © 2011 American Chemical Society
Aromatic residues engineered into the beta-turn nucleation site of ubiquitin lead to a complex folding landscape, non-native side-chain interactions, and kinetic traps.

PubMed

Rea, Anita M; Simpson, Emma R; Meldrum, Jill K; Williams, Huw E L; Searle, Mark S

2008-12-02

The fast folding of small proteins is likely to be the product of evolutionary pressures that balance the search for native-like contacts in the transition state with the minimum number of stable non-native interactions that could lead to partially folded states prone to aggregation and amyloid formation. We have investigated the effects of non-native interactions on the folding landscape of yeast ubiquitin by introducing aromatic substitutions into the beta-turn region of the N-terminal beta-hairpin, using both the native G-bulged type I turn sequence (TXTGK) as well as an engineered 2:2 XNGK type I' turn sequence. The N-terminal beta-hairpin is a recognized folding nucleation site in ubiquitin. The folding kinetics for wt-Ub (TLTGK) and the type I' turn mutant (TNGK) reveal only a weakly populated intermediate, however, substitution with X = Phe or Trp in either context results in a high propensity to form a stable compact intermediate where the initial U-->I collapse is visible as a distinct kinetic phase. The introduction of Trp into either of the two host turn sequences results in either complex multiphase kinetics with the possibility of parallel folding pathways, or formation of a highly compact I-state stabilized by non-native interactions that must unfold before refolding. Sequence substitutions with aromatic residues within a localized beta-turn capable of forming non-native hydrophobic contacts in both the native state and partially folded states has the undesirable consequence that folding is frustrated by the formation of stable compact intermediates that evolutionary pressures at the sequence level may have largely eliminated.
Topography of funneled landscapes determines the thermodynamics and kinetics of protein folding

PubMed Central

Wang, Jin; Oliveira, Ronaldo J.; Chu, Xiakun; Whitford, Paul C.; Chahine, Jorge; Han, Wei; Wang, Erkang; Onuchic, José N.; Leite, Vitor B.P.

2012-01-01

The energy landscape approach has played a fundamental role in advancing our understanding of protein folding. Here, we quantify protein folding energy landscapes by exploring the underlying density of states. We identify three quantities essential for characterizing landscape topography: the stabilizing energy gap between the native and nonnative ensembles δE, the energetic roughness ΔE, and the scale of landscape measured by the entropy S. We show that the dimensionless ratio between the gap, roughness, and entropy of the system accurately predicts the thermodynamics, as well as the kinetics of folding. Large Λ implies that the energy gap (or landscape slope towards the native state) is dominant, leading to more funneled landscapes. We investigate the role of topological and energetic roughness for proteins of different sizes and for proteins of the same size, but with different structural topologies. The landscape topography ratio Λ is shown to be monotonically correlated with the thermodynamic stability against trapping, as characterized by the ratio of folding temperature versus trapping temperature. Furthermore, Λ also monotonically correlates with the folding kinetic rates. These results provide the quantitative bridge between the landscape topography and experimental folding measurements. PMID:23019359
Course 12: Proteins: Structural, Thermodynamic and Kinetic Aspects

NASA Astrophysics Data System (ADS)

Finkelstein, A. V.

1 Introduction 2 Overview of protein architectures and discussion of physical background of their natural selection 2.1 Protein structures 2.2 Physical selection of protein structures 3 Thermodynamic aspects of protein folding 3.1 Reversible denaturation of protein structures 3.2 What do denatured proteins look like? 3.3 Why denaturation of a globular protein is the first-order phase transition 3.4 "Gap" in energy spectrum: The main characteristic that distinguishes protein chains from random polymers 4 Kinetic aspects of protein folding 4.1 Protein folding in vivo 4.2 Protein folding in vitro (in the test-tube) 4.3 Theory of protein folding rates and solution of the Levinthal paradox
General Mechanism of Two-State Protein Folding Kinetics

PubMed Central

Rollins, Geoffrey C.; Dill, Ken A.

2016-01-01

We describe here a general model of the kinetic mechanism of protein folding. In the Foldon Funnel Model, proteins fold in units of secondary structures, which form sequentially along the folding pathway, stabilized by tertiary interactions. The model predicts that the free energy landscape has a volcano shape, rather than a simple funnel, that folding is two-state (single-exponential) when secondary structures are intrinsically unstable, and that each structure along the folding path is a transition state for the previous structure. It shows how sequential pathways are consistent with multiple stochastic routes on funnel landscapes, and it gives good agreement with the 9 order of magnitude dependence of folding rates on protein size for a set of 93 proteins, at the same time it is consistent with the near independence of folding equilibrium constant on size. This model gives estimates of folding rates of proteomes, leading to a median folding time in Escherichia coli of about 5 s. PMID:25056406
Impact of hydrodynamic interactions on protein folding rates depends on temperature

NASA Astrophysics Data System (ADS)

Zegarra, Fabio C.; Homouz, Dirar; Eliaz, Yossi; Gasic, Andrei G.; Cheung, Margaret S.

2018-03-01

We investigated the impact of hydrodynamic interactions (HI) on protein folding using a coarse-grained model. The extent of the impact of hydrodynamic interactions, whether it accelerates, retards, or has no effect on protein folding, has been controversial. Together with a theoretical framework of the energy landscape theory (ELT) for protein folding that describes the dynamics of the collective motion with a single reaction coordinate across a folding barrier, we compared the kinetic effects of HI on the folding rates of two protein models that use a chain of single beads with distinctive topologies: a 64-residue α /β chymotrypsin inhibitor 2 (CI2) protein, and a 57-residue β -barrel α -spectrin Src-homology 3 domain (SH3) protein. When comparing the protein folding kinetics simulated with Brownian dynamics in the presence of HI to that in the absence of HI, we find that the effect of HI on protein folding appears to have a "crossover" behavior about the folding temperature. This means that at a temperature greater than the folding temperature, the enhanced friction from the hydrodynamic solvents between the beads in an unfolded configuration results in lowered folding rate; conversely, at a temperature lower than the folding temperature, HI accelerates folding by the backflow of solvent toward the folded configuration of a protein. Additionally, the extent of acceleration depends on the topology of a protein: for a protein like CI2, where its folding nucleus is rather diffuse in a transition state, HI channels the formation of contacts by favoring a major folding pathway in a complex free energy landscape, thus accelerating folding. For a protein like SH3, where its folding nucleus is already specific and less diffuse, HI matters less at a temperature lower than the folding temperature. Our findings provide further theoretical insight to protein folding kinetic experiments and simulations.
An overlapping region between the two terminal folding units of the outer surface protein A (OspA) controls its folding behavior.

PubMed

Makabe, Koki; Nakamura, Takashi; Dhar, Debanjan; Ikura, Teikichi; Koide, Shohei; Kuwajima, Kunihiro

2018-04-27

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions. Copyright © 2018. Published by Elsevier Ltd.
Renormalized Hamiltonian for a peptide chain: Digitalizing the protein folding problem

NASA Astrophysics Data System (ADS)

Fernández, Ariel; Colubri, Andrés

2000-05-01

A renormalized Hamiltonian for a flexible peptide chain is derived to generate the long-time limit dynamics compatible with a coarsening of torsional conformation space. The renormalization procedure is tailored taking into account the coarse graining imposed by the backbone torsional constraints due to the local steric hindrance and the local backbone-side-group interactions. Thus, the torsional degrees of freedom for each residue are resolved modulo basins of attraction in its so-called Ramachandran map. This Ramachandran renormalization (RR) procedure is implemented so that the chain is energetically driven to form contact patterns as their respective collective topological constraints are fulfilled within the coarse description. In this way, the torsional dynamics are digitalized and become codified as an evolving pattern in a binary matrix. Each accepted Monte Carlo step in a canonical ensemble simulation is correlated with the real mean first passage time it takes to reach the destination coarse topological state. This real-time correlation enables us to test the RR dynamics by comparison with experimentally probed kinetic bottlenecks along the dominant folding pathway. Such intermediates are scarcely populated at any given time, but they determine the kinetic funnel leading to the active structure. This landscape region is reached through kinetically controlled steps needed to overcome the conformational entropy of the random coil. The results are specialized for the bovine pancreatic trypsin inhibitor, corroborating the validity of our method.
Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics.

PubMed

Bisaria, Namita; Greenfeld, Max; Limouse, Charles; Mabuchi, Hideo; Herschlag, Daniel

2017-09-12

Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity-that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔG align , the probability of aligning tertiary contact partners, and ΔG tert , the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔG HJH ) or from changes in the electrostatic environment (ΔG +/- ) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔG tert ). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔG tert values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.
Quantifying the topography of the intrinsic energy landscape of flexible biomolecular recognition

PubMed Central

Chu, Xiakun; Gan, Linfeng; Wang, Erkang; Wang, Jin

2013-01-01

Biomolecular functions are determined by their interactions with other molecules. Biomolecular recognition is often flexible and associated with large conformational changes involving both binding and folding. However, the global and physical understanding for the process is still challenging. Here, we quantified the intrinsic energy landscapes of flexible biomolecular recognition in terms of binding–folding dynamics for 15 homodimers by exploring the underlying density of states, using a structure-based model both with and without considering energetic roughness. By quantifying three individual effective intrinsic energy landscapes (one for interfacial binding, two for monomeric folding), the association mechanisms for flexible recognition of 15 homodimers can be classified into two-state cooperative “coupled binding–folding” and three-state noncooperative “folding prior to binding” scenarios. We found that the association mechanism of flexible biomolecular recognition relies on the interplay between the underlying effective intrinsic binding and folding energy landscapes. By quantifying the whole global intrinsic binding–folding energy landscapes, we found strong correlations between the landscape topography measure Λ (dimensionless ratio of energy gap versus roughness modulated by the configurational entropy) and the ratio of the thermodynamic stable temperature versus trapping temperature, as well as between Λ and binding kinetics. Therefore, the global energy landscape topography determines the binding–folding thermodynamics and kinetics, crucial for the feasibility and efficiency of realizing biomolecular function. We also found “U-shape” temperature-dependent kinetic behavior and a dynamical cross-over temperature for dividing exponential and nonexponential kinetics for two-state homodimers. Our study provides a unique way to bridge the gap between theory and experiments. PMID:23754431
Redesigning the type II' β-turn in green fluorescent protein to type I': implications for folding kinetics and stability.

PubMed

Madan, Bharat; Sokalingam, Sriram; Raghunathan, Govindan; Lee, Sun-Gu

2014-10-01

Both Type I' and Type II' β-turns have the same sense of the β-turn twist that is compatible with the β-sheet twist. They occur predominantly in two residue β-hairpins, but the occurrence of Type I' β-turns is two times higher than Type II' β-turns. This suggests that Type I' β-turns may be more stable than Type II' β-turns, and Type I' β-turn sequence and structure can be more favorable for protein folding than Type II' β-turns. Here, we redesigned the native Type II' β-turn in GFP to Type I' β-turn, and investigated its effect on protein folding and stability. The Type I' β-turns were designed based on the statistical analysis of residues in natural Type I' β-turns. The substitution of the native "GD" sequence of i+1 and i+2 residues with Type I' preferred "(N/D)G" sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β-turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β-turns in natural β-hairpins can be further optimized by converting the sequence to Type I'. © 2014 Wiley Periodicals, Inc.
How interfaces affect hydrophobically driven polymer folding.

PubMed

Jamadagni, Sumanth N; Godawat, Rahul; Dordick, Jonathan S; Garde, Shekhar

2009-04-02

Studies of folding-unfolding of hydrophobic polymers in water provide an excellent starting point to probe manybody hydrophobic interactions in the context of realistic self-assembly processes. Such studies in bulk water have highlighted the similarities between thermodynamics of polymer collapse and of protein folding, and emphasized the role of hydration-water structure, density, and fluctuations-in the folding kinetics. Hydrophobic polymers are interfacially active-that is, they prefer locations at aqueous interfaces relative to bulk water-consistent with their low solubility. How does the presence of a hydrophobic solid surface or an essentially hydrophobic vapor-water interface affect the structural, thermodynamic, and kinetic aspects of polymer folding? Using extensive molecular dynamics simulations, we show that the large hydrophobic driving force for polymer collapse in bulk water is reduced at a solid alkane-water interface and further reduced at a vapor-water interface. As a result, at the solid-water interface, folded structures are marginally stable, whereas the vapor-liquid interface unfolds polymers completely. Structural sampling is also significantly affected by the interface. For example, at the solid-water interface, polymer conformations are quasi-2- dimensional, with folded states being pancake-like structures. At the vapor-water interface, the hydrophobic polymer is significantly excluded from the water phase and freely samples a broad range of compact to extended structures. Interestingly, although the driving force for folding is considerably lower, kinetics of folding are faster at both interfaces, highlighting the role of enhanced water fluctuations and dynamics at a hydrophobic interface.
Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

PubMed Central

Ganguly, Debabani; Zhang, Weihong; Chen, Jianhan

2013-01-01

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation. PMID:24278008
Thermodynamics and kinetics of protein folding on the ribosome: Alteration in energy landscapes, denatured state, and transition state ensembles

NASA Astrophysics Data System (ADS)

O'Brien, Edward; Vendruscolo, Michele; Dobson, Christopher

2010-03-01

In vitro experiments examining cotranslational folding utilize ribosome-nascent chain complexes (RNCs) in which the nascent chain is stalled at different points of its biosynthesis on the ribosome. We investigate the thermodynamics, kinetics, and structural properties of RNCs containing five different globular and repeat proteins stalled at ten different nascent chain lengths using coarse grained replica exchange simulations. We find that when the proteins are stalled near the ribosome exit tunnel opening they exhibit altered folding coopserativity, quantified by the van't Hoff enthalpy criterion; a significantly altered denatured state ensemble, in terms of Rg and shape parameters (Rg tensor); and the appearance of partially folded intermediates during cotranslation, evidenced by the appearance of a third basin in the free energy profile. These trends are due in part to excluded volume (crowding) interactions between the ribosome and nascent chain. We perform in silico temperature-jump experiments on the RNCs and examine nascent chain folding kinetics and structural changes in the transition state ensemble at various stall lengths.
Structural analysis of kinetic folding intermediates for a TIM barrel protein, indole-3-glycerol phosphate synthase, by hydrogen exchange mass spectrometry and Gō-model simulation

PubMed Central

Gu, Zhenyu; Rao, Maithreyi K.; Forsyth, William R.

2009-01-01

The structures of partially-folded states appearing during the folding of a (βα)8 TIM barrel protein, the indole-3-glycerol phosphate synthase from S. solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō-model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (βα)4 region, modest protection in the neighboring (βα)1–3 and (βα)5β6 segments and no significant protection in the remaining N- and C-terminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (βα)2–5β6 region after 5 s of folding demonstrates that these species represent kinetically-distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a Cα Gō-model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of structure offering protection against exchange in the on-pathway intermediate(s). Because the native-centric Gō-model simulations do not explicitly include sequence-specific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō-model simulation. PMID:17942114
Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks.

PubMed Central

Downs, W D; Cech, T R

1996-01-01

The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures. PMID:8756414
Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains

PubMed Central

Daidone, Isabella; Neuweiler, Hannes; Doose, Sören; Sauer, Markus; Smith, Jeremy C.

2010-01-01

Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20–100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient β-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events. PMID:20098498
Prediction of RNA secondary structures: from theory to models and real molecules

NASA Astrophysics Data System (ADS)

Schuster, Peter

2006-05-01

RNA secondary structures are derived from RNA sequences, which are strings built form the natural four letter nucleotide alphabet, {AUGC}. These coarse-grained structures, in turn, are tantamount to constrained strings over a three letter alphabet. Hence, the secondary structures are discrete objects and the number of sequences always exceeds the number of structures. The sequences built from two letter alphabets form perfect structures when the nucleotides can form a base pair, as is the case with {GC} or {AU}, but the relation between the sequences and structures differs strongly from the four letter alphabet. A comprehensive theory of RNA structure is presented, which is based on the concepts of sequence space and shape space, being a space of structures. It sets the stage for modelling processes in ensembles of RNA molecules like evolutionary optimization or kinetic folding as dynamical phenomena guided by mappings between the two spaces. The number of minimum free energy (mfe) structures is always smaller than the number of sequences, even for two letter alphabets. Folding of RNA molecules into mfe energy structures constitutes a non-invertible mapping from sequence space onto shape space. The preimage of a structure in sequence space is defined as its neutral network. Similarly the set of suboptimal structures is the preimage of a sequence in shape space. This set represents the conformation space of a given sequence. The evolutionary optimization of structures in populations is a process taking place in sequence space, whereas kinetic folding occurs in molecular ensembles that optimize free energy in conformation space. Efficient folding algorithms based on dynamic programming are available for the prediction of secondary structures for given sequences. The inverse problem, the computation of sequences for predefined structures, is an important tool for the design of RNA molecules with tailored properties. Simultaneous folding or cofolding of two or more RNA molecules can be modelled readily at the secondary structure level and allows prediction of the most stable (mfe) conformations of complexes together with suboptimal states. Cofolding algorithms are important tools for efficient and highly specific primer design in the polymerase chain reaction (PCR) and help to explain the mechanisms of small interference RNA (si-RNA) molecules in gene regulation. The evolutionary optimization of RNA structures is illustrated by the search for a target structure and mimics aptamer selection in evolutionary biotechnology. It occurs typically in steps consisting of short adaptive phases interrupted by long epochs of little or no obvious progress in optimization. During these quasi-stationary epochs the populations are essentially confined to neutral networks where they search for sequences that allow a continuation of the adaptive process. Modelling RNA evolution as a simultaneous process in sequence and shape space provides answers to questions of the optimal population size and mutation rates. Kinetic folding is a stochastic process in conformation space. Exact solutions are derived by direct simulation in the form of trajectory sampling or by solving the master equation. The exact solutions can be approximated straightforwardly by Arrhenius kinetics on barrier trees, which represent simplified versions of conformational energy landscapes. The existence of at least one sequence forming any arbitrarily chosen pair of structures is granted by the intersection theorem. Folding kinetics is the key to understanding and designing multistable RNA molecules or RNA switches. These RNAs form two or more long lived conformations, and conformational changes occur either spontaneously or are induced through binding of small molecules or other biopolymers. RNA switches are found in nature where they act as elements in genetic and metabolic regulation. The reliability of RNA secondary structure prediction is limited by the accuracy with which the empirical parameters can be determined and by principal deficiencies, for example by the lack of energy contributions resulting from tertiary interactions. In addition, native structures may be determined by folding kinetics rather than by thermodynamics. We address the first problem by considering base pair probabilities or base pairing entropies, which are derived from the partition function of conformations. A high base pair probability corresponding to a low pairing entropy is taken as an indicator of a high reliability of prediction. Pseudoknots are discussed as an example of a tertiary interaction that is highly important for RNA function. Moreover, pseudoknot formation is readily incorporated into structure prediction algorithms. Some examples of experimental data on RNA secondary structures that are readily explained using the landscape concept are presented. They deal with (i) properties of RNA molecules with random sequences, (ii) RNA molecules from restricted alphabets, (iii) existence of neutral networks, (iv) shape space covering, (v) riboswitches and (vi) evolution of non-coding RNAs as an example of evolution restricted to neutral networks.

The H2A-H2B dimeric kinetic intermediate is stabilized by widespread hydrophobic burial with few fully native interactions.

PubMed

Guyett, Paul J; Gloss, Lisa M

2012-01-20

The H2A-H2B histone heterodimer folds via monomeric and dimeric kinetic intermediates. Within ∼5 ms, the H2A and H2B polypeptides associate in a nearly diffusion limited reaction to form a dimeric ensemble, denoted I₂ and I₂*, the latter being a subpopulation characterized by a higher content of nonnative structure (NNS). The I₂ ensemble folds to the native heterodimer, N₂, through an observable, first-order kinetic phase. To determine the regions of structure in the I₂ ensemble, we characterized 26 Ala mutants of buried hydrophobic residues, spanning the three helices of the canonical histone folds of H2A and H2B and the H2B C-terminal helix. All but one targeted residue contributed significantly to the stability of I₂, the transition state and N₂; however, only residues in the hydrophobic core of the dimer interface perturbed the I₂* population. Destabilization of I₂* correlated with slower folding rates, implying that NNS is not a kinetic trap but rather accelerates folding. The pattern of Φ values indicated that residues forming intramolecular interactions in the peripheral helices contributed similar stability to I₂ and N₂, but residues involved in intermolecular interactions in the hydrophobic core are only partially folded in I₂. These findings suggest a dimerize-then-rearrange model. Residues throughout the histone fold contribute to the stability of I₂, but after the rapid dimerization reaction, the hydrophobic core of the dimer interface has few fully native interactions. In the transition state leading to N₂, more native-like interactions are developed and nonnative interactions are rearranged. Copyright © 2011 Elsevier Ltd. All rights reserved.
Combination of Markov state models and kinetic networks for the analysis of molecular dynamics simulations of peptide folding.

PubMed

Radford, Isolde H; Fersht, Alan R; Settanni, Giovanni

2011-06-09

Atomistic molecular dynamics simulations of the TZ1 beta-hairpin peptide have been carried out using an implicit model for the solvent. The trajectories have been analyzed using a Markov state model defined on the projections along two significant observables and a kinetic network approach. The Markov state model allowed for an unbiased identification of the metastable states of the system, and provided the basis for commitment probability calculations performed on the kinetic network. The kinetic network analysis served to extract the main transition state for folding of the peptide and to validate the results from the Markov state analysis. The combination of the two techniques allowed for a consistent and concise characterization of the dynamics of the peptide. The slowest relaxation process identified is the exchange between variably folded and denatured species, and the second slowest process is the exchange between two different subsets of the denatured state which could not be otherwise identified by simple inspection of the projected trajectory. The third slowest process is the exchange between a fully native and a partially folded intermediate state characterized by a native turn with a proximal backbone H-bond, and frayed side-chain packing and termini. The transition state for the main folding reaction is similar to the intermediate state, although a more native like side-chain packing is observed.
PREFACE Protein folding: lessons learned and new frontiers Protein folding: lessons learned and new frontiers

NASA Astrophysics Data System (ADS)

Pappu, Rohit V.; Nussinov, Ruth

2009-03-01

In appropriate physiological milieux proteins spontaneously fold into their functional three-dimensional structures. The amino acid sequences of functional proteins contain all the information necessary to specify the folds. This remarkable observation has spawned research aimed at answering two major questions. (1) Of all the conceivable structures that a protein can adopt, why is the ensemble of native-like structures the most favorable? (2) What are the paths by which proteins manage to robustly and reproducibly fold into their native structures? Anfinsen's thermodynamic hypothesis has guided the pursuit of answers to the first question whereas Levinthal's paradox has influenced the development of models for protein folding dynamics. Decades of work have led to significant advances in the folding problem. Mean-field models have been developed to capture our current, coarse grain understanding of the driving forces for protein folding. These models are being used to predict three-dimensional protein structures from sequence and stability profiles as a function of thermodynamic and chemical perturbations. Impressive strides have also been made in the field of protein design, also known as the inverse folding problem, thereby testing our understanding of the determinants of the fold specificities of different sequences. Early work on protein folding pathways focused on the specific sequence of events that could lead to a simplification of the search process. However, unifying principles proved to be elusive. Proteins that show reversible two-state folding-unfolding transitions turned out to be a gift of natural selection. Focusing on these simple systems helped researchers to uncover general principles regarding the origins of cooperativity in protein folding thermodynamics and kinetics. On the theoretical front, concepts borrowed from polymer physics and the physics of spin glasses led to the development of a framework based on energy landscape theories. These theories predict that evolved sequences (functional proteins as opposed to random sequences) find their native folds by minimizing geometric (topological) frustration (i.e. avoiding entropic bottlenecks/kinetic traps). In some cases, following a dominant pathway is the optimal way to minimize frustration, whereas in extreme cases, proteins may fold without encountering bottlenecks. Experimental studies of two-state proteins led in turn to the development of quantitative descriptors that have allowed specific testing of theoretical predictions. These include methods such as phi value analysis to characterize transition state ensembles and descriptors that measure the effects of geometry/topology on folding rates. Interestingly, there exists a striking inverse correlation between the relative contact order (the distance in sequence space between spatially proximal contacts made in the native state) and the folding rates of several two-state proteins. The relative contact order provides a rough estimate of the net entropic cost associated with realizing the folded state, and theories have been developed to explain the observed correlation between the contact order and folding rates. Despite its maturity as a field, there are several areas that come under the rubric of protein folding that are just beginning to receive attention. For example, how do complications in vivo such as macromolecular crowding, confinement, the presence of cosolutes, membrane anchoring, and tethering to surfaces influence protein stabilities and folding dynamics? While we are accustomed to studying proteins at concentrations that are amenable to investigation via probes whose signal intensities grow with protein concentration, this does not make these readouts relevant to the in vivo setting. In cells, protein concentrations are tightly regulated and are likely to be orders of magnitude lower than what we are accustomed to using within in vitro experimental setups. Protein folding in vivo is a complex multi-scale dynamical problem when one considers the synergies between protein expression, spontaneous folding, chaperonin-assisted folding, protein targeting, the kinetics of post-translational modifications, protein degradation, and of course the drive to avoid aggregation. Further, there is growing recognition that cells not only tolerate but select for proteins that are intrinsically disordered. These proteins are essential for many crucial activities, and yet their inability to fold in isolation makes them prone to proteolytic processing and aggregation. In the series of papers that make up this special focus on protein folding in physical biology, leading researchers provide insights into diverse cross-sections of problems in protein folding. Barrick provides a concise review of what we have learned from the study of two-state folders and draws attention to how several unanswered questions are being approached using studies on large repeat proteins. Dissecting the contribution of hydration-mediated interactions to driving forces for protein folding and assembly has been extremely challenging. There is renewed interest in using hydrostatic pressure as a tool to access folding intermediates and decipher the role of partially hydrated states in folding, misfolding, and aggregation. Silva and Foguel review many of the nuances that have been uncovered by perturbing hydrostatic pressure as a thermodynamic parameter. As noted above, protein folding in vivo is expected to be considerably more complex than the folding of two-state proteins in dilute solutions. Lucent et al review the state-of-the-art in the development of quantitative theories to explain chaperonin-assisted folding in vivo. Additionally, they highlight unanswered questions pertaining to the processing of unfolded/misfolded proteins by the chaperone machinery. Zhuang et al present results that focus on the effects of surface tethering on transition state ensembles and folding mechanisms of a model two-state protein. Their results are important because several proteins in vivo fold while being anchored to membranes. Finally, several neurodegenerative and systemic diseases are associated with the aggregation of intrinsically disordered polypeptides. The search for cures in these debilitating and fatal diseases has focused attention on shared attributes in aggregation mechanisms of different proteins and the possibility of identifying druggable targets from mechanistic studies. Abedini and Raleigh review common features gleaned from mechanistic studies of the aggregation of several intrinsically disordered proteins. They propose that the population of helical intermediates and their stabilization via interactions with membranes might be an important route by which the process of aggregation leads to toxicity. The five papers that form this protein folding focus cover specific sub-topics within the larger field of protein folding. They address current questions and emphasize the importance of the growing and productive interface between the physical sciences and biology. We hope that these papers will stimulate much discussion and more importantly advances in the areas highlighted by the contributors.
Approaching the thermodynamic view of protein folding through the reproduction of Anfinsen's experiment by undergraduate physical biochemistry students.

PubMed

Fernandez-Reche, Andres; Cobos, Eva S; Luque, Irene; Ruiz-Sanz, Javier; Martinez, Jose C

2018-01-04

In 1972 Christian B. Anfinsen received the Nobel Prize in Chemistry for "…his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation." The understanding of this principle is crucial for physical biochemistry students, since protein folding studies, bio-computing sciences and protein design approaches are founded on such a well-demonstrated connection. Herein, we describe a detailed and easy-to-follow experiment to reproduce the most relevant assays carried out at Anfinsen's laboratory in the 60s. This experiment provides students with a platform to interpret by themselves the structural and kinetic experiments conceived to understand the protein folding problem. In addition, this three-day experiment brings students a nice opportunity for protein manipulation as well as for the setting up of spectroscopic and chromatographic techniques. © 2018 by The International Union of Biochemistry and Molecular Biology, 2018. © 2018 The International Union of Biochemistry and Molecular Biology.
Solvent Effects on Protein Folding/Unfolding

NASA Astrophysics Data System (ADS)

García, A. E.; Hillson, N.; Onuchic, J. N.

Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.
Force feedback effects on single molecule hopping and pulling experiments

NASA Astrophysics Data System (ADS)

Rico-Pasto, M.; Pastor, I.; Ritort, F.

2018-03-01

Single-molecule experiments with optical tweezers have become an important tool to study the properties and mechanisms of biological systems, such as cells and nucleic acids. In particular, force unzipping experiments have been used to extract the thermodynamics and kinetics of folding and unfolding reactions. In hopping experiments, a molecule executes transitions between the unfolded and folded states at a preset value of the force [constant force mode (CFM) under force feedback] or trap position [passive mode (PM) without feedback] and the force-dependent kinetic rates extracted from the lifetime of each state (CFM) and the rupture force distributions (PM) using the Bell-Evans model. However, hopping experiments in the CFM are known to overestimate molecular distances and folding free energies for fast transitions compared to the response time of the feedback. In contrast, kinetic rate measurements from pulling experiments have been mostly done in the PM while the CFM is seldom implemented in pulling protocols. Here, we carry out hopping and pulling experiments in a short DNA hairpin in the PM and CFM at three different temperatures (6 °C, 25 °C, and 45 °C) exhibiting largely varying kinetic rates. As expected, we find that equilibrium hopping experiments in the CFM and PM perform well at 6 °C (where kinetics are slow), whereas the CFM overestimates molecular parameters at 45 °C (where kinetics are fast). In contrast, nonequilibrium pulling experiments perform well in both modes at all temperatures. This demonstrates that the same kind of feedback algorithm in the CFM leads to more reliable determination of the folding reaction parameters in irreversible pulling experiments.
Force feedback effects on single molecule hopping and pulling experiments.

PubMed

Rico-Pasto, M; Pastor, I; Ritort, F

2018-03-28

Single-molecule experiments with optical tweezers have become an important tool to study the properties and mechanisms of biological systems, such as cells and nucleic acids. In particular, force unzipping experiments have been used to extract the thermodynamics and kinetics of folding and unfolding reactions. In hopping experiments, a molecule executes transitions between the unfolded and folded states at a preset value of the force [constant force mode (CFM) under force feedback] or trap position [passive mode (PM) without feedback] and the force-dependent kinetic rates extracted from the lifetime of each state (CFM) and the rupture force distributions (PM) using the Bell-Evans model. However, hopping experiments in the CFM are known to overestimate molecular distances and folding free energies for fast transitions compared to the response time of the feedback. In contrast, kinetic rate measurements from pulling experiments have been mostly done in the PM while the CFM is seldom implemented in pulling protocols. Here, we carry out hopping and pulling experiments in a short DNA hairpin in the PM and CFM at three different temperatures (6 °C, 25 °C, and 45 °C) exhibiting largely varying kinetic rates. As expected, we find that equilibrium hopping experiments in the CFM and PM perform well at 6 °C (where kinetics are slow), whereas the CFM overestimates molecular parameters at 45 °C (where kinetics are fast). In contrast, nonequilibrium pulling experiments perform well in both modes at all temperatures. This demonstrates that the same kind of feedback algorithm in the CFM leads to more reliable determination of the folding reaction parameters in irreversible pulling experiments.
How Four Scientists Integrate Thermodynamic and Kinetic Theory, Context, Analogies, and Methods in Protein-Folding and Dynamics Research: Implications for Biochemistry Instruction

ERIC Educational Resources Information Center

Jeffery, Kathleen A.; Pelaez, Nancy; Anderson, Trevor R.

2018-01-01

To keep biochemistry instruction current and relevant, it is crucial to expose students to cutting-edge scientific research and how experts reason about processes governed by thermodynamics and kinetics such as protein folding and dynamics. This study focuses on how experts explain their research into this topic with the intention of informing…
Circuit topology of self-interacting chains: implications for folding and unfolding dynamics.

PubMed

Mugler, Andrew; Tans, Sander J; Mashaghi, Alireza

2014-11-07

Understanding the relationship between molecular structure and folding is a central problem in disciplines ranging from biology to polymer physics and DNA origami. Topology can be a powerful tool to address this question. For a folded linear chain, the arrangement of intra-chain contacts is a topological property because rearranging the contacts requires discontinuous deformations. Conversely, the topology is preserved when continuously stretching the chain while maintaining the contact arrangement. Here we investigate how the folding and unfolding of linear chains with binary contacts is guided by the topology of contact arrangements. We formalize the topology by describing the relations between any two contacts in the structure, which for a linear chain can either be in parallel, in series, or crossing each other. We show that even when other determinants of folding rate such as contact order and size are kept constant, this 'circuit' topology determines folding kinetics. In particular, we find that the folding rate increases with the fractions of parallel and crossed relations. Moreover, we show how circuit topology constrains the conformational phase space explored during folding and unfolding: the number of forbidden unfolding transitions is found to increase with the fraction of parallel relations and to decrease with the fraction of series relations. Finally, we find that circuit topology influences whether distinct intermediate states are present, with crossed contacts being the key factor. The approach presented here can be more generally applied to questions on molecular dynamics, evolutionary biology, molecular engineering, and single-molecule biophysics.
Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy

PubMed Central

Borgia, Alessandro; Wensley, Beth G.; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B.; Hoffmann, Armin; Pfeil, Shawn H.; Lipman, Everett A.; Clarke, Jane; Schuler, Benjamin

2012-01-01

Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes. PMID:23149740
Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy.

PubMed

Borgia, Alessandro; Wensley, Beth G; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B; Hoffmann, Armin; Pfeil, Shawn H; Lipman, Everett A; Clarke, Jane; Schuler, Benjamin

2012-01-01

Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes.
A novel microfluidic mixer based on dual-hydrodynamic focusing for interrogating the kinetics of DNA-protein interaction.

PubMed

Li, Ying; Xu, Fei; Liu, Chao; Xu, Youzhi; Feng, Xiaojun; Liu, Bi-Feng

2013-08-21

Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA-protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to <0.55 μL min⁻¹, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules.
Slow Proton Transfer Coupled to Unfolding Explains the Puzzling Results of Single-Molecule Experiments on BBL, a Paradigmatic Downhill Folding Protein

PubMed Central

Cerminara, Michele; Campos, Luis A.; Ramanathan, Ravishankar; Muñoz, Victor

2013-01-01

A battery of thermodynamic, kinetic, and structural approaches has indicated that the small α-helical protein BBL folds-unfolds via the one-state downhill scenario. Yet, single-molecule fluorescence spectroscopy offers a more conflicting view. Single-molecule experiments at pH 6 show a unique half-unfolded conformational ensemble at mid denaturation, whereas other experiments performed at higher pH show a bimodal distribution, as expected for two-state folding. Here we use thermodynamic and laser T-jump kinetic experiments combined with theoretical modeling to investigate the pH dependence of BBL stability, folding kinetics and mechanism within the pH 6–11 range. We find that BBL unfolding is tightly coupled to the protonation of one of its residues with an apparent pKa of ∼7. Therefore, in chemical denaturation experiments around neutral pH BBL unfolds gradually, and also converts in binary fashion to the protonated species. Moreover, under the single-molecule experimental conditions (denaturant midpoint and 279 K), we observe that proton transfer is much slower than the ∼15 microseconds folding-unfolding kinetics of BBL. The relaxation kinetics is distinctly biphasic, and the overall relaxation time (i.e. 0.2–0.5 ms) becomes controlled by the proton transfer step. We then show that a simple theoretical model of protein folding coupled to proton transfer explains quantitatively all these results as well as the two sets of single-molecule experiments, including their more puzzling features. Interestingly, this analysis suggests that BBL unfolds following a one-state downhill folding mechanism at all conditions. Accordingly, the source of the bimodal distributions observed during denaturation at pH 7–8 is the splitting of the unique conformational ensemble of BBL onto two slowly inter-converting protonation species. Both, the unprotonated and protonated species unfold gradually (one-state downhill), but they exhibit different degree of unfolding at any given condition because the native structure is less stable for the protonated form. PMID:24205082
Folding and unfolding single RNA molecules under tension

PubMed Central

Woodside, Michael T; García-García, Cuauhtémoc; Block, Steven M

2010-01-01

Single-molecule force spectroscopy constitutes a powerful method for probing RNA folding: it allows the kinetic, energetic, and structural properties of intermediate and transition states to be determined quantitatively, yielding new insights into folding pathways and energy landscapes. Recent advances in experimental and theoretical methods, including fluctuation theorems, kinetic theories, novel force clamps, and ultrastable instruments, have opened new avenues for study. These tools have been used to probe folding in simple model systems, for example, RNA and DNA hairpins. Knowledge gained from such systems is helping to build our understanding of more complex RNA structures composed of multiple elements, as well as how nucleic acids interact with proteins involved in key cellular activities, such as transcription and translation. PMID:18786653
New approach to the study of transient protein conformations: the formation of a semiburied salt link in the folding pathway of barnase.

PubMed

Oliveberg, M; Fersht, A R

1996-05-28

We use in this study a novel kinetic approach to determine the H+ titration properties of a semiburied salt link in the transition state for unfolding of barnase. The approach is based on changes in the pH dependence of the kinetics upon mutation of a target residue. This makes it relatively insensitive to the absolute value of the stability and, thereby, to artifacts caused by structural rearrangements around the site of mutation. The semiburied salt bridge studied here is between Asp93 and Arg69. Mutation of either residue significantly destabilized the protein, and the pKa value of Asp93 is severely lowered in the native state to below 1 because of the ionic interaction with Arg69. The Asp93-Arg69 salt link appears to be formed early in the folding process; the pKa value of Asp93 in the transition state (approximately 1) is similar to that in the native state, and deletion of the ionic interaction with Arg69 substantially destabilizes the folding intermediate and changes the kinetic behavior from multistate to two-state or close to two-state, depending on the conditions. The results suggest that the formation of ionic interactions within clusters of hydrophobic residues can be important for early folding events and can control kinetically the folding pathway. This is not because of the inherent stability of the salt link but because the presence of two unpaired charges is very unfavorable. The data reveal also that fractional phi values are consistent with a uniformly expanded transition state or one with closely spaced energy levels and not with parallel folding pathways.
Refolding dynamics of stretched biopolymers upon force quench

PubMed Central

Hyeon, Changbong; Morrison, Greg; Pincus, David L.; Thirumalai, D.

2009-01-01

Single-molecule force spectroscopy methods can be used to generate folding trajectories of biopolymers from arbitrary regions of the folding landscape. We illustrate the complexity of the folding kinetics and generic aspects of the collapse of RNA and proteins upon force quench by using simulations of an RNA hairpin and theory based on the de Gennes model for homopolymer collapse. The folding time, τF, depends asymmetrically on δfS = f S − f m and δf Q = f m − f Q where f S (f Q) is the stretch (quench) force and f m is the transition midforce of the RNA hairpin. In accord with experiments, the relaxation kinetics of the molecular extension, R(t), occurs in three stages: A rapid initial decrease in the extension is followed by a plateau and finally, an abrupt reduction in R(t) occurs as the native state is approached. The duration of the plateau increases as λ = τ Q/τ F decreases (where τ Q is the time in which the force is reduced from f S to f Q). Variations in the mechanisms of force-quench relaxation as λ is altered are reflected in the experimentally measurable time-dependent entropy, which is computed directly from the folding trajectories. An analytical solution of the de Gennes model under tension reproduces the multistage stage kinetics in R(t). The prediction that the initial stages of collapse should also be a generic feature of polymers is validated by simulation of the kinetics of toroid (globule) formation in semiflexible (flexible) homopolymers in poor solvents upon quenching the force from a fully stretched state. Our findings give a unified explanation for multiple disparate experimental observations of protein folding. PMID:19915145
Electrostatically Accelerated Coupled Binding and Folding of Intrinsically Disordered Proteins

PubMed Central

Ganguly, Debabani; Otieno, Steve; Waddell, Brett; Iconaru, Luigi; Kriwacki, Richard W.; Chen, Jianhan

2012-01-01

Intrinsically disordered proteins (IDPs) are now recognized to be prevalent in biology, and many potential functional benefits have been discussed. However, the frequent requirement of peptide folding in specific interactions of IDPs could impose a kinetic bottleneck, which could be overcome only by efficient folding upon encounter. Intriguingly, existing kinetic data suggest that specific binding of IDPs is generally no slower than that of globular proteins. Here, we exploited the cell cycle regulator p27Kip1 (p27) as a model system to understand how IDPs might achieve efficient folding upon encounter for facile recognition. Combining experiments and coarse-grained modeling, we demonstrate that long-range electrostatic interactions between enriched charges on p27 and near its binding site on cyclin A not only enhance the encounter rate (i.e., electrostatic steering), but also promote folding-competent topologies in the encounter complexes, allowing rapid subsequent formation of short-range native interactions en route to the specific complex. In contrast, nonspecific hydrophobic interactions, while hardly affecting the encounter rate, can significantly reduce the efficiency of folding upon encounter and lead to slower binding kinetics. Further analysis of charge distributions in a set of known IDP complexes reveals that, although IDP binding sites tend to be more hydrophobic compared to the rest of the target surface, their vicinities are frequently enriched with charges to complement those on IDPs. This observation suggests that electrostatically accelerated encounter and induced folding might represent a prevalent mechanism for promoting facile IDP recognition. PMID:22721951
Impact of ion binding on poly-L-lysine (un)folding energy landscape and kinetics.

PubMed

Xiong, Kan; Asher, Sanford A

2012-06-21

We utilize T-jump UV resonance Raman spectroscopy (UVRR) to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix, and turns) of PLL are slower than those of short alanine-based peptides. The PLL pure α-helix folding time is similar to that of short alanine-based peptides. We for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO(4)(-) binding to the Lys side chain -NH(3)(+) groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO(4)(-) binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate(s) differs from the unfolding coordinate(s). The-π helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process.
Folding pathway of a multidomain protein depends on its topology of domain connectivity

PubMed Central

Inanami, Takashi; Terada, Tomoki P.; Sasai, Masaki

2014-01-01

How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant. PMID:25267632
The Energy Landscape, Folding Pathways and the Kinetics of a Knotted Protein

PubMed Central

Prentiss, Michael C.; Wales, David J.; Wolynes, Peter G.

2010-01-01

The folding pathway and rate coefficients of the folding of a knotted protein are calculated for a potential energy function with minimal energetic frustration. A kinetic transition network is constructed using the discrete path sampling approach, and the resulting potential energy surface is visualized by constructing disconnectivity graphs. Owing to topological constraints, the low-lying portion of the landscape consists of three distinct regions, corresponding to the native knotted state and to configurations where either the N or C terminus is not yet folded into the knot. The fastest folding pathways from denatured states exhibit early formation of the N terminus portion of the knot and a rate-determining step where the C terminus is incorporated. The low-lying minima with the N terminus knotted and the C terminus free therefore constitute an off-pathway intermediate for this model. The insertion of both the N and C termini into the knot occurs late in the folding process, creating large energy barriers that are the rate limiting steps in the folding process. When compared to other protein folding proteins of a similar length, this system folds over six orders of magnitude more slowly. PMID:20617197

On the Role of Entropy in the Protein Folding Process

NASA Astrophysics Data System (ADS)

Hoppe, Travis

2011-12-01

A protein's ultimate function and activity is determined by the unique three-dimensional structure taken by the folding process. Protein malfunction due to misfolding is the culprit of many clinical disorders, such as abnormal protein aggregations. This leads to neurodegenerative disorders like Huntington's and Alzheimer's disease. We focus on a subset of the folding problem, exploring the role and effects of entropy on the process of protein folding. Four major concepts and models are developed and each pertains to a specific aspect of the folding process: entropic forces, conformational states under crowding, aggregation, and macrostate kinetics from microstate trajectories. The exclusive focus on entropy is well-suited for crowding studies, as many interactions are nonspecific. We show how a stabilizing entropic force can arise purely from the motion of crowders in solution. In addition we are able to make a a quantitative prediction of the crowding effect with an implicit crowding approximation using an aspherical scaled-particle theory. In order to investigate the effects of aggregation, we derive a new operator expansion method to solve the Ising/Potts model with external fields over an arbitrary graph. Here the external fields are representative of the entropic forces. We show that this method reduces the problem of calculating the partition function to the solution of recursion relations. Many of the methods employed are coarse-grained approximations. As such, it is useful to have a viable method for extracting macrostate information from time series data. We develop a method to cluster the microstates into physically meaningful macrostates by grouping similar relaxation times from a transition matrix. Overall, the studied topics allow us to understand deeper the complicated process involving proteins.
Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

PubMed Central

Gruber, Tobias; Balbach, Jochen

2015-01-01

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state. PMID:26368922
Discrete kinetic models from funneled energy landscape simulations.

PubMed

Schafer, Nicholas P; Hoffman, Ryan M B; Burger, Anat; Craig, Patricio O; Komives, Elizabeth A; Wolynes, Peter G

2012-01-01

A general method for facilitating the interpretation of computer simulations of protein folding with minimally frustrated energy landscapes is detailed and applied to a designed ankyrin repeat protein (4ANK). In the method, groups of residues are assigned to foldons and these foldons are used to map the conformational space of the protein onto a set of discrete macrobasins. The free energies of the individual macrobasins are then calculated, informing practical kinetic analysis. Two simple assumptions about the universality of the rate for downhill transitions between macrobasins and the natural local connectivity between macrobasins lead to a scheme for predicting overall folding and unfolding rates, generating chevron plots under varying thermodynamic conditions, and inferring dominant kinetic folding pathways. To illustrate the approach, free energies of macrobasins were calculated from biased simulations of a non-additive structure-based model using two structurally motivated foldon definitions at the full and half ankyrin repeat resolutions. The calculated chevrons have features consistent with those measured in stopped flow chemical denaturation experiments. The dominant inferred folding pathway has an "inside-out", nucleation-propagation like character.
Kinetics from Replica Exchange Molecular Dynamics Simulations.

PubMed

Stelzl, Lukas S; Hummer, Gerhard

2017-08-08

Transitions between metastable states govern many fundamental processes in physics, chemistry and biology, from nucleation events in phase transitions to the folding of proteins. The free energy surfaces underlying these processes can be obtained from simulations using enhanced sampling methods. However, their altered dynamics makes kinetic and mechanistic information difficult or impossible to extract. Here, we show that, with replica exchange molecular dynamics (REMD), one can not only sample equilibrium properties but also extract kinetic information. For systems that strictly obey first-order kinetics, the procedure to extract rates is rigorous. For actual molecular systems whose long-time dynamics are captured by kinetic rate models, accurate rate coefficients can be determined from the statistics of the transitions between the metastable states at each replica temperature. We demonstrate the practical applicability of the procedure by constructing master equation (Markov state) models of peptide and RNA folding from REMD simulations.
Using enzyme folding to explore the mechanism of therapeutic touch: a feasibility study.

PubMed

Strickland, Mallory L; Boylan, Helen M

2010-07-01

The goal of this research is to design a novel model using protein folding to study Therapeutic Touch, a noncontact form of energy manipulation healing. Presented is a feasibility study suggesting that the denaturation path of ribonuclease A may be a useful model to study the energy exchange underlying therapeutic touch. The folding of ribonuclease A serves as a controlled energy-requiring system in which energy manipulation can be measured by the degree of folding achieved. A kinetic assay and fluorescence spectroscopy are used to assess the enzyme-folding state. The data suggest that the kinetic assay is a useful means of assessing the degree of refolding, and specifically, the enzyme function. However, fluorescence spectroscopy was not shown to be an effective measurement of enzyme structure for the purposes of this work. More research is needed to assess the underlying mechanism of therapeutic touch to complement the existing studies. An enzyme-folding model may provide a useful means of studying the energy exchange in therapeutic touch.
Navigating ligand protein binding free energy landscapes: universality and diversity of protein folding and molecular recognition mechanisms

NASA Astrophysics Data System (ADS)

Verkhivker, Gennady M.; Rejto, Paul A.; Bouzida, Djamal; Arthurs, Sandra; Colson, Anthony B.; Freer, Stephan T.; Gehlhaar, Daniel K.; Larson, Veda; Luty, Brock A.; Marrone, Tami; Rose, Peter W.

2001-03-01

Thermodynamic and kinetic aspects of ligand-protein binding are studied for the methotrexate-dihydrofolate reductase system from the binding free energy profile constructed as a function of the order parameter. Thermodynamic stability of the native complex and a cooperative transition to the unique native structure suggest the nucleation kinetic mechanism at the equilibrium transition temperature. Structural properties of the transition state ensemble and the ensemble of nucleation conformations are determined by kinetic simulations of the transmission coefficient and ligand-protein association pathways. Structural analysis of the transition states and the nucleation conformations reconciles different views on the nucleation mechanism in protein folding.
Hidden complexity of free energy surfaces for peptide (protein) folding.

PubMed

Krivov, Sergei V; Karplus, Martin

2004-10-12

An understanding of the thermodynamics and kinetics of protein folding requires a knowledge of the free energy surface governing the motion of the polypeptide chain. Because of the many degrees of freedom involved, surfaces projected on only one or two progress variables are generally used in descriptions of the folding reaction. Such projections result in relatively smooth surfaces, but they could mask the complexity of the unprojected surface. Here we introduce an approach to determine the actual (unprojected) free energy surface and apply it to the second beta-hairpin of protein G, which has been used as a model system for protein folding. The surface is represented by a disconnectivity graph calculated from a long equilibrium folding-unfolding trajectory. The denatured state is found to have multiple low free energy basins. Nevertheless, the peptide shows exponential kinetics in folding to the native basin. Projected surfaces obtained from the present analysis have a simple form in agreement with other studies of the beta-hairpin. The hidden complexity found for the beta-hairpin surface suggests that the standard funnel picture of protein folding should be revisited.
Expression and refolding of tobacco anionic peroxidase from E. coli inclusion bodies.

PubMed

Hushpulian, D M; Savitski, P A; Rojkova, A M; Chubar, T A; Fechina, V A; Sakharov, I Yu; Lagrimini, L M; Tishkov, V I; Gazaryan, I G

2003-11-01

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.
Supersymmetric quantum mechanics method for the Fokker-Planck equation with applications to protein folding dynamics

NASA Astrophysics Data System (ADS)

Polotto, Franciele; Drigo Filho, Elso; Chahine, Jorge; Oliveira, Ronaldo Junio de

2018-03-01

This work developed analytical methods to explore the kinetics of the time-dependent probability distributions over thermodynamic free energy profiles of protein folding and compared the results with simulation. The Fokker-Planck equation is mapped onto a Schrödinger-type equation due to the well-known solutions of the latter. Through a semi-analytical description, the supersymmetric quantum mechanics formalism is invoked and the time-dependent probability distributions are obtained with numerical calculations by using the variational method. A coarse-grained structure-based model of the two-state protein Tm CSP was simulated at a Cα level of resolution and the thermodynamics and kinetics were fully characterized. Analytical solutions from non-equilibrium conditions were obtained with the simulated double-well free energy potential and kinetic folding times were calculated. It was found that analytical folding time as a function of temperature agrees, quantitatively, with simulations and experiments from the literature of Tm CSP having the well-known 'U' shape of the Chevron Plots. The simple analytical model developed in this study has a potential to be used by theoreticians and experimentalists willing to explore, quantitatively, rates and the kinetic behavior of their system by informing the thermally activated barrier. The theory developed describes a stochastic process and, therefore, can be applied to a variety of biological as well as condensed-phase two-state systems.
Rapid three-dimensional microfluidic mixer for high viscosity solutions to unravel earlier folding kinetics of G-quadruplex under molecular crowding conditions.

PubMed

Liu, Chao; Li, Ying; Li, Yiwei; Chen, Peng; Feng, Xiaojun; Du, Wei; Liu, Bi-Feng

2016-01-01

Rapid mixing of highly viscous solutions is a great challenge, which helps to analyze the reaction kinetics in viscous liquid phase, particularly to discover the folding kinetics of macromolecules under molecular crowding conditions mimicking the conditions inside cells. Here, we demonstrated a novel microfluidic mixer based on Dean flows with three-dimensional (3D) microchannel configuration for fast mixing of high-viscosity fluids. The main structure contained three consecutive subunits, each consisting of a "U"-type channel followed by a chamber with different width and height. Thus, the two solutions injected from the two inlets would undergo a mixing in the first "U"-type channel due to the Dean flow effect, and simultaneous vortices expansions in both horizontal and vertical directions in the following chamber. Numerical simulations and experimental characterizations confirmed that the micromixer could achieve a mixing time of 122.4μs for solutions with viscosities about 33.6 times that of pure water. It was the fastest micromixer for high viscosity solutions compared with previous reports. With this highly efficient 3D microfluidic mixer, we further characterized the early folding kinetics of human telomere G-quadruplex under molecular crowding conditions, and unravelled a new folding process within 550μs. Copyright © 2015 Elsevier B.V. All rights reserved.
Structural Transitions of Confined Model Proteins: Molecular Dynamics Simulation and Experimental Validation

PubMed Central

Lu, Diannan; Liu, Zheng; Wu, Jianzhong

2006-01-01

Proteins fold in a confined space not only in vivo, i.e., folding assisted by molecular chaperons and chaperonins in a crowded cellular medium, but also in vitro as in production of recombinant proteins. Despite extensive work on protein folding in bulk, little is known about how and to what extent the thermodynamics and kinetics of protein folding are altered by confinement. In this work, we use a Gō-like off-lattice model to investigate the folding and stability of an all β-sheet protein in spherical cages of different sizes and surface hydrophobicity. We find whereas extreme confinement inhibits correct folding, a hydrophilic cage stabilizes the protein due to restriction of the unfolded configurations. In a hydrophobic cage, however, strong attraction from the cage surface destabilizes the confined protein because of competition between self-aggregation and adsorption of hydrophobic residues. We show that the kinetics of protein collapse and folding is strongly correlated with both the cage size and the surface hydrophobicity. It is demonstrated that a cage of moderate size and hydrophobicity optimizes both the folding yield and kinetics of structural transitions. To support the simulation results, we have also investigated the refolding of hen-egg lysozyme in the presence of cetyltrimethylammoniumbromide (CTAB) surfactants that provide an effective confinement of the proteins by micellization. The influence of the surfactant hydrophobicity on the structural and biological activity of the protein is determined with circular dichroism spectrum, fluorescence emission spectrum, and biological activity assay. It is shown that, as predicted by coarse-grained simulations, CTAB micelles facilitate the collapse of denatured lysozyme, whereas the addition of β-cyclodextrin-grafted-PNIPAAm, a weakly hydrophobic stripper, dissociates CTAB micelles and promotes the conformational rearrangement and thereby gives an improved recovery of lysozyme activity. PMID:16461405
Quantum Transmission Conditions for Diffusive Transport in Graphene with Steep Potentials

NASA Astrophysics Data System (ADS)

Barletti, Luigi; Negulescu, Claudia

2018-05-01

We present a formal derivation of a drift-diffusion model for stationary electron transport in graphene, in presence of sharp potential profiles, such as barriers and steps. Assuming the electric potential to have steep variations within a strip of vanishing width on a macroscopic scale, such strip is viewed as a quantum interface that couples the classical regions at its left and right sides. In the two classical regions, where the potential is assumed to be smooth, electron and hole transport is described in terms of semiclassical kinetic equations. The diffusive limit of the kinetic model is derived by means of a Hilbert expansion and a boundary layer analysis, and consists of drift-diffusion equations in the classical regions, coupled by quantum diffusive transmission conditions through the interface. The boundary layer analysis leads to the discussion of a four-fold Milne (half-space, half-range) transport problem.
Protein folding: Over half a century lasting quest. Comment on "There and back again: Two views on the protein folding puzzle" by Alexei V. Finkelstein et al.

NASA Astrophysics Data System (ADS)

Krokhotin, Andrey; Dokholyan, Nikolay V.

2017-07-01

Most proteins fold into unique three-dimensional (3D) structures that determine their biological functions, such as catalytic activity or macromolecular binding. Misfolded proteins can pose a threat through aberrant interactions with other proteins leading to a number of diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [1,2]. What does determine 3D structure of proteins? The first clue to this question came more than fifty years ago when Anfinsen demonstrated that unfolded proteins can spontaneously fold to their native 3D structures [3,4]. Anfinsen's experiments lead to the conclusion that proteins fold to unique native structure corresponding to the stable and kinetically accessible free energy minimum, and protein native structure is solely determined by its amino acid sequence. The question of how exactly proteins find their free energy minimum proved to be a difficult problem. One of the puzzles, initially pointed out by Levinthal, was an inconsistency between observed protein folding times and theoretical estimates. A self-avoiding polymer model of a globular protein of 100-residues length on a cubic lattice can sample at least 1047 states. Based on the assumption that conformational sampling occurs at the highest vibrational mode of proteins (∼picoseconds), predicted folding time by searching among all the possible conformations leads to ∼1027 years (much larger than the age of the universe) [5]. In contrast, observed protein folding time range from microseconds to minutes. Due to tremendous theoretical progress in protein folding field that has been achieved in past decades, the source of this inconsistency is currently understood that is thoroughly described in the review by Finkelstein et al. [6].
Energy landscape of knotted protein folding

PubMed Central

Sułkowska, Joanna I.; Noel, Jeffrey K.; Onuchic, Jose N.

2012-01-01

Recent experiments have conclusively shown that proteins are able to fold from an unknotted, denatured polypeptide to the knotted, native state without the aid of chaperones. These experiments are consistent with a growing body of theoretical work showing that a funneled, minimally frustrated energy landscape is sufficient to fold small proteins with complex topologies. Here, we present a theoretical investigation of the folding of a knotted protein, 2ouf, engineered in the laboratory by a domain fusion that mimics an evolutionary pathway for knotted proteins. Unlike a previously studied knotted protein of similar length, we see reversible folding/knotting and a surprising lack of deep topological traps with a coarse-grained structure-based model. Our main interest is to investigate how evolution might further select the geometry and stiffness of the threading region of the newly fused protein. We compare the folding of the wild-type protein to several mutants. Similarly to the wild-type protein, all mutants show robust and reversible folding, and knotting coincides with the transition state ensemble. As observed experimentally, our simulations show that the knotted protein folds about ten times slower than an unknotted construct with an identical contact map. Simulated folding kinetics reflect the experimentally observed rollover in the folding limbs of chevron plots. Successful folding of the knotted protein is restricted to a narrow range of temperature as compared to the unknotted protein and fits of the kinetic folding data below folding temperature suggest slow, nondiffusive dynamics for the knotted protein. PMID:22891304
An adaptive bias - hybrid MD/kMC algorithm for protein folding and aggregation.

PubMed

Peter, Emanuel K; Shea, Joan-Emma

2017-07-05

In this paper, we present a novel hybrid Molecular Dynamics/kinetic Monte Carlo (MD/kMC) algorithm and apply it to protein folding and aggregation in explicit solvent. The new algorithm uses a dynamical definition of biases throughout the MD component of the simulation, normalized in relation to the unbiased forces. The algorithm guarantees sampling of the underlying ensemble in dependency of one average linear coupling factor 〈α〉 τ . We test the validity of the kinetics in simulations of dialanine and compare dihedral transition kinetics with long-time MD-simulations. We find that for low 〈α〉 τ values, kinetics are in good quantitative agreement. In folding simulations of TrpCage and TrpZip4 in explicit solvent, we also find good quantitative agreement with experimental results and prior MD/kMC simulations. Finally, we apply our algorithm to study growth of the Alzheimer Amyloid Aβ 16-22 fibril by monomer addition. We observe two possible binding modes, one at the extremity of the fibril (elongation) and one on the surface of the fibril (lateral growth), on timescales ranging from ns to 8 μs.
A one-dimensional free energy surface does not account for two-probe folding kinetics of protein alpha(3)D.

PubMed

Liu, Feng; Dumont, Charles; Zhu, Yongjin; DeGrado, William F; Gai, Feng; Gruebele, Martin

2009-02-14

We present fluorescence-detected measurements of the temperature-jump relaxation kinetics of the designed three-helix bundle protein alpha(3)D taken under solvent conditions identical to previous infrared-detected kinetics. The fluorescence-detected rate is similar to the IR-detected rate only at the lowest temperature where we could measure it (326 K). The fluorescence-detected rate decreases by a factor of 3 over the 326-344 K temperature range, whereas the IR-detected rate remains nearly constant over the same range. To investigate this probe dependence, we tested an extensive set of physically reasonable one-dimensional (1D) free energy surfaces by Langevin dynamics simulation. The simulations included coordinate- and temperature-dependent roughness, diffusion coefficients, and IR/fluorescence spectroscopic signatures. None of these can reproduce the IR and fluorescence data simultaneously, forcing us to the conclusion that a 1D free energy surface cannot accurately describe the folding of alpha(3)D. This supports the hypothesis that alpha(3)D has a multidimensional free energy surface conducive to downhill folding at 326 K, and that it is already an incipient downhill folder with probe-dependent kinetics near its melting point.
Phi-value analysis of apo-azurin folding: comparison between experiment and theory.

PubMed

Zong, Chenghang; Wilson, Corey J; Shen, Tongye; Wolynes, Peter G; Wittung-Stafshede, Pernilla

2006-05-23

Pseudomonas aeruginosa azurin is a 128-residue beta-sandwich metalloprotein; in vitro kinetic experiments have shown that it folds in a two-state reaction. Here, we used a variational free energy functional to calculate the characteristics of the transition state ensemble (TSE) for folding of the apo-form of P. aeruginosa azurin and investigate how it responds to thermal and mutational changes. The variational method directly yields predicted chevron plots for wild-type and mutant apo-forms of azurin. In parallel, we performed in vitro kinetic-folding experiments on the same set of azurin variants using chemical perturbation. Like the wild-type protein, all apo-variants fold in apparent two-state reactions both in calculations and in stopped-flow mixing experiments. Comparisons of phi (phi) values determined from the experimental and theoretical chevron parameters reveal an excellent agreement for most positions, indicating a polarized, highly structured TSE for folding of P. aeruginosa apo-azurin. We also demonstrate that careful analysis of side-chain interactions is necessary for appropriate theoretical description of core mutants.
Principles of protein folding--a perspective from simple exact models.

PubMed Central

Dill, K. A.; Bromberg, S.; Yue, K.; Fiebig, K. M.; Yee, D. P.; Thomas, P. D.; Chan, H. S.

1995-01-01

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse. PMID:7613459
Equilibrium thermodynamics and folding kinetics of a short, fast-folding, beta-hairpin.

PubMed

Jimenez-Cruz, Camilo A; Garcia, Angel E

2014-04-14

Equilibrium thermodynamics of a short beta-hairpin are studied using unbiased all-atom replica exchange molecular dynamics simulations in explicit solvent. An exploratory analysis of the free energy landscape of the system is provided in terms of various structural characteristics, for both the folded and unfolded ensembles. We find that the favorable interactions between the ends introduced by the tryptophan cap, along with the flexibility of the turn region, explain the remarkable stability of the folded state. Charging of the N termini results in effective roughening of the free energy landscape and stabilization of non-native contacts. Folding-unfolding dynamics are further discussed using a set of 2413 independent molecular dynamics simulations, 2 ns to 20 ns long, at the melting temperature of the beta-hairpin. A novel method for the construction of Markov models consisting of an iterative refinement of the discretization in reduced dimensionality is presented and used to generate a detailed kinetic network of the system. The hairpin is found to fold heterogeneously on sub-microsecond timescales, with the relative position of the tryptophan side chains driving the selection of the specific pathway.
A Kinetic Model of Trp-Cage Folding from Multiple Biased Molecular Dynamics Simulations

PubMed Central

Marinelli, Fabrizio; Pietrucci, Fabio; Laio, Alessandro; Piana, Stefano

2009-01-01

Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins. Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For the Trp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap. Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at of 4.4 Å from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the and chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data. PMID:19662155

Proline 54 trans-cis isomerization is responsible for the kinetic partitioning at the last-step photocycle of photoactive yellow protein

PubMed Central

Lee, Byoung-Chul; Hoff, Wouter D.

2008-01-01

Photoactive yellow protein (PYP), a blue-light photoreceptor for Ectothiorhodospira halophila, has provided a unique system for studying protein folding that is coupled with a photocycle. Upon receptor activation by blue light, PYP proceeds through a photocycle that includes a partially folded signaling state. The last-step photocycle is a thermal recovery reaction from the signaling state to the native state. Bi-exponential kinetics had been observed for the last-step photocycle; however, the slow phase of the bi-exponential kinetics has not been extensively studied. Here we analyzed both fast and slow phases of the last-step photocycle in PYP. From the analysis of the denaturant dependence of the fast and slow phases, we found that the last-step photocycle proceeds through parallel channels of the folding pathway. The burial of the solvent-accessible area was responsible for the transition state of the fast phase, while structural rearrangement from the compact state to the native state was responsible for the transition state of the slow phase. The photocycle of PYP was linked to the thermodynamic cycle that includes both unfolding and refolding of the fast- and slow-phase intermediates. In order to test the hypothesis of proline-limited folding for the slow phase, we constructed two proline mutants: P54A and P68A. We found that only a single phase of the last-step photocycle was observed in P54A. This suggests that there is a low energy barrier between trans to cis conformation in P54 in the light-induced state of PYP, and the resulting cis conformation of P54 generates a slow-phase kinetic trap during the photocycle-coupled folding pathway of PYP. PMID:18794212
A kinetic model of trp-cage folding from multiple biased molecular dynamics simulations.

PubMed

Marinelli, Fabrizio; Pietrucci, Fabio; Laio, Alessandro; Piana, Stefano

2009-08-01

Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins.Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For theTrp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap.Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at Calpha-RMSD of 4.4 A from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the Pro12-delta3 and Gly11-alpha3 chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data.
Functional modulation of a protein folding landscape via side-chain distortion

PubMed Central

Kelch, Brian A.; Salimi, Neema L.; Agard, David A.

2012-01-01

Ultrahigh-resolution (< 1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ∼6° distortion along the side-chain, estimated to cost 4 kcal/mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions. PMID:22635267
Hydrophobic folding units derived from dissimilar monomer structures and their interactions.

PubMed

Tsai, C J; Nussinov, R

1997-01-01

We have designed an automated procedure to cut a protein into compact hydrophobic folding units. The hydrophobic units are large enough to contain tertiary non-local interactions, reflecting potential nucleation sites during protein folding. The quality of a hydrophobic folding unit is evaluated by four criteria. The first two correspond to visual characterization of a structural domain, namely, compactness and extent of isolation. We use the definition of Zehfus and Rose (Zehfus MH, Rose GD, 1986, Biochemistry 25:35-340) to calculate the compactness of a cut protein unit. The isolation of a unit is based on the solvent accessible surface area (ASA) originally buried in the interior and exposed to the solvent after cutting. The third quantity is the hydrophobicity, equivalent to the fraction of the buried non-polar ASA with respect to the total non-polar ASA. The last criterion in the evaluation of a folding unit is the number of segments it includes. To conform with the rationale of obtaining hydrophobic units, which may relate to early folding events, the hydrophobic interactions are implicitly and explicitly applied in their generation and assessment. We follow Holm and Sander (Holm L, Sander C, 1994, Proteins 19:256-268) to reduce the multiple cutting-point problem to a one-dimensional search for all reasonable trial cuts. However, as here we focus on the hydrophobic cores, the contact matrix used to obtain the first non-trivial eigenvector contains only hydrophobic contracts, rather than all, hydrophobic and hydrophilic, interactions. This dataset of hydrophobic folding units, derived from structurally dissimilar single chain monomers, is particularly useful for investigations of the mechanism of protein folding. For cases where there are kinetic data, the one or more hydrophobic folding units generated for a protein correlate with the two or with the three-state folding process observed. We carry out extensive amino acid sequence order independent structural comparisons to generate a structurally non-redundant set of hydrophobic folding units for fold recognition and for statistical purposes.
Computational design of RNA parts, devices, and transcripts with kinetic folding algorithms implemented on multiprocessor clusters.

PubMed

Thimmaiah, Tim; Voje, William E; Carothers, James M

2015-01-01

With progress toward inexpensive, large-scale DNA assembly, the demand for simulation tools that allow the rapid construction of synthetic biological devices with predictable behaviors continues to increase. By combining engineered transcript components, such as ribosome binding sites, transcriptional terminators, ligand-binding aptamers, catalytic ribozymes, and aptamer-controlled ribozymes (aptazymes), gene expression in bacteria can be fine-tuned, with many corollaries and applications in yeast and mammalian cells. The successful design of genetic constructs that implement these kinds of RNA-based control mechanisms requires modeling and analyzing kinetically determined co-transcriptional folding pathways. Transcript design methods using stochastic kinetic folding simulations to search spacer sequence libraries for motifs enabling the assembly of RNA component parts into static ribozyme- and dynamic aptazyme-regulated expression devices with quantitatively predictable functions (rREDs and aREDs, respectively) have been described (Carothers et al., Science 334:1716-1719, 2011). Here, we provide a detailed practical procedure for computational transcript design by illustrating a high throughput, multiprocessor approach for evaluating spacer sequences and generating functional rREDs. This chapter is written as a tutorial, complete with pseudo-code and step-by-step instructions for setting up a computational cluster with an Amazon, Inc. web server and performing the large numbers of kinefold-based stochastic kinetic co-transcriptional folding simulations needed to design functional rREDs and aREDs. The method described here should be broadly applicable for designing and analyzing a variety of synthetic RNA parts, devices and transcripts.
A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo.

PubMed

Ruminski, Dana J; Watson, Peter Y; Mahen, Elisabeth M; Fedor, Martha J

2016-03-01

RNAs must assemble into specific structures in order to carry out their biological functions, but in vitro RNA folding reactions produce multiple misfolded structures that fail to exchange with functional structures on biological time scales. We used carefully designed self-cleaving mRNAs that assemble through well-defined folding pathways to identify factors that differentiate intracellular and in vitro folding reactions. Our previous work showed that simple base-paired RNA helices form and dissociate with the same rate and equilibrium constants in vivo and in vitro. However, exchange between adjacent secondary structures occurs much faster in vivo, enabling RNAs to quickly adopt structures with the lowest free energy. We have now used this approach to probe the effects of an extensively characterized DEAD-box RNA helicase, Mss116p, on a series of well-defined RNA folding steps in yeast. Mss116p overexpression had no detectable effect on helix formation or dissociation kinetics or on the stability of interdomain tertiary interactions, consistent with previous evidence that intracellular factors do not affect these folding parameters. However, Mss116p overexpression did accelerate exchange between adjacent helices. The nonprocessive nature of RNA duplex unwinding by DEAD-box RNA helicases is consistent with a branch migration mechanism in which Mss116p lowers barriers to exchange between otherwise stable helices by the melting and annealing of one or two base pairs at interhelical junctions. These results suggest that the helicase activity of DEAD-box proteins like Mss116p distinguish intracellular RNA folding pathways from nonproductive RNA folding reactions in vitro and allow RNA structures to overcome kinetic barriers to thermodynamic equilibration in vivo. © 2016 Ruminski et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
Determining the folding and unfolding rate constants of nucleic acids by biosensor. Application to telomere G-quadruplex.

PubMed

Zhao, Yong; Kan, Zhong-yuan; Zeng, Zhi-xiong; Hao, Yu-hua; Chen, Hua; Tan, Zheng

2004-10-20

Nucleic acid molecules may fold into secondary structures, and the formation of such structures is involved in many biological processes and technical applications. The folding and unfolding rate constants define the kinetics of conformation interconversion and the stability of these structures and is important in realizing their functions. We developed a method to determine these kinetic parameters using an optical biosensor based on surface plasmon resonance. The folding and unfolding of a nucleic acid is coupled with a hybridization reaction by immobilization of the target nucleic acid on a sensor chip surface and injection of a complementary probe nucleic acid over the sensor chip surface. By monitoring the time course of duplex formation, both the folding and unfolding rate constants for the target nucleic acid and the association and dissociation rate constants for the target-probe duplex can all be derived from the same measurement. We applied this method to determine the folding and unfolding rate constants of the G-quadruplex of human telomere sequence (TTAGGG)(4) and its association and dissociation rate constants with the complementary strand (CCCTAA)(4). The results show that both the folding and unfolding occur on the time scale of minutes at physiological concentration of K(+). We speculate that this property might be important for telomere elongation. A complete set of the kinetic parameters for both of the structures allows us to study the competition between the formation of the quadruplex and the duplex. Calculations indicate that the formation of both the quadruplex and the duplex is strand concentration-dependent, and the quadruplex can be efficiently formed at low strand concentration. This property may provide the basis for the formation of the quadruplex in vivo in the presence of a complementary strand.
Ultrafast microfluidic mixer for tracking the early folding kinetics of human telomere G-quadruplex.

PubMed

Li, Ying; Liu, Chao; Feng, Xiaojun; Xu, Youzhi; Liu, Bi-Feng

2014-05-06

The folding of G-quadruplex is hypothesized to undergo a complex process, from the formation of a hairpin structure to a triplex intermediate and to the final G-quadruplex. Currently, no experimental evidence has been found for the hairpin formation, because it folds in the time regime of 10-100 μs, entailing the development of microfluidic mixers with a mixing time of less than 10 μs. In this paper, we reported an ultrarapid micromixer with a mixing time of 5.5 μs, which represents the fastest turbulent micromixer to our best knowledge. Evaluations of the micromixer were conducted to confirm its mixing efficiency for small molecules and macromolecules. This new micromixer enabled us to interrogate the hairpin formation in the early folding process of human telomere G-quadruplex. The experimental kinetic evidence for the formation of hairpin was obtained for the first time.
Mutational Studies Uncover Non-Native Structure in the Dimeric Kinetic Intermediate of the H2A-H2B Heterodimer

PubMed Central

Stump, Matthew R.; Gloss, Lisa M.

2010-01-01

The folding pathway of the histone H2A-H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I2. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below ~0.4 M urea (concentrations inaccessible from the 4 M urea unfolded state), a roll-over in the rates was observed; this suggests that a component of the I2 ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I2 ensemble was assessed with a set of H2A-H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central α2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I2 and the transition state between I2 and N2. This limited mutational study indicated that residues in the α2 helices of H2A and H2B, as well as α1 of H2B and both the C-terminus of α3 and the short αC helix of H2A contribute to the stability of the I2 burst phase species. Interestingly, at least eight of the nine targeted residues stabilize I2 by interactions that are non-native to some extent. Given that destabilizing I2 and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structure present in the I2 ensemble enables efficient folding of H2A-H2B. PMID:20600120
A lattice protein with an amyloidogenic latent state: stability and folding kinetics.

PubMed

Palyanov, Andrey Yu; Krivov, Sergei V; Karplus, Martin; Chekmarev, Sergei F

2007-03-15

We have designed a model lattice protein that has two stable folded states, the lower free energy native state and a latent state of somewhat higher energy. The two states have a sizable part of their structures in common (two "alpha-helices") and differ in the content of "alpha-helices" and "beta-strands" in the rest of their structures; i.e. for the native state, this part is alpha-helical, and for the latent state it is composed of beta-strands. Thus, the lattice protein free energy surface mimics that of amyloidogenic proteins that form well organized fibrils under appropriate conditions. A Go-like potential was used and the folding process was simulated with a Monte Carlo method. To gain insight into the equilibrium free energy surface and the folding kinetics, we have combined standard approaches (reduced free energy surfaces, contact maps, time-dependent populations of the characteristic states, and folding time distributions) with a new approach. The latter is based on a principal coordinate analysis of the entire set of contacts, which makes possible the introduction of unbiased reaction coordinates and the construction of a kinetic network for the folding process. The system is found to have four characteristic basins, namely a semicompact globule, an on-pathway intermediate (the bifurcation basin), and the native and latent states. The bifurcation basin is shallow and consists of the structure common to the native and latent states, with the rest disorganized. On the basis of the simulation results, a simple kinetic model describing the transitions between the characteristic states was developed, and the rate constants for the essential transitions were estimated. During the folding process the system dwells in the bifurcation basin for a relatively short time before it proceeds to the native or latent state. We suggest that such a bifurcation may occur generally for proteins in which native and latent states have a sizable part of their structures in common. Moreover, there is the possibility of introducing changes in the system (e.g., mutations), which guide the system toward the native or misfolded state.
The folding pathways and thermodynamics of semiflexible polymers

NASA Astrophysics Data System (ADS)

Wu, Jing; Cheng, Chenqian; Liu, Gaoyuan; Zhang, Ping; Chen, Tao

2018-05-01

Inspired by the protein folding and DNA packing, we have systematically studied the thermodynamic and kinetic behaviors of single semiflexible homopolymers by Langevin dynamics simulations. In line with experiments, a rich variety of folding products, such as rod-like bundles, hairpins, toroids, and a mixture of them, are observed in the complete diagram of states. Moreover, knotted structures with a significant population are found in a certain range of bending stiffness in thermal equilibrium. As the solvent quality becomes poorer, the population of the intermediate occurring in the folding process increases, which leads to a severe chevron rollover for the folding arm. However, the population of the intermediates in the unfolding process is very low, insufficient to induce unfolding arm rollover. The total types of folding pathways from the coil state to the toroidal state for a semiflexible polymer chain remain unchanged by varying the solvent quality or temperature, whereas the kinetic partitioning into different folding events can be tuned significantly. In the process of knotting, three types of mechanisms, namely, plugging, slipknotting, and sliding, are discovered. Along the folding evolution, a semiflexible homopolymer chain can knot at any stage of folding upon leaving the extended coil state, and the probability to find a knot increases with chain compactness. In addition, we find rich types of knotted topologies during the folding of a semiflexible homopolymer chain. This study should be helpful in gaining insight into the general principles of biopolymer folding.
Folding of apomyoglobin: Analysis of transient intermediate structure during refolding using quick hydrogen deuterium exchange and NMR

PubMed Central

NISHIMURA, Chiaki

2017-01-01

The structures of apomyoglobin folding intermediates have been widely analyzed using physical chemistry methods including fluorescence, circular dichroism, small angle X-ray scattering, NMR, mass spectrometry, and rapid mixing. So far, at least two intermediates (on sub-millisecond- and millisecond-scales) have been demonstrated for apomyoglobin folding. The combination of pH-pulse labeling and NMR is a useful tool for analyzing the kinetic intermediates at the atomic level. Its use has revealed that the latter-phase kinetic intermediate of apomyoglobin (6 ms) was composed of helices A, B, G and H, whereas the equilibrium intermediate, called the pH 4 molten-globule intermediate, was composed mainly of helices A, G and H. The improved strategy for the analysis of the kinetic intermediate was developed to include (1) the dimethyl sulfoxide method, (2) data processing with the various labeling times, and (3) a new in-house mixer. Particularly, the rapid mixing revealed that helices A and G were significantly more protected at the earlier stage (400 µs) of the intermediate (former-phase intermediate) than the other helices. Mutation studies, where each hydrophobic residue was replaced with an alanine in helices A, B, E, F, G and H, indicated that both non-native and native-like structures exist in the latter-phase folding intermediate. The N-terminal part of helix B is a weak point in the intermediate, and the docking of helix E residues to the core of the A, B, G and H helices was interrupted by a premature helix B, resulting in the accumulation of the intermediate composed of helices A, B, G and H. The prediction-based protein engineering produced important mutants: Helix F in a P88K/A90L/S92K/A94L mutant folded in the latter-phase intermediate, although helix F in the wild type does not fold even at the native state. Furthermore, in the L11G/W14G/A70L/G73W mutant, helix A did not fold but helix E did, which is similar to what was observed in the kinetic intermediate of apoleghemoglobin. Thus, this protein engineering resulted in a changed structure for the apomyoglobin folding intermediate. PMID:28077807
Folding of apomyoglobin: Analysis of transient intermediate structure during refolding using quick hydrogen deuterium exchange and NMR.

PubMed

Nishimura, Chiaki

2017-01-01

The structures of apomyoglobin folding intermediates have been widely analyzed using physical chemistry methods including fluorescence, circular dichroism, small angle X-ray scattering, NMR, mass spectrometry, and rapid mixing. So far, at least two intermediates (on sub-millisecond- and millisecond-scales) have been demonstrated for apomyoglobin folding. The combination of pH-pulse labeling and NMR is a useful tool for analyzing the kinetic intermediates at the atomic level. Its use has revealed that the latter-phase kinetic intermediate of apomyoglobin (6 ms) was composed of helices A, B, G and H, whereas the equilibrium intermediate, called the pH 4 molten-globule intermediate, was composed mainly of helices A, G and H. The improved strategy for the analysis of the kinetic intermediate was developed to include (1) the dimethyl sulfoxide method, (2) data processing with the various labeling times, and (3) a new in-house mixer. Particularly, the rapid mixing revealed that helices A and G were significantly more protected at the earlier stage (400 µs) of the intermediate (former-phase intermediate) than the other helices. Mutation studies, where each hydrophobic residue was replaced with an alanine in helices A, B, E, F, G and H, indicated that both non-native and native-like structures exist in the latter-phase folding intermediate. The N-terminal part of helix B is a weak point in the intermediate, and the docking of helix E residues to the core of the A, B, G and H helices was interrupted by a premature helix B, resulting in the accumulation of the intermediate composed of helices A, B, G and H. The prediction-based protein engineering produced important mutants: Helix F in a P88K/A90L/S92K/A94L mutant folded in the latter-phase intermediate, although helix F in the wild type does not fold even at the native state. Furthermore, in the L11G/W14G/A70L/G73W mutant, helix A did not fold but helix E did, which is similar to what was observed in the kinetic intermediate of apoleghemoglobin. Thus, this protein engineering resulted in a changed structure for the apomyoglobin folding intermediate.
The regulation mechanism of yitJ and metF riboswitches

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gong, Sha; Wang, Yujie; Zhang, Wenbing, E-mail: wbzhang@whu.edu.cn

2015-07-28

Riboswitches which function at the transcriptional level are sensitive to cotranscriptional folding. Based on the recently proposed theory of cotranscriptional folding, we developed a transition node approximation method to effectively decrease the conformation space of long RNA chains. Our results indicate that this approximation is reliable for calculating the cotranscriptional folding kinetics of long mRNA chains. We theoretically studied the cotranscriptional folding behavior of the yitJ and metF riboswitches in the absence/presence of S-adenosylmethionine. Although the two S-box riboswitches have similar OFF-state structures and share common features of riboswitches operated at the transcriptional level, their regulation mechanisms are different. Themore » yitJ riboswitch is regulated by a combination of thermodynamic and kinetic mechanisms, while the metF riboswitch is solely kinetically controlled. For the yitJ riboswitch, transcriptional pausing at the U-stretch directly following the terminator decreases the amount of ligand required to trigger the switch. The different regulation mechanisms and binding affinities of the two riboswitches result from the different lengths of the anti-terminator helix, which in yitJ is short and only disrupts helix P1 of the riboswitch aptamer, but in metF is long and breaks both the helices P1 and P4.« less
First Passage Times, Lifetimes, and Relaxation Times of Unfolded Proteins

NASA Astrophysics Data System (ADS)

Dai, Wei; Sengupta, Anirvan M.; Levy, Ronald M.

2015-07-01

The dynamics of proteins in the unfolded state can be quantified in computer simulations by calculating a spectrum of relaxation times which describes the time scales over which the population fluctuations decay to equilibrium. If the unfolded state space is discretized, we can evaluate the relaxation time of each state. We derive a simple relation that shows the mean first passage time to any state is equal to the relaxation time of that state divided by the equilibrium population. This explains why mean first passage times from state to state within the unfolded ensemble can be very long but the energy landscape can still be smooth (minimally frustrated). In fact, when the folding kinetics is two-state, all of the unfolded state relaxation times within the unfolded free energy basin are faster than the folding time. This result supports the well-established funnel energy landscape picture and resolves an apparent contradiction between this model and the recently proposed kinetic hub model of protein folding. We validate these concepts by analyzing a Markov state model of the kinetics in the unfolded state and folding of the miniprotein NTL9 (where NTL9 is the N -terminal domain of the ribosomal protein L9), constructed from a 2.9 ms simulation provided by D. E. Shaw Research.
First Passage Times, Lifetimes, and Relaxation Times of Unfolded Proteins.

PubMed

Dai, Wei; Sengupta, Anirvan M; Levy, Ronald M

2015-07-24

The dynamics of proteins in the unfolded state can be quantified in computer simulations by calculating a spectrum of relaxation times which describes the time scales over which the population fluctuations decay to equilibrium. If the unfolded state space is discretized, we can evaluate the relaxation time of each state. We derive a simple relation that shows the mean first passage time to any state is equal to the relaxation time of that state divided by the equilibrium population. This explains why mean first passage times from state to state within the unfolded ensemble can be very long but the energy landscape can still be smooth (minimally frustrated). In fact, when the folding kinetics is two-state, all of the unfolded state relaxation times within the unfolded free energy basin are faster than the folding time. This result supports the well-established funnel energy landscape picture and resolves an apparent contradiction between this model and the recently proposed kinetic hub model of protein folding. We validate these concepts by analyzing a Markov state model of the kinetics in the unfolded state and folding of the miniprotein NTL9 (where NTL9 is the N-terminal domain of the ribosomal protein L9), constructed from a 2.9 ms simulation provided by D. E. Shaw Research.
Enhanced picture of protein-folding intermediates using organic solvents in H/D exchange and quench-flow experiments

PubMed Central

Nishimura, Chiaki; Dyson, H. Jane; Wright, Peter E.

2005-01-01

Hydrogen/deuterium exchange followed by trapping of the labeled species in the aprotic solvent DMSO has been used to elucidate structure in both the burst-phase molten globule-folding intermediate of apomyoglobin and in an equilibrium intermediate that models the kinetic intermediate. Precise estimates can be made of exchange times in an interrupted exchange-out experiment at pH 4 followed by analysis in DMSO solution, giving extensive sequence-specific information about the structure of the equilibrium intermediate. In addition, the use of DMSO as a solvent for NMR measurements after quench-flow pH-pulse labeling experiments gives a greatly increased data set for the elucidation of the kinetic folding pathway. Interestingly, differences are observed in some regions of apomyoglobin between the equilibrium and kinetic intermediates. These differences are quantitative rather than qualitative; that is, the overall patterns of labeling and secondary structure formation remain similar between the two species. However, local differences are observed, which probably reflect the difference in the solution conditions for the equilibrium experiment (pH 4) vs. the kinetic experiment (pH 6) and the change in the status of the stabilizing hydrogen bond between the side chains of His-24 and His-119. PMID:15769860
Microsecond Unfolding Kinetics of Sheep Prion Protein Reveals an Intermediate that Correlates with Susceptibility to Classical Scrapie

PubMed Central

Chen, Kai-Chun; Xu, Ming; Wedemeyer, William J.; Roder, Heinrich

2011-01-01

The microsecond folding and unfolding kinetics of ovine prion proteins (ovPrP) were measured under various solution conditions. A fragment comprising residues 94–233 of the full-length ovPrP was studied for four variants with differing susceptibilities to classical scrapie in sheep. The observed biexponential unfolding kinetics of ovPrP provides evidence for an intermediate species. However, in contrast to previous results for human PrP, there is no evidence for an intermediate under refolding conditions. Global analysis of the kinetic data, based on a sequential three-state mechanism, quantitatively accounts for all folding and unfolding data as a function of denaturant concentration. The simulations predict that an intermediate accumulates under both folding and unfolding conditions, but is observable only in unfolding experiments because the intermediate is optically indistinguishable from the native state. The relative population of intermediates in two ovPrP variants, both transiently and under destabilizing equilibrium conditions, correlates with their propensities for classical scrapie. The variant susceptible to classical scrapie has a larger population of the intermediate state than the resistant variant. Thus, the susceptible variant should be favored to undergo the PrPC to PrPSc conversion and oligomerization. PMID:21889460
Peptide chain dynamics in light and heavy water: zooming in on internal friction.

PubMed

Schulz, Julius C F; Schmidt, Lennart; Best, Robert B; Dzubiella, Joachim; Netz, Roland R

2012-04-11

Frictional effects due to the chain itself, rather than the solvent, may have a significant effect on protein dynamics. Experimentally, such "internal friction" has been investigated by studying folding or binding kinetics at varying solvent viscosity; however, the molecular origin of these effects is hard to pinpoint. We consider the kinetics of disordered glycine-serine and α-helix forming alanine peptides and a coarse-grained protein folding model in explicit-solvent molecular dynamics simulations. By varying the solvent mass over more than two orders of magnitude, we alter only the solvent viscosity and not the folding free energy. Folding dynamics at the near-vanishing solvent viscosities accessible by this approach suggests that solvent and internal friction effects are intrinsically entangled. This finding is rationalized by calculation of the polymer end-to-end distance dynamics from a Rouse model that includes internal friction. An analysis of the friction profile along different reaction coordinates, extracted from the simulation data, demonstrates that internal as well as solvent friction varies substantially along the folding pathways and furthermore suggests a connection between friction and the formation of hydrogen bonds upon folding. © 2012 American Chemical Society
Orientating lipase molecules through surface chemical control for enhanced activity: A QCM-D and ToF-SIMS investigation.

PubMed

Joyce, Paul; Kempson, Ivan; Prestidge, Clive A

2016-06-01

Bio-active materials consisting of lipase encapsulated within porous silica particles were engineered to control the adsorption kinetics and molecular orientation of lipase, which play critical roles in the digestion kinetics of triglycerides. The adsorption kinetics of Candida antartica lipase A (CalA) was monitored using quartz crystal microbalance with dissipation (QCM-D) and controlled by altering the hydrophobicity of a silica binding support. The extent of adsorption was 2-fold greater when CalA was adsorbed onto hydrophobic silica compared to hydrophilic silica. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) fragmentation patterns, in conjunction with multivariate statistics, demonstrated enhanced exposure of the lipase's catalytic domain, specifically the histidine group responsible for activity, when CalA was adsorbed on hydrophilic silica. Consequently, lipid digestion kinetics were enhanced when CalA was loaded in hydrophilic porous silica particles, i.e., a 2-fold increase in the pseudo-first-order rate constant for digestion when compared to free lipase. In contrast, digestion kinetics were inhibited when CalA was hosted in hydrophobic porous silica, i.e., a 5-fold decrease in pseudo-first-order rate constant for digestion when compared to free lipase. These findings provide valuable insights into the mechanism of lipase action which can be exploited to develop smarter food and drug delivery systems consisting of porous lipid-based materials. Copyright © 2016 Elsevier B.V. All rights reserved.

Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding.

PubMed

Kleinschmidt, J H; Tamm, L K

1999-04-20

The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers. Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center. The first folding intermediate is stable at 2 degrees C for at least 1 h. A second intermediate has been isolated at temperatures between 7 and 20 degrees C. The Trps move 4-5 A closer to the center of the bilayer at this stage. Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer. The final (native) structure is observed at higher temperatures of refolding. In this structure, the Trps are located on average about 9-10 A from the bilayer center. Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.
Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation

PubMed Central

Nguyen, Houbi; Jäger, Marcus; Moretto, Alessandro; Gruebele, Martin; Kelly, Jeffery W.

2003-01-01

The equilibrium unfolding of the Formin binding protein 28 (FBP) WW domain, a stable three-stranded β-sheet protein, can be described as reversible apparent two-state folding. Kinetics studied by laser temperature jump reveal a third state at temperatures below the midpoint of unfolding. The FBP free-energy surface can be tuned between three-state and two-state kinetics by changing the temperature, by truncation of the C terminus, or by selected point mutations. FBP WW domain is the smallest three-state folder studied to date and the only one that can be freely tuned between three-state and apparent two-state folding by several methods (temperature, truncation, and mutation). Its small size (28–37 residues), the availability of a quantitative reaction coordinate (φT), the fast folding time scale (10s of μs), and the tunability of the folding routes by small temperature or sequence changes make this system the ideal prototype for studying more subtle features of the folding free-energy landscape by simulations or analytical theory. PMID:12651955
Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation.

PubMed

Nguyen, Houbi; Jager, Marcus; Moretto, Alessandro; Gruebele, Martin; Kelly, Jeffery W

2003-04-01

The equilibrium unfolding of the Formin binding protein 28 (FBP) WW domain, a stable three-stranded beta-sheet protein, can be described as reversible apparent two-state folding. Kinetics studied by laser temperature jump reveal a third state at temperatures below the midpoint of unfolding. The FBP free-energy surface can be tuned between three-state and two-state kinetics by changing the temperature, by truncation of the C terminus, or by selected point mutations. FBP WW domain is the smallest three-state folder studied to date and the only one that can be freely tuned between three-state and apparent two-state folding by several methods (temperature, truncation, and mutation). Its small size (28-37 residues), the availability of a quantitative reaction coordinate (phi(T)), the fast folding time scale (10s of micros), and the tunability of the folding routes by small temperature or sequence changes make this system the ideal prototype for studying more subtle features of the folding free-energy landscape by simulations or analytical theory.
The role of proline-containing peptide triads in β-sheet formation: A kinetic study.

PubMed

Takor, Gaius A; Higashiya, Seiichiro; Sikirzhytski, Vitali K; Seeley, Jason P; Lednev, Igor K; Welch, John T

2015-06-01

The design of biomimetic materials through molecular self-assembly is a growing area of modern nanotechnology. With problems of protein folding, self-assembly, and sequence-structure relationships as essential in nanotechnology as in biology, the effect of the nucleation of β-hairpin formation by proline on the folding process has been investigated in model studies. Previously such studies were limited to investigations of the influence of proline on the formation of turns in short peptide sequences. The effect of proline-based triads on the folding of an 11-kDa amyloidogenic peptide GH6[(GA)3GY(GA)3GE]8 GAH6 (YE8) was investigated by selective substitution of the proline-substituted triads at the γ-turn sites. The folding and fibrillation of the singly proline-substituted polypeptides, e.g., GH6-[(GA)3GY(GA)3GE]7(GA)3GY(GA)3PD-GAH6 (8PD), and doubly proline-substituted polypeptides, e.g., GH6-[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD-GAH6 (4,8PD), were directly monitored by circular dichroism and deep UV resonance Raman and fluorescence spectroscopies. These findings were used to identify the essential folding domains, i.e., the minimum number of β-strands necessary for stable folding. These experimental findings may be especially useful in the design and construction of peptidic materials for a wide range of applications as well as in understanding the mechanisms of folding critical to fibril formation. © 2015 Wiley Periodicals, Inc.
Selected mode of dendritic growth with n-fold symmetry in the presence of a forced flow

NASA Astrophysics Data System (ADS)

Alexandrov, D. V.; Galenko, P. K.

2017-07-01

The effect of n-fold crystal symmetry is investigated for a two-dimensional stable dendritic growth in the presence of a forced convective flow. We consider dendritic growth in a one-component undercooled liquid. The theory is developed for the parabolic solid-liquid surface of dendrite growing at arbitrary growth Péclet numbers keeping in mind small anisotropies of surface energy and growth kinetics. The selection criterion determining the stable growth velocity of the dendritic tip and its stable tip diameter is found on the basis of solvability analysis. The obtained criterion includes previously developed theories of thermally and kinetically controlled dendritic growth with convection for the case of four-fold crystal symmetry. The obtained nonlinear system of equations (representing the selection criterion and undercooling balance) for the determination of dendrite tip velocity and dendrite tip diameter is analytically solved in a parametric form. These exact solutions clearly demonstrate a transition between thermally and kinetically controlled growth regimes. In addition, we show that the dendrites with larger crystal symmetry grow faster than those with smaller symmetry.
Further optimization of a hybrid united-atom and coarse-grained force field for folding simulations: Improved backbone hydration and interactions between charged side chains

PubMed Central

Han, Wei; Schulten, Klaus

2012-01-01

PACE, a hybrid force field which couples united-atom protein models with coarse-grained (CG) solvent, has been further optimized, aiming to improve itse ciency for folding simulations. Backbone hydration parameters have been re-optimized based on hydration free energies of polyalanyl peptides through atomistic simulations. Also, atomistic partial charges from all-atom force fields were combined with PACE in order to provide a more realistic description of interactions between charged groups. Using replica exchange molecular dynamics (REMD), ab initio folding using the new PACE has been achieved for seven small proteins (16 – 23 residues) with different structural motifs. Experimental data about folded states, such as their stability at room temperature, melting point and NMR NOE constraints, were also well reproduced. Moreover, a systematic comparison of folding kinetics at room temperature has been made with experiments, through standard MD simulations, showing that the new PACE may speed up the actual folding kinetics 5-10 times. Together with the computational speedup benefited from coarse-graining, the force field provides opportunities to study folding mechanisms. In particular, we used the new PACE to fold a 73-residue protein, 3D, in multiple 10 – 30 μs simulations, to its native states (Cα RMSD ~ 0.34 nm). Our results suggest the potential applicability of the new PACE for the study of folding and dynamics of proteins. PMID:23204949
Rough energy landscapes in protein folding: dimeric E. coli Trp repressor folds through three parallel channels.

PubMed

Gloss, L M; Simler, B R; Matthews, C R

2001-10-05

The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development. Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration. Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species. A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer. The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations. These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain. One, perhaps both, of these packing modes contains non-native contacts. Copyright 2001 Academic Press.
Folding of guanine quadruplex molecules-funnel-like mechanism or kinetic partitioning? An overview from MD simulation studies.

PubMed

Šponer, Jiří; Bussi, Giovanni; Stadlbauer, Petr; Kührová, Petra; Banáš, Pavel; Islam, Barira; Haider, Shozeb; Neidle, Stephen; Otyepka, Michal

2017-05-01

Guanine quadruplexes (GQs) play vital roles in many cellular processes and are of much interest as drug targets. In contrast to the availability of many structural studies, there is still limited knowledge on GQ folding. We review recent molecular dynamics (MD) simulation studies of the folding of GQs, with an emphasis paid to the human telomeric DNA GQ. We explain the basic principles and limitations of all types of MD methods used to study unfolding and folding in a way accessible to non-specialists. We discuss the potential role of G-hairpin, G-triplex and alternative GQ intermediates in the folding process. We argue that, in general, folding of GQs is fundamentally different from funneled folding of small fast-folding proteins, and can be best described by a kinetic partitioning (KP) mechanism. KP is a competition between at least two (but often many) well-separated and structurally different conformational ensembles. The KP mechanism is the only plausible way to explain experiments reporting long time-scales of GQ folding and the existence of long-lived sub-states. A significant part of the natural partitioning of the free energy landscape of GQs comes from the ability of the GQ-forming sequences to populate a large number of syn-anti patterns in their G-tracts. The extreme complexity of the KP of GQs typically prevents an appropriate description of the folding landscape using just a few order parameters or collective variables. We reconcile available computational and experimental studies of GQ folding and formulate basic principles characterizing GQ folding landscapes. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.
Molecular dynamics simulation of phosphorylated KID post-translational modification.

PubMed

Chen, Hai-Feng

2009-08-05

Kinase-inducible domain (KID) as transcriptional activator can stimulate target gene expression in signal transduction by associating with KID interacting domain (KIX). NMR spectra suggest that apo-KID is an unstructured protein. After post-translational modification by phosphorylation, KID undergoes a transition from disordered to well folded protein upon binding to KIX. However, the mechanism of folding coupled to binding is poorly understood. To get an insight into the mechanism, we have performed ten trajectories of explicit-solvent molecular dynamics (MD) for both bound and apo phosphorylated KID (pKID). Ten MD simulations are sufficient to capture the average properties in the protein folding and unfolding. Room-temperature MD simulations suggest that pKID becomes more rigid and stable upon the KIX-binding. Kinetic analysis of high-temperature MD simulations shows that bound pKID and apo-pKID unfold via a three-state and a two-state process, respectively. Both kinetics and free energy landscape analyses indicate that bound pKID folds in the order of KIX access, initiation of pKID tertiary folding, folding of helix alpha(B), folding of helix alpha(A), completion of pKID tertiary folding, and finalization of pKID-KIX binding. Our data show that the folding pathways of apo-pKID are different from the bound state: the foldings of helices alpha(A) and alpha(B) are swapped. Here we also show that Asn139, Asp140 and Leu141 with large Phi-values are key residues in the folding of bound pKID. Our results are in good agreement with NMR experimental observations and provide significant insight into the general mechanisms of binding induced protein folding and other conformational adjustment in post-translational modification.
The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces

PubMed Central

Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D; Johnson, Christopher M; Fersht, Alan R

2010-01-01

Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution. PMID:20662005
Folding and Unfolding Pathways of the Human Telomeric G-Quadruplex

PubMed Central

Gray, Robert D.; Trent, John O.; Chaires, Jonathan B.

2014-01-01

Sequence analogues of human telomeric DNA such as d[AGGG(TTAGGG)3] (Tel22) fold into monomeric quadruplex structures in the presence of a suitable cation. To investigate the pathway for unimolecular quadruplex formation, we monitored the kinetics of K+-induced folding of Tel22 by circular dichroism (CD), intrinsic 2-aminopurine fluorescence, and fluorescence resonance energy transfer (FRET). The results are consistent with a four-step pathway U ↔ I1 ↔ I2 ↔ I3 ↔ F where U and F represent unfolded and folded conformational ensembles, and I1, I2, and I3 are intermediates. Previous kinetic studies have shown that I1 is formed in a rapid pre-equilibrium and may consist of an ensemble of “prefolded” hairpin structures brought about by cation-induced electrostatic collapse of the DNA. The current study shows that I1 converts to I2 with a relaxation time τ1 = 0.1 s at 25 °C in 25 mM KCl. The CD spectrum of I2 is characteristic of an antiparallel quadruplex that could form as a result of intra-molecular fold-over of the I1 hairpins. I3 is relatively slowly formed (τ2 ≈ 3700 s) and has CD and FRET properties consistent with those expected of a triplex structure as previously observed in equilibrium melting studies. I3 converts to F with τ3 ≈ 750 s. Identical pathways with different kinetic constants involving a rapidly formed antiparallel intermediate were observed with oligonucleotides forming mixed parallel/antiparallel hybrid-1 and hybrid-2 topologies (e.g. d[TTGGG(TTAGGG)3A and d[TAGGG(TTAGGG)3TT]). Aspects of the kinetics of unfolding were also monitored by the spectroscopic methods listed above and by time-resolved fluorescence lifetime measurements using a complementary strand trap assay. These experiments reveal a slow, rate-limiting step along the unfolding pathway. PMID:24487181
Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap.

PubMed Central

Thoden, J. B.; Holden, H. M.; Fisher, A. J.; Sinclair, J. F.; Wesenberg, G.; Baldwin, T. O.; Rayment, I.

1997-01-01

Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas. PMID:9007973
Computational study of RNA folding kinetics and thermodynamics

NASA Astrophysics Data System (ADS)

Morgan, Steven Robert

RNA in its many forms is involved in the processes of protein manufacture, gene splicing, catalysis and gene regulation. It is also the store of genetic information in some viruses. The function of the RNA is determined by its structure, and it is the purpose of this thesis to investigate kinetic and thermodynamic properties of RNA secondary structures in order to obtain a better understanding of their formation and function. Our main tenet is that kinetic formation of RNA structure is necessary to explain features found in natural RNA structures, as well as aspects of the biological function of RNA. Firstly we show that examination of the energies of fragments of RNA secondary structure provides evidence for kinetic formation of structure. Local regions of RNA of length less than about 100 nucleotides adopt a conformation with energy near or equal to the minimum possible for those regions, whilst the energies of larger domains are much further from the their respective minima. This is consistent with the patterns that would be expected if RNA structure is folded Idneticatic during transcription. A Monte-Carlo algorithm is then used to model the kinetic folding of RNA during transcriptional growth. The algorithm is capable of finding the correct structure of a natural RNA for which the minimum free energy approach is unsuccessful. In the viral phage MS2 Idneticatic formed RNA structure plays an important role in the regulation of gene expression. The folding algorithm can accurately model this by IdneticaUy controlling access to the gene initiation region. The algorithm is also successfully used to model the control of replication in the ColEl plasmid. Taking a different approach, we then use a simplified model of RNA secondary structure to investigate the size of energy barriers between degenerate minimum energy structures. This model has much in common with physical systems such as spin glasses, and in fact shows similar behaviour to these systems in that energy barriers between structures grow quickly with the length of the RNA sequence. These barriers will serve to trap RNA in non-optimal structures. Together these studies demonstrate the necessity of studying RNA secondary structure from a kinetic point of view, and provide clear directions in which further work may be taken. Kinetic models of RNA secondary structure should continue to prove useful in modelling the structure and function of RNA.
A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)

NASA Astrophysics Data System (ADS)

Bergasa-Caceres, Fernando; Rabitz, Herschel A.

2013-06-01

A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.
Kinetic network study of the diversity and temperature dependence of Trp-Cage folding pathways: combining transition path theory with stochastic simulations.

PubMed

Zheng, Weihua; Gallicchio, Emilio; Deng, Nanjie; Andrec, Michael; Levy, Ronald M

2011-02-17

We present a new approach to study a multitude of folding pathways and different folding mechanisms for the 20-residue mini-protein Trp-Cage using the combined power of replica exchange molecular dynamics (REMD) simulations for conformational sampling, transition path theory (TPT) for constructing folding pathways, and stochastic simulations for sampling the pathways in a high dimensional structure space. REMD simulations of Trp-Cage with 16 replicas at temperatures between 270 and 566 K are carried out with an all-atom force field (OPLSAA) and an implicit solvent model (AGBNP). The conformations sampled from all temperatures are collected. They form a discretized state space that can be used to model the folding process. The equilibrium population for each state at a target temperature can be calculated using the weighted-histogram-analysis method (WHAM). By connecting states with similar structures and creating edges satisfying detailed balance conditions, we construct a kinetic network that preserves the equilibrium population distribution of the state space. After defining the folded and unfolded macrostates, committor probabilities (P(fold)) are calculated by solving a set of linear equations for each node in the network and pathways are extracted together with their fluxes using the TPT algorithm. By clustering the pathways into folding "tubes", a more physically meaningful picture of the diversity of folding routes emerges. Stochastic simulations are carried out on the network, and a procedure is developed to project sampled trajectories onto the folding tubes. The fluxes through the folding tubes calculated from the stochastic trajectories are in good agreement with the corresponding values obtained from the TPT analysis. The temperature dependence of the ensemble of Trp-Cage folding pathways is investigated. Above the folding temperature, a large number of diverse folding pathways with comparable fluxes flood the energy landscape. At low temperature, however, the folding transition is dominated by only a few localized pathways.
Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry

PubMed Central

Vander Meulen, Kirk A.; Butcher, Samuel E.

2012-01-01

A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop–receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH‡) and Eyring transition state entropies (ΔS‡). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH25°C = −41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH‡ = −0.6 ± 0.5). These parameters are sigificantly positively shifted in magnesium (ΔH25°C = −20.5 ± 2.1 kcal/mol, ΔH‡ = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl2). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake. PMID:22058128
Determination of protein folding kinetic types using sequence and predicted secondary structure and solvent accessibility.

PubMed

Zhang, Hua; Zhang, Tuo; Gao, Jianzhao; Ruan, Jishou; Shen, Shiyi; Kurgan, Lukasz

2012-01-01

Proteins fold through a two-state (TS), with no visible intermediates, or a multi-state (MS), via at least one intermediate, process. We analyze sequence-derived factors that determine folding types by introducing a novel sequence-based folding type predictor called FOKIT. This method implements a logistic regression model with six input features which hybridize information concerning amino acid composition and predicted secondary structure and solvent accessibility. FOKIT provides predictions with average Matthews correlation coefficient (MCC) between 0.58 and 0.91 measured using out-of-sample tests on four benchmark datasets. These results are shown to be competitive or better than results of four modern predictors. We also show that FOKIT outperforms these methods when predicting chains that share low similarity with the chains used to build the model, which is an important advantage given the limited number of annotated chains. We demonstrate that inclusion of solvent accessibility helps in discrimination of the folding kinetic types and that three of the features constitute statistically significant markers that differentiate TS and MS folders. We found that the increased content of exposed Trp and buried Leu are indicative of the MS folding, which implies that the exposure/burial of certain hydrophobic residues may play important role in the formation of the folding intermediates. Our conclusions are supported by two case studies.
How cooperative are protein folding and unfolding transitions?

PubMed Central

Malhotra, Pooja

2016-01-01

Abstract A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two‐state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non‐cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier‐less “downhill” folding, as well as for continuous “uphill” unfolding transitions, indicate that gradual non‐cooperative processes may be ubiquitous features on the free energy landscape of protein folding. PMID:27522064
Label-enhanced surface plasmon resonance applied to label-free interaction analysis of small molecules and fragments.

PubMed

Eng, Lars; Nygren-Babol, Linnéa; Hanning, Anders

2016-10-01

Surface plasmon resonance (SPR) is a well-established method for studying interactions between small molecules and biomolecules. In particular, SPR is being increasingly applied within fragment-based drug discovery; however, within this application area, the limited sensitivity of SPR may constitute a problem. This problem can be circumvented by the use of label-enhanced SPR that shows a 100-fold higher sensitivity as compared with conventional SPR. Truly label-free interaction data for small molecules can be obtained by applying label-enhanced SPR in a surface competition assay format. The enhanced sensitivity is accompanied by an increased specificity and inertness toward disturbances (e.g., bulk refractive index disturbances). Label-enhanced SPR can be used for fragment screening in a competitive assay format; the competitive format has the added advantage of confirming the specificity of the molecular interaction. In addition, label-enhanced SPR extends the accessible kinetic regime of SPR to the analysis of very fast fragment binding kinetics. In this article, we demonstrate the working principles and benchmark the performance of label-enhanced SPR in a model system-the interaction between carbonic anhydrase II and a number of small-molecule sulfonamide-based inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.
Unfolding of globular polymers by external force

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bell, Samuel; Terentjev, Eugene M., E-mail: emt1000@cam.ac.uk

2015-11-14

We examine the problem of a polymer chain, folded into a globule in poor solvent, subjected to a constant tensile force. Such a situation represents a Gibbs thermodynamic ensemble and is useful for analysing force-clamp atomic force microscopy measurements, now very common in molecular biophysics. Using a basic Flory mean-field theory, we account for surface interactions of monomers with solvent. Under an increasing tensile force, a first-order phase transition occurs from a compact globule to a fully extended chain, in an “all-or-nothing” unfolding event. This contrasts with the regime of imposed extension, first studied by Halperin and Zhulina [Europhys. Lett.more » 15, 417 (1991)], where there is a regime of coexistence of a partial globule with an extended chain segment. We relate the transition forces in this problem to the solvent quality and degree of polymerisation, and also find analytical expressions for the energy barriers present in the problem. Using these expressions, we analyse the kinetic problem of a force-ramp experiment and show that the force at which a globule ruptures depends on the rate of loading.« less

Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme

PubMed Central

Xie, Zheng; Srividya, Narayanan; Sosnick, Tobin R.; Pan, Tao; Scherer, Norbert F.

2004-01-01

The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET). Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-Ieq-to-N, and focused on the Ieq-to-N transition. The present study focuses on the U-to-Ieq transition. Comparative ensemble measurements of the ribozyme construct labeled with fluorescein at the 5′ end and Cy3 at the 3′ end show that modifications required for labeling do not interfere with folding and help to define the Mg2+ concentration range for the U-to-Ieq transition. Histogram analysis of the Mg2+-dependent single-molecule FRET efficiency reveals two previously undetermined folding intermediates. The single-molecule FRET trajectories exhibit non-two-state and nonergodic behaviors at intermediate Mg2+ concentrations on the time scale of seconds. The trajectories at intermediate Mg2+ concentrations are classified into five classes based on three FRET levels and their dynamics of interconversion within the measured time range. This heterogeneity, together with the observation of “nonsudden jump” FRET transitions, indicates that the early folding steps of this ribozyme involve a series of intermediates with different degrees of kinetic isolation and that folding occurs under kinetic control and involves many “local” conformational switches. A free energy contour is constructed to illustrate the complex folding surface. PMID:14704266
Kinetic Network Study of the Diversity and Temperature Dependence of Trp-Cage Folding Pathways: Combining Transition Path Theory with Stochastic Simulations

PubMed Central

Zheng, Weihua; Gallicchio, Emilio; Deng, Nanjie; Andrec, Michael; Levy, Ronald M.

2011-01-01

We present a new approach to study a multitude of folding pathways and different folding mechanisms for the 20-residue mini-protein Trp-Cage using the combined power of replica exchange molecular dynamics (REMD) simulations for conformational sampling, Transition Path Theory (TPT) for constructing folding pathways and stochastic simulations for sampling the pathways in a high dimensional structure space. REMD simulations of Trp-Cage with 16 replicas at temperatures between 270K and 566K are carried out with an all-atom force field (OPLSAA) and an implicit solvent model (AGBNP). The conformations sampled from all temperatures are collected. They form a discretized state space that can be used to model the folding process. The equilibrium population for each state at a target temperature can be calculated using the Weighted-Histogram-Analysis Method (WHAM). By connecting states with similar structures and creating edges satisfying detailed balance conditions, we construct a kinetic network that preserves the equilibrium population distribution of the state space. After defining the folded and unfolded macrostates, committor probabilities (Pfold) are calculated by solving a set of linear equations for each node in the network and pathways are extracted together with their fluxes using the TPT algorithm. By clustering the pathways into folding “tubes”, a more physically meaningful picture of the diversity of folding routes emerges. Stochastic simulations are carried out on the network and a procedure is developed to project sampled trajectories onto the folding tubes. The fluxes through the folding tubes calculated from the stochastic trajectories are in good agreement with the corresponding values obtained from the TPT analysis. The temperature dependence of the ensemble of Trp-Cage folding pathways is investigated. Above the folding temperature, a large number of diverse folding pathways with comparable fluxes flood the energy landscape. At low temperature, however, the folding transition is dominated by only a few localized pathways. PMID:21254767
Swellix: a computational tool to explore RNA conformational space.

PubMed

Sloat, Nathan; Liu, Jui-Wen; Schroeder, Susan J

2017-11-21

The sequence of nucleotides in an RNA determines the possible base pairs for an RNA fold and thus also determines the overall shape and function of an RNA. The Swellix program presented here combines a helix abstraction with a combinatorial approach to the RNA folding problem in order to compute all possible non-pseudoknotted RNA structures for RNA sequences. The Swellix program builds on the Crumple program and can include experimental constraints on global RNA structures such as the minimum number and lengths of helices from crystallography, cryoelectron microscopy, or in vivo crosslinking and chemical probing methods. The conceptual advance in Swellix is to count helices and generate all possible combinations of helices rather than counting and combining base pairs. Swellix bundles similar helices and includes improvements in memory use and efficient parallelization. Biological applications of Swellix are demonstrated by computing the reduction in conformational space and entropy due to naturally modified nucleotides in tRNA sequences and by motif searches in Human Endogenous Retroviral (HERV) RNA sequences. The Swellix motif search reveals occurrences of protein and drug binding motifs in the HERV RNA ensemble that do not occur in minimum free energy or centroid predicted structures. Swellix presents significant improvements over Crumple in terms of efficiency and memory use. The efficient parallelization of Swellix enables the computation of sequences as long as 418 nucleotides with sufficient experimental constraints. Thus, Swellix provides a practical alternative to free energy minimization tools when multiple structures, kinetically determined structures, or complex RNA-RNA and RNA-protein interactions are present in an RNA folding problem.
A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.

PubMed Central

Shastry, M C; Luck, S D; Roder, H

1998-01-01

A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R. M. Clegg, M. J. Fulwyler, F. J. Barrantes, and T. M. Jovin. 1985. Rev. Sci. Instrum. 56:283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance downstream from the mixer, using an Hg(Xe) arc lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 micros of its initiation and that the dead time of the measurement is 45 +/- 5 micros, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera have been instrumental in obtaining artifact-free kinetic data over the time window from approximately 45 micros to a few milliseconds with signal-to-noise levels comparable to those of conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction. PMID:9591695
Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase.

PubMed

Beassoni, Paola R; Berti, Federico Pérez de; Otero, Lisandro H; Risso, Valeria A; Ferreyra, Raul G; Lisa, Angela T; Domenech, Carlos E; Ermácora, Mario R

2010-06-01

Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure.
Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

PubMed Central

Bisaria, Namita; Greenfeld, Max; Limouse, Charles; Pavlichin, Dmitri S.; Mabuchi, Hideo; Herschlag, Daniel

2016-01-01

The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations. PMID:27493222
How Fast is Collapse of Proteins During Folding?

NASA Astrophysics Data System (ADS)

Chahine, J.; Onuchic, J. N.; Socci, N. D.

1998-03-01

Recent experiments in fast folding proteins are now starting to address the question of how fast is collapse relative to the total folding time. Using minimalist models, we are able to investigate the way in which different scenarios of folding can arise depending on the interplay between the collapse order parameter and the order parameter sensitive to specific tertiary contacts. Most of our earlier studies have focused on the limit that collapse is very fast compared to the total folding time. In this work we focus on the opposite limit, i.e., at the folding temperature, collapse and folding occurs simultaneously. The folding mechanism becomes very different in this limit. Particularly, the non-specific collapse transition, that occurs at temperatures higher than the folding temperature for the fast collapse limit, now occurs between the folding and the glass temperature. We show how this transition can be identified and its consequences for the folding kinetics.
Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

NASA Astrophysics Data System (ADS)

Bergasa-Caceres, Fernando; Rabitz, Herschel A.

2014-01-01

A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.
Toxicity and kinetic parameters of the aerobic biodegradation of the phenol and alkylphenols by a mixed culture.

PubMed

Acuña-Argüelles, M E; Olguin-Lora, P; Razo-Flores, E

2003-04-01

A mixed culture aerobically metabolized phenol, cresol isomers (o-,m-,p-), 2-ethylphenol and xylenol isomers (2,5-DMP and 3,4-DMP) as the sole carbon and energy source. This culture had a high tolerance towards phenol with values of maximum degradation rate (Vmax) of 47 microM phenol mg-1 protein h-1 and inhibition substrate constant (Ki) of 10 mM. These kinetic parameters were considerably diminished and the toxicity increased with the alkylphenols. For example with 2,5-xylenol, Vmax and Ki values of 0.8 microM 2,5-xylenol mg-1 protein h-1 and 1.3 mM, respectively, were obtained. The cresols were 5-fold more toxic than phenol, whereas 2-ethylphenol and 3,4-xylenol were 11-fold more toxic, and 2,5-xylenol was 34-fold more toxic than phenol.
Cleavage Mapping the Topology of Protein Folding Intermediates

DTIC Science & Technology

1995-03-13

1993). 29. Nakano, T., Antonino , L. C, Fox, R. O. & Fink, A. L. Effect of proline mutation on the stability and kinetics of folding of...Biochem. 62, 653-683. Nakano, T., Antonino , L. C., Fox, R. O., & Fink, A. L. (1993) Biochemistry 32, 2534-2541. Nozaki, Y. (1972) Methods Enzymol. 26, 43
Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta

PubMed Central

Brown, Jessica A.; Zhang, Likui; Sherrer, Shanen M.; Taylor, John-Stephen; Burgers, Peter M. J.; Suo, Zucai

2010-01-01

Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase η (yPolη), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPolη which contains only the polymerase domain. In the absence of yPolη's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPolη may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPolη discriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide 5′-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides. PMID:20798853
Biosorption of landfill leachate by Phanerochaete sp. ISTL01: isotherms, kinetics and toxicological assessment.

PubMed

Ghosh, Pooja; Thakur, Indu Shekhar

2017-07-01

The study investigates the ability of fungus Phanerochaete sp. ISTL01 for biosorption of color from landfill leachate. Batch mode experiments were conducted to study the effects of pH, temperature, adsorbent dose, contact time and initial leachate concentration on biosorption. Maximum biosorption capacity was determined as 17.73 mg g -1 of biomass. Equilibrium isotherms and kinetics were further studied. The biosorption data were found to fit well to the Freundlich isotherm and pseudo-second-order kinetic model. The value of activation energy suggested that chemisorption mechanism was involved. Biosorption efficiency was also evaluated by the Methyltetrazolium (MTT) assay for cytotoxicity and alkaline comet assay in HepG2 human hepato-carcinoma cells. The fungus reduced toxicity as shown by 1.3-fold increase in MTT EC 50 and 1.5- and 1.1-fold reduction in Tail moment and Olive tail moment, respectively, after 12 h biosorption. The fungus showed good biosorption characteristics in terms of contaminant-level reduction per unit mass of adsorbent, process kinetics and toxicity reduction, envisaging its application in leachate treatment.
The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones

PubMed Central

Topping, Traci B; Gloss, Lisa M

2011-01-01

The goal of this study was to examine fibril formation by the heterodimeric eukaryotic histones (H2A-H2B and H3-H4) and homodimeric archaeal histones (hMfB and hPyA1). The histone fold dimerization motif is an obligatorily domain-swapped structure comprised of two fused helix:β-loop:helix motifs. Domain swapping has been proposed as a mechanism for the evolution of protein oligomers as well as a means to form precursors in the formation of amyloid-like fibrils. Despite sharing a common fold, the eukaryotic histones of the core nucleosome and archaeal histones fold by kinetic mechanisms of differing complexity with transient population of partially folded monomeric and/or dimeric species. No relationship was apparent between fibrillation propensity and equilibrium stability or population of kinetic intermediates. Only H3 and H4, as isolated monomers and as a heterodimer, readily formed fibrils at room temperature, and this propensity correlates with the significantly lower solubility of these polypeptides. The fibrils were characterized by ThT fluorescence, FTIR, and far-UV CD spectroscopies and electron microscopy. The helical histone fold comprises the protease-resistant core of the fibrils, with little or no protease protection of the poorly structured N-terminal tails. The highly charged tails inhibit fibrillation through electrostatic repulsion. Kinetic studies indicate that H3 and H4 form a co-fibril, with simultaneous incorporation of both histones. The potential impact of H3 and H4 fibrillation on the cytotoxicity of extracellular histones and α-synuclein-mediated neurotoxicity and fibrillation is considered. PMID:21953551
How long does it take to equilibrate the unfolded state of a protein?

PubMed Central

Levy, Ronald M; Dai, Wei; Deng, Nan-Jie; Makarov, Dmitrii E

2013-01-01

How long does it take to equilibrate the unfolded state of a protein? The answer to this question has important implications for our understanding of why many small proteins fold with two state kinetics. When the equilibration within the unfolded state U is much faster than the folding, the folding kinetics will be two state even if there are many folding pathways with different barriers. Yet the mean first passage times (MFPTs) between different regions of the unfolded state can be much longer than the folding time. This seems to imply that the equilibration within U is much slower than the folding. In this communication we resolve this paradox. We present a formula for estimating the time to equilibrate the unfolded state of a protein. We also present a formula for the MFPT to any state within U, which is proportional to the average lifetime of that state divided by the state population. This relation is valid when the equilibration within U is very fast as compared with folding as it often is for small proteins. To illustrate the concepts, we apply the formulas to estimate the time to equilibrate the unfolded state of Trp-cage and MFPTs within the unfolded state based on a Markov State Model using an ultra-long 208 microsecond trajectory of the miniprotein to parameterize the model. The time to equilibrate the unfolded state of Trp-cage is ∼100 ns while the typical MFPTs within U are tens of microseconds or longer. PMID:23963761
Chevron Behavior and Isostable Enthalpic Barriers in Protein Folding: Successes and Limitations of Simple Gō-like Modeling

PubMed Central

Kaya, Hüseyin; Liu, Zhirong; Chan, Hue Sun

2005-01-01

It has been demonstrated that a “near-Levinthal” cooperative mechanism, whereby the common Gō interaction scheme is augmented by an extra favorability for the native state as a whole, can lead to apparent two-state folding/unfolding kinetics over a broad range of native stabilities in lattice models of proteins. Here such a mechanism is shown to be generalizable to a simplified continuum (off-lattice) Langevin dynamics model with a Cα protein chain representation, with the resulting chevron plots exhibiting an extended quasilinear regime reminiscent of that of apparent two-state real proteins. Similarly high degrees of cooperativity are possible in Gō-like continuum models with rudimentary pairwise desolvation barriers as well. In these models, cooperativity increases with increasing desolvation barrier height, suggesting strongly that two-state-like folding/unfolding kinetics would be achievable when the pairwise desolvation barrier becomes sufficiently high. Besides cooperativity, another generic folding property of interest that has emerged from published experiments on several apparent two-state proteins is that their folding relaxation under constant native stability (isostability) conditions is essentially Arrhenius, entailing high intrinsic enthalpic folding barriers of ∼17–30 kcal/mol. Based on a new analysis of published data on barnase, here we propose that a similar property should also apply to a certain class of non-two-state proteins that fold with chevron rollovers. However, several continuum Gō-like constructs considered here fail to predict any significant intrinsic enthalpic folding barrier under isostability conditions; thus the physical origin of such barriers in real proteins remains to be elucidated. PMID:15863486
Equilibrium and kinetic folding of rabbit muscle triosephosphate isomerase by hydrogen exchange mass spectrometry.

PubMed

Pan, Hai; Raza, Ashraf S; Smith, David L

2004-03-05

Unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM), a model for (betaalpha)8-barrel proteins, has been studied by amide hydrogen exchange/mass spectrometry. Unfolding was studied by destabilizing the protein in guanidine hydrochloride (GdHCl) or urea, pulse-labeling with 2H2O and analyzing the intact protein by HPLC electrospray ionization mass spectrometry. Bimodal isotope patterns were found in the mass spectra of the labeled protein, indicating two-state unfolding behavior. Refolding experiments were performed by diluting solutions of TIM unfolded in GdHCl or urea and pulse-labeling with 2H2O at different times. Mass spectra of the intact protein labeled after one to two minutes had three envelopes of isotope peaks, indicating population of an intermediate. Kinetic modeling indicates that the stability of the folding intermediate in water is only 1.5 kcal/mol. Failure to detect the intermediate in the unfolding experiments was attributed to its low stability and the high concentrations of denaturant required for unfolding experiments. The folding status of each segment of the polypeptide backbone was determined from the deuterium levels found in peptic fragments of the labeled protein. Analysis of these spectra showed that the C-terminal half folds to form the intermediate, which then forms native TIM with folding of the N-terminal half. These results show that TIM folding fits the (4+4) model for folding of (betaalpha)8-barrel proteins. Results of a double-jump experiment indicate that proline isomerization does not contribute to the rate-limiting step in the folding of TIM.
Numerical impact simulation of gradually increased kinetic energy transfer has the potential to break up folded protein structures resulting in cytotoxic brain tissue edema.

PubMed

von Holst, Hans; Li, Xiaogai

2013-07-01

Although the consequences of traumatic brain injury (TBI) and its treatment have been improved, there is still a substantial lack of understanding the mechanisms. Numerical simulation of the impact can throw further lights on site and mechanism of action. A finite element model of the human head and brain tissue was used to simulate TBI. The consequences of gradually increased kinetic energy transfer was analyzed by evaluating the impact intracranial pressure (ICP), strain level, and their potential influences on binding forces in folded protein structures. The gradually increased kinetic energy was found to have the potential to break apart bonds of Van der Waals in all impacts and hydrogen bonds at simulated impacts from 6 m/s and higher, thereby superseding the energy in folded protein structures. Further, impacts below 6 m/s showed none or very slight increase in impact ICP and strain levels, whereas impacts of 6 m/s or higher showed a gradual increase of the impact ICP and strain levels reaching over 1000 KPa and over 30%, respectively. The present simulation study shows that the free kinetic energy transfer, impact ICP, and strain levels all have the potential to initiate cytotoxic brain tissue edema by unfolding protein structures. The definition of mild, moderate, and severe TBI should thus be looked upon as the same condition and separated only by a gradual severity of impact.
PyFolding: Open-Source Graphing, Simulation, and Analysis of the Biophysical Properties of Proteins.

PubMed

Lowe, Alan R; Perez-Riba, Albert; Itzhaki, Laura S; Main, Ewan R G

2018-02-06

For many years, curve-fitting software has been heavily utilized to fit simple models to various types of biophysical data. Although such software packages are easy to use for simple functions, they are often expensive and present substantial impediments to applying more complex models or for the analysis of large data sets. One field that is reliant on such data analysis is the thermodynamics and kinetics of protein folding. Over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open-source, and extensible Python framework for graphing, analysis, and simulation of the biophysical properties of proteins. To demonstrate the utility of PyFolding, we have used it to analyze and model experimental protein folding and thermodynamic data. Examples include: 1) multiphase kinetic folding fitted to linked equations, 2) global fitting of multiple data sets, and 3) analysis of repeat protein thermodynamics with Ising model variants. Moreover, we demonstrate how PyFolding is easily extensible to novel functionality beyond applications in protein folding via the addition of new models. Example scripts to perform these and other operations are supplied with the software, and we encourage users to contribute notebooks and models to create a community resource. Finally, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and among research teams. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Protein folding and misfolding: mechanism and principles

PubMed Central

Englander, S. Walter; Mayne, Leland; Krishna, Mallela M. G.

2012-01-01

Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding. PMID:18405419
Carbamate and Pyrethroid Resistance in the Akron Strain of Anopheles gambiae

PubMed Central

Mutunga, James M.; Anderson, Troy D.; Craft, Derek T.; Gross, Aaron D.; Swale, Daniel R.; Tong, Fan; Wong, Dawn M.; Carlier, Paul R.; Bloomquist, Jeffrey R.

2015-01-01

Insecticide resistance in the malaria vector, Anopheles gambiae is a serious problem, epitomized by the multi-resistant Akron strain, originally isolated in the country of Benin. Here we report resistance in this strain to pyrethroids and DDT (13-fold to 35-fold compared to the susceptible G3 strain), but surprisingly little resistance to etofenprox, a compound sometimes described as a “pseudo-pyrethroid.” There was also strong resistance to topically-applied commercial carbamates (45-fold to 81-fold), except for the oximes aldicarb and methomyl. Biochemical assays showed enhanced cytochrome P450 monooxygenase and carboxylesterase activity, but not that of glutathione-S-transferase. A series of substituted α,α,α,-trifluoroacetophenone oxime methylcarbamates were evaluated for enzyme inhibition potency and toxicity against G3 and Akron mosquitoes. The compound bearing an unsubstituted phenyl ring showed the greatest toxicity to mosquitoes of both strains. Low cross resistance in Akron was retained by all analogs in the series. Kinetic analysis of acetylcholinesterase activity and its inhibition by insecticides in the G3 strain showed inactivation rate constants greater than that of propoxur, and against Akron enzyme inactivation rate constants similar to that of aldicarb. However, inactivation rate constants against recombinant human AChE were essentially identical to that of the G3 strain. Thus, the acetophenone oxime carbamates described here, though potent insecticides that control resistant Akron mosquitoes, require further structural modification to attain acceptable selectivity and human safety. PMID:26047119

Programmed folding of DNA origami structures through single-molecule force control.

PubMed

Bae, Wooli; Kim, Kipom; Min, Duyoung; Ryu, Je-Kyung; Hyeon, Changbong; Yoon, Tae-Young

2014-12-03

Despite the recent development in the design of DNA origami, its folding yet relies on thermal or chemical annealing methods. We here demonstrate mechanical folding of the DNA origami structure via a pathway that has not been accessible to thermal annealing. Using magnetic tweezers, we stretch a single scaffold DNA with mechanical tension to remove its secondary structures, followed by base pairing of the stretched DNA with staple strands. When the force is subsequently quenched, folding of the DNA nanostructure is completed through displacement between the bound staple strands. Each process in the mechanical folding is well defined and free from kinetic traps, enabling us to complete folding within 10 min. We also demonstrate parallel folding of DNA nanostructures through multiplexed manipulation of the scaffold DNAs. Our results suggest a path towards programmability of the folding pathway of DNA nanostructures.
Free energy profiles from single-molecule pulling experiments.

PubMed

Hummer, Gerhard; Szabo, Attila

2010-12-14

Nonequilibrium pulling experiments provide detailed information about the thermodynamic and kinetic properties of molecules. We show that unperturbed free energy profiles as a function of molecular extension can be obtained rigorously from such experiments without using work-weighted position histograms. An inverse Weierstrass transform is used to relate the system free energy obtained from the Jarzynski equality directly to the underlying molecular free energy surface. An accurate approximation for the free energy surface is obtained by using the method of steepest descent to evaluate the inverse transform. The formalism is applied to simulated data obtained from a kinetic model of RNA folding, in which the dynamics consists of jumping between linker-dominated folded and unfolded free energy surfaces.
Free Energy Landscape - Settlements of Key Residues.

NASA Astrophysics Data System (ADS)

Aroutiounian, Svetlana

2007-03-01

FEL perspective in studies of protein folding transitions reflects notion that since there are ˜10^N conformations to scan in search of lowest free energy state, random search is beyond biological timescale. Protein folding must follow certain fel pathways and folding kinetics of evolutionary selected proteins dominates kinetic traps. Good model for functional robustness of natural proteins - coarse-grained model protein is not very accurate but affords bringing simulations closer to biological realm; Go-like potential secures the fel funnel shape; biochemical contacts signify the funnel bottleneck. Boltzmann-weighted ensemble of protein conformations and histogram method are used to obtain from MC sampling of protein conformational space the approximate probability distribution. The fel is F(rmsd) = -1/βLn[Hist(rmsd)], β=kBT and rmsd is root-mean-square-deviation from native conformation. The sperm whale myoglobin has rich dynamic behavior, is small and large - on computational scale, has a symmetry in architecture and unusual sextet of residue pairs. Main idea: there is a mathematical relation between protein fel and a key residues set providing stability to folding transition. Is the set evolutionary conserved also for functional reasons? Hypothesis: primary sequence determines the key residues positions conserved as stabilizers and the fel is the battlefield for the folding stability. Preliminary results: primary sequence - not the architecture, is the rule settler, indeed.
Frnakenstein: multiple target inverse RNA folding.

PubMed

Lyngsø, Rune B; Anderson, James W J; Sizikova, Elena; Badugu, Amarendra; Hyland, Tomas; Hein, Jotun

2012-10-09

RNA secondary structure prediction, or folding, is a classic problem in bioinformatics: given a sequence of nucleotides, the aim is to predict the base pairs formed in its three dimensional conformation. The inverse problem of designing a sequence folding into a particular target structure has only more recently received notable interest. With a growing appreciation and understanding of the functional and structural properties of RNA motifs, and a growing interest in utilising biomolecules in nano-scale designs, the interest in the inverse RNA folding problem is bound to increase. However, whereas the RNA folding problem from an algorithmic viewpoint has an elegant and efficient solution, the inverse RNA folding problem appears to be hard. In this paper we present a genetic algorithm approach to solve the inverse folding problem. The main aims of the development was to address the hitherto mostly ignored extension of solving the inverse folding problem, the multi-target inverse folding problem, while simultaneously designing a method with superior performance when measured on the quality of designed sequences. The genetic algorithm has been implemented as a Python program called Frnakenstein. It was benchmarked against four existing methods and several data sets totalling 769 real and predicted single structure targets, and on 292 two structure targets. It performed as well as or better at finding sequences which folded in silico into the target structure than all existing methods, without the heavy bias towards CG base pairs that was observed for all other top performing methods. On the two structure targets it also performed well, generating a perfect design for about 80% of the targets. Our method illustrates that successful designs for the inverse RNA folding problem does not necessarily have to rely on heavy biases in base pair and unpaired base distributions. The design problem seems to become more difficult on larger structures when the target structures are real structures, while no deterioration was observed for predicted structures. Design for two structure targets is considerably more difficult, but far from impossible, demonstrating the feasibility of automated design of artificial riboswitches. The Python implementation is available at http://www.stats.ox.ac.uk/research/genome/software/frnakenstein.
Frnakenstein: multiple target inverse RNA folding

PubMed Central

2012-01-01

Background RNA secondary structure prediction, or folding, is a classic problem in bioinformatics: given a sequence of nucleotides, the aim is to predict the base pairs formed in its three dimensional conformation. The inverse problem of designing a sequence folding into a particular target structure has only more recently received notable interest. With a growing appreciation and understanding of the functional and structural properties of RNA motifs, and a growing interest in utilising biomolecules in nano-scale designs, the interest in the inverse RNA folding problem is bound to increase. However, whereas the RNA folding problem from an algorithmic viewpoint has an elegant and efficient solution, the inverse RNA folding problem appears to be hard. Results In this paper we present a genetic algorithm approach to solve the inverse folding problem. The main aims of the development was to address the hitherto mostly ignored extension of solving the inverse folding problem, the multi-target inverse folding problem, while simultaneously designing a method with superior performance when measured on the quality of designed sequences. The genetic algorithm has been implemented as a Python program called Frnakenstein. It was benchmarked against four existing methods and several data sets totalling 769 real and predicted single structure targets, and on 292 two structure targets. It performed as well as or better at finding sequences which folded in silico into the target structure than all existing methods, without the heavy bias towards CG base pairs that was observed for all other top performing methods. On the two structure targets it also performed well, generating a perfect design for about 80% of the targets. Conclusions Our method illustrates that successful designs for the inverse RNA folding problem does not necessarily have to rely on heavy biases in base pair and unpaired base distributions. The design problem seems to become more difficult on larger structures when the target structures are real structures, while no deterioration was observed for predicted structures. Design for two structure targets is considerably more difficult, but far from impossible, demonstrating the feasibility of automated design of artificial riboswitches. The Python implementation is available at http://www.stats.ox.ac.uk/research/genome/software/frnakenstein. PMID:23043260
Tracking polypeptide folds on the free energy surface: effects of the chain length and sequence.

PubMed

Brukhno, Andrey V; Ricchiuto, Piero; Auer, Stefan

2012-07-26

Characterization of the folding transition in polypeptides and assessing the thermodynamic stability of their structured folds are of primary importance for approaching the problem of protein folding. We use molecular dynamics simulations for a coarse grained polypeptide model in order to (1) obtain the equilibrium conformation diagram of homopolypeptides in a broad range of the chain lengths, N = 10, ..., 100, and temperatures, T (in a multicanonical ensemble), and (2) determine free energy profiles (FEPs) projected onto an optimal, so-called "natural", reaction coordinate that preserves the height of barriers and the diffusion coefficients on the underlying free energy hyper-surface. We then address the following fundamental questions. (i) How well does a kinetically determined free energy landscape of a single chain represent the polypeptide equilibrium (ensemble) behavior? In particular, under which conditions might the correspondence be lost, and what are the possible implications for the folding processes? (ii) How does the free energy landscape depend on the chain length (homopolypeptides) and the monomer interaction sequence (heteropolypeptides)? Our data reveal that at low T values equilibrium structures adopted by relatively short homopolypeptides (N < 60) are dominated by α-helical folds which correspond to the primary and secondary minima of the FEP. In contrast, longer homopolypeptides (N > 70), upon quasi-equilibrium cooling, fold preferentially in β-bundles with small helical portions, while the FEPs exhibit no distinct global minima. Moreover, subject to the choice of the initial configuration, at sufficiently low T, essentially metastable structures can be found and prevail far from the true thermodynamic equilibrium. We also show that, by sequence-enabling the polypeptide model, it is possible to restrict the chain to a very specific part of the configuration space, which results in substantial simplification and smoothing of the free energy landscape as compared to the case of the corresponding homopolypeptide.
Inflation Rates, Car Devaluation, and Chemical Kinetics.

ERIC Educational Resources Information Center

Pogliani, Lionello; Berberan-Santos, Mario N.

1996-01-01

Describes the inflation rate problem and offers an interesting analogy with chemical kinetics. Presents and solves the car devaluation problem as a normal chemical kinetic problem where the order of the rate law and the value of the rate constant are derived. (JRH)
Slowest kinetic modes revealed by metabasin renormalization

NASA Astrophysics Data System (ADS)

Okushima, Teruaki; Niiyama, Tomoaki; Ikeda, Kensuke S.; Shimizu, Yasushi

2018-02-01

Understanding the slowest relaxations of complex systems, such as relaxation of glass-forming materials, diffusion in nanoclusters, and folding of biomolecules, is important for physics, chemistry, and biology. For a kinetic system, the relaxation modes are determined by diagonalizing its transition rate matrix. However, for realistic systems of interest, numerical diagonalization, as well as extracting physical understanding from the diagonalization results, is difficult due to the high dimensionality. Here, we develop an alternative and generally applicable method of extracting the long-time scale relaxation dynamics by combining the metabasin analysis of Okushima et al. [Phys. Rev. E 80, 036112 (2009), 10.1103/PhysRevE.80.036112] and a Jacobi method. We test the method on an illustrative model of a four-funnel model, for which we obtain a renormalized kinematic equation of much lower dimension sufficient for determining slow relaxation modes precisely. The method is successfully applied to the vacancy transport problem in ionic nanoparticles [Niiyama et al., Chem. Phys. Lett. 654, 52 (2016), 10.1016/j.cplett.2016.04.088], allowing a clear physical interpretation that the final relaxation consists of two successive, characteristic processes.
Molecular and Cellular Biophysics

NASA Astrophysics Data System (ADS)

Jackson, Meyer B.

2006-01-01

Molecular and Cellular Biophysics provides advanced undergraduate and graduate students with a foundation in the basic concepts of biophysics. Students who have taken physical chemistry and calculus courses will find this book an accessible and valuable aid in learning how these concepts can be used in biological research. The text provides a rigorous treatment of the fundamental theories in biophysics and illustrates their application with examples. Conformational transitions of proteins are studied first using thermodynamics, and subsequently with kinetics. Allosteric theory is developed as the synthesis of conformational transitions and association reactions. Basic ideas of thermodynamics and kinetics are applied to topics such as protein folding, enzyme catalysis and ion channel permeation. These concepts are then used as the building blocks in a treatment of membrane excitability. Through these examples, students will gain an understanding of the general importance and broad applicability of biophysical principles to biological problems. Offers a unique synthesis of concepts across a wide range of biophysical topics Provides a rigorous theoretical treatment, alongside applications in biological systems Author has been teaching biophysics for nearly 25 years
Problem Solving through Paper Folding

ERIC Educational Resources Information Center

Wares, Arsalan

2014-01-01

The purpose of this article is to describe a couple of challenging mathematical problems that involve paper folding. These problem-solving tasks can be used to foster geometric and algebraic thinking among students. The context of paper folding makes some of the abstract mathematical ideas involved relatively concrete. When implemented…
Folding mechanism of an extremely thermostable (βα)(8)-barrel enzyme: a high kinetic barrier protects the protein from denaturation.

PubMed

Carstensen, Linn; Zoldák, Gabriel; Schmid, Franz-Xaver; Sterner, Reinhard

2012-04-24

HisF, the cyclase subunit of imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima, is an extremely thermostable (βα)(8)-barrel protein. We elucidated the unfolding and refolding mechanism of HisF. Its unfolding transition is reversible and adequately described by the two-state model, but 6 weeks is necessary to reach equilibrium (at 25 °C). During refolding, initially a burst-phase off-pathway intermediate is formed. The subsequent productive folding occurs in two kinetic phases with time constants of ~3 and ~20 s. They reflect a sequential process via an on-pathway intermediate, as revealed by stopped-flow double-mixing experiments. The final step leads to native HisF, which associates with the glutaminase subunit HisH to form the functional ImGPS complex. The conversion of the on-pathway intermediate to the native protein results in a 10(6)-fold increase of the time constant for unfolding from 89 ms to 35 h (at 4.0 M GdmCl) and thus establishes a high energy barrier to denaturation. We conclude that the extra stability of HisF is used for kinetic protection against unfolding. In its refolding mechanism, HisF resembles other (βα)(8)-barrel proteins.
Forced unfolding of single-chain polymeric nanoparticles.

PubMed

Hosono, Nobuhiko; Kushner, Aaron M; Chung, Jaeyoon; Palmans, Anja R A; Guan, Zhibin; Meijer, E W

2015-06-03

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is applied to single-chain polymeric nanoparticles (SCPNs) to acquire information about the internal folding structure of SCPNs and inherent kinetic parameters of supramolecular self-assembling motifs embedded into the SCPNs. The SCPNs used here are polyacrylate-based polymers carrying 2-ureido-4-[1H]-pyrimidinone (UPy) or benzene-1,3,5-tricarboxamide (BTA) pendants that induce an intramolecular chain collapse into nanoparticles consisting of one polymer chain only via internal supramolecular cross-linking. The SCPN is stretched by an AFM cantilever to unfold mechanically, which allows measuring of force-extension profiles of the SCPNs. Consecutive peaks observed in the force profiles are attributed to rupture events of self-assembled UPy/BTA units in the SCPNs. The force profiles have been analyzed statistically for a series of polymers with different UPy/BTA incorporation densities. The results provide insights into the internal conformation of SCPNs, where the folding structure can be changed with the incorporation density of UPy/BTA. In addition, dynamic loading rate analysis allows the determination of kinetic parameters of BTA self-assembly, which has not been accessible by any other method. This study offers a rational tool for understanding the folding structure, kinetics, and pathway of two series of SCPNs.
Thermodynamic properties of an extremely rapid protein folding reaction.

PubMed

Schindler, T; Schmid, F X

1996-12-24

The cold-shock protein CspB from Bacillus subtilis is a very small beta-barrel protein, which folds with a time constant of 1 ms (at 25 degrees C) in a U reversible N two-state reaction. To elucidate the energetics of this extremely fast reaction we investigated the folding kinetics of CspB as a function of both temperature and denaturant concentration between 2 and 45 degrees C and between 1 and 8 M urea. Under all these conditions unfolding and refolding were reversible monoexponential reactions. By using transition state theory, data from 327 kinetic curves were jointly analyzed to determine the thermodynamic activation parameters delta H H2O++, delta S H2O++, delta G H2O++, and delta C p H2O++ for unfolding and refolding and their dependences on the urea concentration. 90% of the total change in heat capacity and 96% of the change in the m value (m = d delta G/d[urea]) occur between the unfolded state and the activated state. This suggests that for CspB the activated state of folding is unusually well structured and almost equivalent to the native protein in its interactions with the solvent. As a consequence of this native-like activated state a strong temperature-dependent enthalpy/entropy compensation is observed for the refolding kinetics, and the barrier to refolding shifts from being largely enthalpic at low temperature to largely entropic at high temperature. This shift originates not from the changes in the folding protein chains itself, but from the changes in the protein-solvent interactions. We speculate that the absence of intermediates and the native-like activated state in the folding of CspB are correlated with the small size and the structural type of this protein. The stabilization of a small beta-sheet as in CspB requires extensive non-local interactions, and therefore incomplete sheets are unstable. As a consequence, the critical activated state is reached only very late in folding. The instability of partially folded structure is a means to avoid misfolding prior to the rate-limiting step, and a native-like activated state reduces the risk of non-productive side reactions during the final steps to the native state.
Disulfide bond rearrangement during formation of the chorionic gonadotropin beta-subunit cystine knot in vivo.

PubMed

Wilken, Jason A; Bedows, Elliott

2004-05-04

The intracellular kinetic folding pathway of the human chorionic gonadotropin beta-subunit (hCG-beta) reveals the presence of a disulfide between Cys residues 38-57 that is not detected by X-ray analysis of secreted hCG-beta. This led us to propose that disulfide rearrangement is an essential feature of cystine knot formation during CG-beta folding. To test this, we used disulfide bond formation to monitor progression of intracellular folding intermediates of a previously uncharacterized protein, the CG-beta subunit of cynomolgous macaque (Macaca fascicularis). Like its human counterpart hCG-beta with which it shares 81% identity, macaque (m)CG-beta is a cystine knot-containing subunit that assembles with an alpha-subunit common to all glycoprotein hormone members of its species to form a biologically active heterodimer, mCG, which, like hCG, is required for pregnancy maintenance. An early mCG-beta folding intermediate, mpbeta1, contained two disulfide bonds, one between Cys34 and Cys88 and the other between Cys38 and Cys57. The subsequent folding intermediate, mpbeta2-early, was represented by an ensemble of folding forms that, in addition to the two disulfides mentioned above, included disulfide linkages between Cys9 and Cys57 and between Cys38 and Cys90. These latter two disulfides are those contained within the beta-subunit cystine knot and reveal that a disulfide exchange occurred during the mpbeta2-early folding step leading to formation of the mCG-beta knot. Thus, while defining the intracellular kinetic protein folding pathway of a monkey homologue of CG-beta, we detected the previously predicted disulfide exchange event crucial for CG-beta cystine knot formation and attainment of CG-beta assembly competence.
Investigation of RNA Hairpin Loop Folding with Time-Resolved Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Stancik, Aaron Lee

Ribonucleic acids (RNAs) are a group of functional biopolymers central to the molecular underpinnings of life. To complete the many processes they mediate, RNAs must fold into precise three-dimensional structures. Hairpin loops are the most ubiquitous and basic structural elements present in all folded RNAs, and are the foundation upon which all complex tertiary structures are built. A hairpin loop forms when a single stranded RNA molecule folds back on itself creating a helical stem of paired bases capped by a loop. This work investigates the formation of UNCG hairpin loops with the sequence 5'-GC(UNCG)GC-3' (N = A, U, G, or C) using both equilibrium infrared (IR) and time-resolved IR spectroscopy. Equilibrium IR melting data were used to determine thermodynamic parameters. Melting temperatures ranged from 50 to 60°C, and enthalpies of unfolding were on the order of 100 kJ/mol. In the time-resolved work, temperature jumps of up to 20°C at 2.5°C increments were obtained with transient relaxation kinetics spanning nanoseconds to hundreds of microseconds. The relaxation kinetics for all of the oligomers studied were fit to first or second order exponentials. Multiple vibrational transitions were probed on each oligomer for fully folded and partially denatured structures. In the time-resolved limit, in contrast to equilibrium melting, RNA does not fold according to two-state behavior. These results are some of the first to show that RNA hairpins fold according to a rugged energy landscape, which contradicts their relatively simple nature. In addition, this work has proven that time-resolved IR spectroscopy is a powerful and novel tool for investigating the earliest events of RNA folding, the formation of the hairpin loop.
Multiple stepwise refolding of immunoglobulin domain I27 upon force quench depends on initial conditions

PubMed Central

Li, Mai Suan; Hu, Chin-Kun; Klimov, Dmitri K.; Thirumalai, D.

2006-01-01

Mechanical folding trajectories for polyproteins starting from initially stretched conformations generated by single-molecule atomic force microscopy experiments [Fernandez, J. M. & Li, H. (2004) Science 303, 1674–1678] show that refolding, monitored by the end-to-end distance, occurs in distinct multiple stages. To clarify the molecular nature of folding starting from stretched conformations, we have probed the folding dynamics, upon force quench, for the single I27 domain from the muscle protein titin by using a Cα-Go model. Upon temperature quench, collapse and folding of I27 are synchronous. In contrast, refolding from stretched initial structures not only increases the folding and collapse time scales but also decouples the two kinetic processes. The increase in the folding times is associated primarily with the stretched state to compact random coil transition. Surprisingly, force quench does not alter the nature of the refolding kinetics, but merely increases the height of the free-energy folding barrier. Force quench refolding times scale as \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\tau}_{F}\\approx {\\tau}_{F}^{0}{\\mathrm{exp}}(f_{q}{\\Delta}x_{f}/k_{{\\mathrm{B}}}T)\\end{equation*}\\end{document}, where Δxf ≈ 0.6 nm is the location of the average transition state along the reaction coordinate given by end-to-end distance. We predict that τF and the folding mechanism can be dramatically altered by the initial and/or final values of force. The implications of our results for design and analysis of experiments are discussed. PMID:16373511
Reversible Aggregation Plays a Crucial Role on the Folding Landscape of p53 Core Domain

PubMed Central

Ishimaru, Daniella; Lima, Luis M. T. R.; Maia, Lenize F.; Lopez, Priscila M.; Ano Bom, Ana P.; Valente, Ana P.; Silva, Jerson L.

2004-01-01

The role of tumor suppressor protein p53 in cell cycle control depends on its flexible and partially unstructured conformation, which makes it crucial to understand its folding landscape. Here we report an intermediate structure of the core domain of the tumor suppressor protein p53 (p53C) during equilibrium and kinetic folding/unfolding transitions induced by guanidinium chloride. This partially folded structure was undetectable when investigated by intrinsic fluorescence. Indeed, the fluorescence data showed a simple two-state transition. On the other hand, analysis of far ultraviolet circular dichroism in 1.0 M guanidinium chloride demonstrated a high content of secondary structure, and the use of an extrinsic fluorescent probe, 4,4′-dianilino-1,1′ binaphthyl-5,5′-disulfonic acid, indicated an increase in exposure of the hydrophobic core at 1 M guanidinium chloride. This partially folded conformation of p53C was plagued by aggregation, as suggested by one-dimensional NMR and demonstrated by light-scattering and gel-filtration chromatography. Dissociation by high pressure of these aggregates reveals the reversibility of the process and that the aggregates have water-excluded cavities. Kinetic measurements show that the intermediate formed in a parallel reaction between unfolded and folded structures and that it is under fine energetic control. They are not only crucial to the folding pathway of p53C but may explain as well the vulnerability of p53C to undergo departure of the native to an inactive state, which makes the cell susceptible to malignant transformation. PMID:15298872
Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation.

PubMed

Vuillard, L; Rabilloud, T; Goldberg, M E

1998-08-15

Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.
Amyloid Polymorphism in the Protein Folding and Aggregation Energy Landscape.

PubMed

Adamcik, Jozef; Mezzenga, Raffaele

2018-02-15

Protein folding involves a large number of steps and conformations in which the folding protein samples different thermodynamic states characterized by local minima. Kinetically trapped on- or off-pathway intermediates are metastable folding intermediates towards the lowest absolute energy minima, which have been postulated to be the natively folded state where intramolecular interactions dominate, and the amyloid state where intermolecular interactions dominate. However, this view largely neglects the rich polymorphism found within amyloid species. We review the protein folding energy landscape in view of recent findings identifying specific transition routes among different amyloid polymorphs. Observed transitions such as twisted ribbon→crystal or helical ribbon→nanotube, and forbidden transitions such helical ribbon↛crystal, are discussed and positioned within the protein folding and aggregation energy landscape. Finally, amyloid crystals are identified as the ground state of the protein folding and aggregation energy landscape. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Engineering diverse changes in beta-turn propensities in the N-terminal beta-hairpin of ubiquitin reveals significant effects on stability and kinetics but a robust folding transition state.

PubMed

Simpson, Emma R; Meldrum, Jill K; Searle, Mark S

2006-04-04

Using the N-terminal 17-residue beta-hairpin of ubiquitin as a "host" for mutational studies, we have investigated the influence of the beta-turn sequence on protein stability and folding kinetics by replacing the native G-bulged turn (TLTGK) with more flexible analogues (TG3K and TG5K) and a series of four-residue type I' beta-turn sequences, commonly found in beta-hairpins. Although a statistical analysis of type I' turns demonstrates residue preferences at specific sites, the frequency of occurrence appears to only broadly correlate with experimentally determined protein stabilities. The subsequent engineering of context-dependent non-native tertiary contacts involving turn residues is shown to produce large changes in stability. Relatively few point mutations have been described that probe secondary structure formation in ubiquitin in a manner that is independent of tertiary contacts. To this end, we have used the more rigorous rate-equilibrium free energy relationship (Leffler analysis), rather than the two-point phi value analysis, to show for a family of engineered beta-turn mutants that stability (range of approximately 20 kJ/mol) and folding kinetics (190-fold variation in refolding rate) are linearly correlated (alpha(f) = 0.74 +/- 0.08). The data are consistent with a transition state that is robust with regard to a wide range of statistically favored and disfavored beta-turn mutations and implicate a loosely assembled beta-hairpin as a key template in transition state stabilization with the beta-turn playing a central role.

Structural and kinetic mapping of side-chain exposure onto the protein energy landscape.

PubMed

Bernstein, Rachel; Schmidt, Kierstin L; Harbury, Pehr B; Marqusee, Susan

2011-06-28

Identification and characterization of structural fluctuations that occur under native conditions is crucial for understanding protein folding and function, but such fluctuations are often rare and transient, making them difficult to study. Native-state hydrogen exchange (NSHX) has been a powerful tool for identifying such rarely populated conformations, but it generally reveals no information about the placement of these species along the folding reaction coordinate or the barriers separating them from the folded state and provides little insight into side-chain packing. To complement such studies, we have performed native-state alkyl-proton exchange, a method analogous to NSHX that monitors cysteine modification rather than backbone amide exchange, to examine the folding landscape of Escherichia coli ribonuclease H, a protein well characterized by hydrogen exchange. We have chosen experimental conditions such that the rate-limiting barrier acts as a kinetic partition: residues that become exposed only upon crossing the unfolding barrier are modified in the EX1 regime (alkylation rates report on the rate of unfolding), while those exposed on the native side of the barrier are modified predominantly in the EX2 regime (alkylation rates report on equilibrium populations). This kinetic partitioning allows for identification and placement of partially unfolded forms along the reaction coordinate. Using this approach we detect previously unidentified, rarely populated conformations residing on the native side of the barrier and identify side chains that are modified only upon crossing the unfolding barrier. Thus, in a single experiment under native conditions, both sides of the rate-limiting barrier are investigated.
Structural and kinetic mapping of side-chain exposure onto the protein energy landscape

PubMed Central

Bernstein, Rachel; Schmidt, Kierstin L.; Harbury, Pehr B.; Marqusee, Susan

2011-01-01

Identification and characterization of structural fluctuations that occur under native conditions is crucial for understanding protein folding and function, but such fluctuations are often rare and transient, making them difficult to study. Native-state hydrogen exchange (NSHX) has been a powerful tool for identifying such rarely populated conformations, but it generally reveals no information about the placement of these species along the folding reaction coordinate or the barriers separating them from the folded state and provides little insight into side-chain packing. To complement such studies, we have performed native-state alkyl-proton exchange, a method analogous to NSHX that monitors cysteine modification rather than backbone amide exchange, to examine the folding landscape of Escherichia coli ribonuclease H, a protein well characterized by hydrogen exchange. We have chosen experimental conditions such that the rate-limiting barrier acts as a kinetic partition: residues that become exposed only upon crossing the unfolding barrier are modified in the EX1 regime (alkylation rates report on the rate of unfolding), while those exposed on the native side of the barrier are modified predominantly in the EX2 regime (alkylation rates report on equilibrium populations). This kinetic partitioning allows for identification and placement of partially unfolded forms along the reaction coordinate. Using this approach we detect previously unidentified, rarely populated conformations residing on the native side of the barrier and identify side chains that are modified only upon crossing the unfolding barrier. Thus, in a single experiment under native conditions, both sides of the rate-limiting barrier are investigated. PMID:21670244
Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.

PubMed

Zhang, Yongli

2017-07-01

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins. © 2017 The Protein Society.
Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding

PubMed Central

Nissley, Daniel A.; Sharma, Ajeet K.; Ahmed, Nabeel; Friedrich, Ulrike A.; Kramer, Günter; Bukau, Bernd; O'Brien, Edward P.

2016-01-01

The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally—a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process. PMID:26887592
Information encoded in non-native states drives substrate-chaperone pairing.

PubMed

Mapa, Koyeli; Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik

2012-09-05

Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones. Copyright © 2012 Elsevier Ltd. All rights reserved.
The rough energy landscape of superfolder GFP is linked to the chromophore

PubMed Central

Andrews, Benjamin T.; Schoenfish, Andrea R.; Roy, Melinda; Waldo, Geoffrey; Jennings, Patricia A.

2009-01-01

Many GFP variants have been developed for use as fluorescent tags, and recently a superfolder GFP (sfGFP) has been developed as a robust folding reporter. This new variant shows increased stability and improved folding kinetics, as well as 100% recovery of native protein after denaturation. Here, we characterize sfGFP, and find that this variant exhibits hysteresis as unfolding and refolding equilibrium titration curves are non-coincident even after equilibration for more than eight half-lives as estimated from kinetic unfolding and refolding studies. This hysteresis is attributed to trapping in a native-like intermediate state. Mutational studies directed towards inhibiting chromophore formation indicate that the novel backbone cyclization is responsible for the hysteresis observed in equilibrium titrations of sfGFP. Slow equilibration and the presence of intermediates imply a rough landscape. However, de novo folding in the absence of the chromophore is dominated by a smoother energy landscape than that sampled during unfolding and refolding of the post-translationally modified polypeptide. PMID:17822714
Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

PubMed Central

2009-01-01

Many studies of RNA folding and catalysis have revealed conformational heterogeneity, metastable folding intermediates, and long-lived states with distinct catalytic activities. We have developed a single-molecule imaging approach for investigating the functional heterogeneity of in vitro-evolved RNA aptamers. Monitoring the association of fluorescently labeled ligands with individual RNA aptamer molecules has allowed us to record binding events over the course of multiple days, thus providing sufficient statistics to quantitatively define the kinetic properties at the single-molecule level. The ligand binding kinetics of the highly optimized RNA aptamer studied here displays a remarkable degree of uniformity and lack of memory. Such homogeneous behavior is quite different from the heterogeneity seen in previous single-molecule studies of naturally derived RNA and protein enzymes. The single-molecule methods we describe may be of use in analyzing the distribution of functional molecules in heterogeneous evolving populations or even in unselected samples of random sequences. PMID:19572753
Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells

PubMed Central

Fischer, Manuel; Horn, Sebastian; Belkacemi, Anouar; Kojer, Kerstin; Petrungaro, Carmelina; Habich, Markus; Ali, Muna; Küttner, Victoria; Bien, Melanie; Kauff, Frank; Dengjel, Jörn; Herrmann, Johannes M.; Riemer, Jan

2013-01-01

Oxidation of cysteine residues to disulfides drives import of many proteins into the intermembrane space of mitochondria. Recent studies in yeast unraveled the basic principles of mitochondrial protein oxidation, but the kinetics under physiological conditions is unknown. We developed assays to follow protein oxidation in living mammalian cells, which reveal that import and oxidative folding of proteins are kinetically and functionally coupled and depend on the oxidoreductase Mia40, the sulfhydryl oxidase augmenter of liver regeneration (ALR), and the intracellular glutathione pool. Kinetics of substrate oxidation depends on the amount of Mia40 and requires tightly balanced amounts of ALR. Mia40-dependent import of Cox19 in human cells depends on the inner membrane potential. Our observations reveal considerable differences in the velocities of mitochondrial import pathways: whereas preproteins with bipartite targeting sequences are imported within seconds, substrates of Mia40 remain in the cytosol for several minutes and apparently escape premature degradation and oxidation. PMID:23676665
Approximate Solutions for a Self-Folding Problem of Carbon Nanotubes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Mikata

2006-08-22

This paper treats approximate solutions for a self-folding problem of carbon nanotubes. It has been observed in the molecular dynamics calculations [1] that a carbon nanotube with a large aspect ratio can self-fold due to van der Waals force between the parts of the same carbon nanotube. The main issue in the self-folding problem is to determine the minimum threshold length of the carbon nanotube at which it becomes possible for the carbon nanotube to self-fold due to the van der Waals force. An approximate mathematical model based on the force method is constructed for the self-folding problem of carbonmore » nanotubes, and it is solved exactly as an elastica problem using elliptic functions. Additionally, three other mathematical models are constructed based on the energy method. As a particular example, the lower and upper estimates for the critical threshold (minimum) length are determined based on both methods for the (5,5) armchair carbon nanotube.« less
Atomic-level description of ubiquitin folding

PubMed Central

Piana, Stefano; Lindorff-Larsen, Kresten; Shaw, David E.

2013-01-01

Equilibrium molecular dynamics simulations, in which proteins spontaneously and repeatedly fold and unfold, have recently been used to help elucidate the mechanistic principles that underlie the folding of fast-folding proteins. The extent to which the conclusions drawn from the analysis of such proteins, which fold on the microsecond timescale, apply to the millisecond or slower folding of naturally occurring proteins is, however, unclear. As a first attempt to address this outstanding issue, we examine here the folding of ubiquitin, a 76-residue-long protein found in all eukaryotes that is known experimentally to fold on a millisecond timescale. Ubiquitin folding has been the subject of many experimental studies, but its slow folding rate has made it difficult to observe and characterize the folding process through all-atom molecular dynamics simulations. Here we determine the mechanism, thermodynamics, and kinetics of ubiquitin folding through equilibrium atomistic simulations. The picture emerging from the simulations is in agreement with a view of ubiquitin folding suggested from previous experiments. Our findings related to the folding of ubiquitin are also consistent, for the most part, with the folding principles derived from the simulation of fast-folding proteins, suggesting that these principles may be applicable to a wider range of proteins. PMID:23503848
Biphasic Kinetic Behavior of Nitrate Reductase from Heterocystous, Nitrogen-Fixing Cyanobacteria 1

PubMed Central

Martin-Nieto, José; Flores, Enrique; Herrero, Antonia

1992-01-01

Nitrate reductase activity from filamentous, heterocyst-forming cyanobacteria showed a biphasic kinetic behavior with respect to nitrate as the variable substrate. Two kinetic components were detected, the first showing a higher affinity for nitrate (Km, 0.05-0.25 mm) and a lower catalytic activity and the second showing a lower affinity for nitrate (Km, 5-25 mm) and a higher (3- to 5-fold) catalytic activity. In contrast, among unicellular cyanobacteria, most representatives studied exhibited a monophasic, Michaelis-Menten kinetic pattern for nitrate reductase activity. Biphasic kinetics remained unchanged with the use of different assay conditions (i.e. cell disruption or permeabilization, two different electron donors) or throughout partial purification of the enzyme. PMID:16652939
There and back again: Two views on the protein folding puzzle.

PubMed

Finkelstein, Alexei V; Badretdin, Azat J; Galzitskaya, Oxana V; Ivankov, Dmitry N; Bogatyreva, Natalya S; Garbuzynskiy, Sergiy O

2017-07-01

The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10-11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: "U-to-N" and "N-to-U". In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the "N-to-U" transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical "detailed balance" principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the "N-to-U" transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the "U-to-N" transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the side of the native state; and both theories agree with experiment. In addition, they predict the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control and explain the observed maximal size of the "foldable" protein domains. Copyright © 2017 Elsevier B.V. All rights reserved.
There and back again: Two views on the protein folding puzzle

NASA Astrophysics Data System (ADS)

Finkelstein, Alexei V.; Badretdin, Azat J.; Galzitskaya, Oxana V.; Ivankov, Dmitry N.; Bogatyreva, Natalya S.; Garbuzynskiy, Sergiy O.

2017-07-01

The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10-11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: ;U-to-N; and ;N-to-U;. In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the ;N-to-U; transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical ;detailed balance; principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the ;N-to-U; transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the ;U-to-N; transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the side of the native state; and both theories agree with experiment. In addition, they predict the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control and explain the observed maximal size of the ;foldable; protein domains.
Revascularization of the graft in obliterative bronchiolitis after heterotopic tracheal transplantation.

PubMed

Nemska, Simona; Daubeuf, François; Frossard, Nelly

2016-02-01

Obliterative bronchiolitis is the principal long-term problem for lung transplant patients. One of the simplest and most reproducible animal models of obliterative bronchiolitis is heterotopic tracheal transplantation in subcutaneous tissue, where the graft is not primarily vascularized. We demonstrate here the rapid graft revascularization and the kinetics of expression of its angiogenic and lymphatic factors. We performed iso- and allotracheal transplantations harvested on day 0-21. The number of functional blood vessels, quantified after intravenous biotinylated dextran administration, increased from D0 (0 for both iso- and allografts) to D21 (44 ± 8 vessels/mm(2) in isografts and 22 ± 3 in allografts, P < 0.001 for both vs. D0). VEGF mRNA expression assessed by qPCR peaked on D1 (4.3-fold increase in isografts and 4.0-fold in allografts, P < 0.0001 for both vs. D0), but receded thereafter. Angiopoietin-1, involved in the maturation of the neoformed vessels, increased later on, by 6.2-fold (P < 0.05) in isografts and 11.5-fold in allografts (P < 0.001) by D21, and angiopoietin-2 by 7.8-fold in isografts (P < 0.05) and 13.8-fold in allografts (P < 0.01). Although always present in the iso- and allografts, there were significantly more and larger LYVE1(+) lymphatic vessels at D21 in allografts than in isografts. Thus, we demonstrate that tracheal grafts are rapidly revascularized by functional blood and lymphatic vessels, due to early VEGF and subsequent angiopoietins expression, which is a new advantage of this model, in addition to its ease of use, reproducibility, and viability in the absence of immunosuppressive treatment. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.
Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

PubMed Central

Reilly, Samantha M.; Lyons, Daniel F.; Wingate, Sara E.; Wright, Robert T.; Correia, John J.; Jameson, David M.; Wadkins, Randy M.

2014-01-01

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs. PMID:25296324
Chemical kinetics as a contract sport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kolb, C.E.

1990-01-01

Earlier in this century chemical kinetics was a basic physical chemistry research topic widely pursued in leading academic chemistry departments. Chemical kinetics now appears to be a discipline practiced chiefly for its applications to societal problems. The chemical kinetics activities directed by D.M. Golden at SRI International are strikingly successful in generating data for key applied problems while at the same time advancing our understanding of chemical kinetics as a scientific discipline. In this talk, the author will contrast the chemical kinetics activities in two contract R D laboratories, one on the right side of the U.S. (ARI) and themore » other on the left (SRI). Their approach to common applied problems ranging from stratospheric heterogeneous kinetics to plasma etching systems for semiconductor processing will be compared and contrasted. Empirically discovered Golden Rules for the pursuit of quality chemical kinetics research in a contract R D environment will be presented and discussed.« less
Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase.

PubMed

Jia, Yan; Liu, Hui; Bao, Wei; Weng, Meizhi; Chen, Wei; Cai, Yongjun; Zheng, Zhongliang; Zou, Guolin

2010-12-01

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Understanding the mechanical properties of DNA origami tiles and controlling the kinetics of their folding and unfolding reconfiguration.

PubMed

Chen, Haorong; Weng, Te-Wei; Riccitelli, Molly M; Cui, Yi; Irudayaraj, Joseph; Choi, Jong Hyun

2014-05-14

DNA origami represents a class of highly programmable macromolecules that can go through conformational changes in response to external signals. Here we show that a two-dimensional origami rectangle can be effectively folded into a short, cylindrical tube by connecting the two opposite edges through the hybridization of linker strands and that this process can be efficiently reversed via toehold-mediated strand displacement. The reconfiguration kinetics was experimentally studied as a function of incubation temperature, initial origami concentration, missing staples, and origami geometry. A kinetic model was developed by introducing the j factor to describe the reaction rates in the cyclization process. We found that the cyclization efficiency (j factor) increases sharply with temperature and depends strongly on the structural flexibility and geometry. A simple mechanical model was used to correlate the observed cyclization efficiency with origami structure details. The mechanical analysis suggests two sources of the energy barrier for DNA origami folding: overcoming global twisting and bending the structure into a circular conformation. It also provides the first semiquantitative estimation of the rigidity of DNA interhelix crossovers, an essential element in structural DNA nanotechnology. This work demonstrates efficient DNA origami reconfiguration, advances our understanding of the dynamics and mechanical properties of self-assembled DNA structures, and should be valuable to the field of DNA nanotechnology.
Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

PubMed

Meuzelaar, Heleen; Vreede, Jocelyne; Woutersen, Sander

2016-06-07

Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Photocatalytic dye degradation with copper-titanium dioxide nanocomposites under sunlight and visible light irradiation

NASA Astrophysics Data System (ADS)

Qayyum Khan, Abdul; Yuan, Shuai; Niu, Sheng; Liu, Fengjiang; Feng, Guang; Jiang, Mengci; Zeng, Heping

2018-01-01

Photocatalytic methalyne blue dye degradation was carried out with copper (Cu)-titanium dioxide (TiO2) nanocomposites under sunlight and visible light irradiation. The Cu-TiO2 nanocomposites were fabricated via femtosecond laser ablation of pressed targets in water. The current method provides a facile route for Cu-TiO2 nanocomposites preparation, which is free from impurities on the catalysts surface. The Cu-TiO2 nanocomposites (with Cu content of 5 wt%) have shown 3 folds faster dye degradation kinetics compared with TiO2 nanoparticles under sunlight irradiation. While under visible light irradiation, the same nanocomposites exhibited 2.6 folds faster kinetics compared with TiO2 nanoparticles. The faster light harvesting efficiency of the catalysts is attributed to more hydroxyl radical generation.

Kinetic contribution to extracellular Na+/K+ selectivity in the Na+/K+ pump.

PubMed

Vleeskens, Elizabeth; Clarke, Ronald J

2018-05-01

The sodium potassium pump (Na + ,K + -ATPase) shows a high selectivity for K + over Na + binding from the extracellular medium. To understand the K + selectivity in the presence of a high concentration of competing Na + ions requires consideration of more than just ion binding affinities. Here, equilibrium-based calculations of the extracellular occupation of the Na + ,K + -ATPase transport sites by Na + and K + are compared to fluxes through Na + and K + transport pathways. The results show that, under physiological conditions, there is a 332-fold selectivity for pumping of K + from the extracellular medium into the cytoplasm relative to Na + , whereas equilibrium calculations alone predict only a 7.5-fold selectivity for K + . Thus, kinetic effects make a major contribution to the determination of extracellular K + selectivity.
Maximizing RNA folding rates: a balancing act.

PubMed Central

Thirumalai, D; Woodson, S A

2000-01-01

Large ribozymes typically require very long times to refold into their active conformation in vitro, because the RNA is easily trapped in metastable misfolded structures. Theoretical models show that the probability of misfolding is reduced when local and long-range interactions in the RNA are balanced. Using the folding kinetics of the Tetrahymena ribozyme as an example, we propose that folding rates are maximized when the free energies of forming independent domains are similar to each other. A prediction is that the folding pathway of the ribozyme can be reversed by inverting the relative stability of the tertiary domains. This result suggests strategies for optimizing ribozyme sequences for therapeutics and structural studies. PMID:10864039
Fast protein folding kinetics

PubMed Central

Gelman, Hannah; Gruebele, Martin

2014-01-01

Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816
Unstirred Water Layers and the Kinetics of Organic Cation Transport

PubMed Central

Shibayama, Takahiro; Morales, Mark; Zhang, Xiaohong; Martinez, Lucy; Berteloot, Alfred; Secomb, Timothy W.; Wright, Stephen H.

2015-01-01

Purpose Unstirred water layers (UWLs) present an unavoidable complication in the measurement of transport kinetics in cultured cells and the high rates of transport achieved by overexpressing heterologous transporters exacerbate the UWL effect. This study examined the correlation between measured Jmax and Kt values and the effect of manipulating UWL thickness or transport Jmax on the accuracy of experimentally determined kinetics of the multidrug transporters, OCT2 and MATE1. Methods Transport of TEA and MPP was measured in CHO cells that stably expressed human OCT2 or MATE1. UWL thickness was manipulated by vigorous reciprocal shaking. Several methods were used to manipulate maximal transport rates. Results Vigorous stirring stimulated uptake of OCT2-mediated transport by decreasing apparent Kt (Ktapp) values. Systematic reduction in transport rates was correlated with reduction in Ktapp values. The slope of these relationships indicated a 1500 µm UWL in multiwell plates. Reducing the influence of UWLs (by decreasing either their thickness or the Jmax of substrate transport) reduced Ktapp by 2-fold to >10-fold. Conclusions Failure to take into account the presence of UWLs in experiments using cultured cells to measure transport kinetics can result in significant underestimates of the affinity of multidrug transporters for substrates. PMID:25791216
Pectin as an Extraordinary Natural Kinetic Hydrate Inhibitor

PubMed Central

Xu, Shurui; Fan, Shuanshi; Fang, Songtian; Lang, Xuemei; Wang, Yanhong; Chen, Jun

2016-01-01

Pectin as a novel natural kinetic hydrate inhibitor, expected to be eco-friendly and sufficiently biodegradable, was studied in this paper. The novel crystal growth inhibition (CGI) and standard induction time methods were used to evaluate its effect as hydrate inhibitor. It could successfully inhibit methane hydrate formation at subcooling temperature up to 12.5 °C and dramatically slowed the hydrate crystal growth. The dosage of pectin decreased by 66% and effective time extended 10 times than typical kinetic inhibitor. Besides, its maximum growth rate was no more than 2.0%/h, which was far less than 5.5%/h of growth rate for PVCap at the same dosage. The most prominent feature was that it totally inhibited methane hydrate crystal rapid growth when hydrate crystalline occurred. Moreover, in terms of typical natural inhibitors, the inhibition activity of pectin increased 10.0-fold in induction time and 2.5-fold in subcooling temperature. The extraordinary inhibition activity is closely related to its hydrogen bonding interaction with water molecules and the hydrophilic structure. Finally, the biodegradability and economical efficiency of pectin were also taken into consideration. The results showed the biodegradability improved 75.0% and the cost reduced by more than 73.3% compared to typical commercial kinetic inhibitors. PMID:26996773
Molecular dynamics studies of protein folding and aggregation

NASA Astrophysics Data System (ADS)

Ding, Feng

This thesis applies molecular dynamics simulations and statistical mechanics to study: (i) protein folding; and (ii) protein aggregation. Most small proteins fold into their native states via a first-order-like phase transition with a major free energy barrier between the folded and unfolded states. A set of protein conformations corresponding to the free energy barrier, Delta G >> kBT, are the folding transition state ensemble (TSE). Due to their evasive nature, TSE conformations are hard to capture (probability ∝ exp(-DeltaG/k BT)) and characterize. A coarse-grained discrete molecular dynamics model with realistic steric constraints is constructed to reproduce the experimentally observed two-state folding thermodynamics. A kinetic approach is proposed to identify the folding TSE. A specific set of contacts, common to the TSE conformations, is identified as the folding nuclei which are necessary to be formed in order for the protein to fold. Interestingly, the amino acids at the site of the identified folding nuclei are highly conserved for homologous proteins sharing the same structures. Such conservation suggests that amino acids that are important for folding kinetics are under selective pressure to be preserved during the course of molecular evolution. In addition, studies of the conformations close to the transition states uncover the importance of topology in the construction of order parameter for protein folding transition. Misfolded proteins often form insoluble aggregates, amyloid fibrils, that deposit in the extracellular space and lead to a type of disease known as amyloidosis. Due to its insoluble and non-crystalline nature, the aggregation structure and, thus the aggregation mechanism, has yet to be uncovered. Discrete molecular dynamics studies reveal an aggregate structure with the same structural signatures as in experimental observations and show a nucleation aggregation scenario. The simulations also suggest a generic aggregation mechanism that globular proteins under a denaturing environment partially unfold and aggregate by forming stabilizing hydrogen bonds between the backbones of the partial folded substructures. Proteins or peptides rich in alpha-helices also aggregate into beta-rich amyloid fibrils. Upon aggregation, the protein or peptide undergoes a conformational transition from alpha-helices to beta-sheets. The transition of alpha-helix to beta-hairpin (two-stranded beta-sheet) is studied in an all-heavy-atom discrete molecular dynamics model of a polyalanine chain. An entropical driving scenario for the alpha-helix to beta-hairpin transition is discovered.
Studies of protein-protein and protein-water interactions by small angle x-ray scattering, terahertz spectroscopy, ASMOS, and computer simulation

NASA Astrophysics Data System (ADS)

Kim, Seung Joong

The protein folding problem has been one of the most challenging subjects in biological physics due to its complexity. Energy landscape theory based on statistical mechanics provides a thermodynamic interpretation of the protein folding process. We have been working to answer fundamental questions about protein-protein and protein-water interactions, which are very important for describing the energy landscape surface of proteins correctly. At first, we present a new method for computing protein-protein interaction potentials of solvated proteins directly from SAXS data. An ensemble of proteins was modeled by Metropolis Monte Carlo and Molecular Dynamics simulations, and the global X-ray scattering of the whole model ensemble was computed at each snapshot of the simulation. The interaction potential model was optimized and iterated by a Levenberg-Marquardt algorithm. Secondly, we report that terahertz spectroscopy directly probes hydration dynamics around proteins and determines the size of the dynamical hydration shell. We also present the sequence and pH-dependence of the hydration shell and the effect of the hydrophobicity. On the other hand, kinetic terahertz absorption (KITA) spectroscopy is introduced to study the refolding kinetics of ubiquitin and its mutants. KITA results are compared to small angle X-ray scattering, tryptophan fluorescence, and circular dichroism results. We propose that KITA monitors the rearrangement of hydrogen bonding during secondary structure formation. Finally, we present development of the automated single molecule operating system (ASMOS) for a high throughput single molecule detector, which levitates a single protein molecule in a 10 microm diameter droplet by the laser guidance. I also have performed supporting calculations and simulations with my own program codes.
An Unusual Salt Effect in an Interfacial Nucleophilic Substitution Reaction.

PubMed

Li, Shuheng; Mrksich, Milan

2018-06-12

This paper reports a kinetic characterization of the interfacial reaction of N-methylpyrrolidine with a self-assembled monolayer presenting an iodoalkyl group. SAMDI (self-assembled monolayers for matrix-assisted laser desorption/ionization) mass spectrometry was used to determine the extent of reaction for monolayers that were treated with a range of concentrations of the nucleophile for a range of times. These data revealed a second-order rate constant for the reaction that was approximately 100-fold greater than that for the analogous solution-phase reaction. However, addition of sodium iodide to the reaction mixture resulted in a 7-fold decrease in the reaction rate. Addition of bromide and chloride salts also gave slower rate constants for the reaction, but only at 100- and 1000-fold higher concentrations than was observed with iodide, respectively. The corresponding solution-phase reactions, by contrast, had rate constants that were unaffected by the concentration of halide salts. This work provides a well-characterized example illustrating the extent to which the kinetics and properties of an interfacial reaction can depart substantially from their better-understood solution-phase counterparts.
Modelling DNA origami self-assembly at the domain level.

PubMed

Dannenberg, Frits; Dunn, Katherine E; Bath, Jonathan; Kwiatkowska, Marta; Turberfield, Andrew J; Ouldridge, Thomas E

2015-10-28

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.
Modelling DNA origami self-assembly at the domain level

NASA Astrophysics Data System (ADS)

Dannenberg, Frits; Dunn, Katherine E.; Bath, Jonathan; Kwiatkowska, Marta; Turberfield, Andrew J.; Ouldridge, Thomas E.

2015-10-01

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.
Concerted dihedral rotations give rise to internal friction in unfolded proteins.

PubMed

Echeverria, Ignacia; Makarov, Dmitrii E; Papoian, Garegin A

2014-06-18

Protein chains undergo conformational diffusion during folding and dynamics, experiencing both thermal kicks and viscous drag. Recent experiments have shown that the corresponding friction can be separated into wet friction, which is determined by the solvent viscosity, and dry friction, where frictional effects arise due to the interactions within the protein chain. Despite important advances, the molecular origins underlying dry friction in proteins have remained unclear. To address this problem, we studied the dynamics of the unfolded cold-shock protein at different solvent viscosities and denaturant concentrations. Using extensive all-atom molecular dynamics simulations we estimated the internal friction time scales and found them to agree well with the corresponding experimental measurements (Soranno et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 17800-17806). Analysis of the reconfiguration dynamics of the unfolded chain further revealed that hops in the dihedral space provide the dominant mechanism of internal friction. Furthermore, the increased number of concerted dihedral moves at physiological conditions suggest that, in such conditions, the concerted motions result in higher frictional forces. These findings have important implications for understanding the folding kinetics of proteins as well as the dynamics of intrinsically disordered proteins.
Conserved nucleation sites reinforce the significance of Phi value analysis in protein-folding studies.

PubMed

Gianni, Stefano; Jemth, Per

2014-07-01

The only experimental strategy to address the structure of folding transition states, the so-called Φ value analysis, relies on the synergy between site directed mutagenesis and the measurement of reaction kinetics. Despite its importance, the Φ value analysis has been often criticized and its power to pinpoint structural information has been questioned. In this hypothesis, we demonstrate that comparing the Φ values between proteins not only allows highlighting the robustness of folding pathways but also provides per se a strong validation of the method. © 2014 International Union of Biochemistry and Molecular Biology.
Structure and Dynamics of the tRNA-like Structure Domain of Brome Mosaic Virus

NASA Astrophysics Data System (ADS)

Vieweger, Mario; Nesbitt, David

2014-03-01

Conformational switching is widely accepted as regulatory mechanism in gene expression in bacterial systems. More recently, similar regulation mechanisms are emerging for viral systems. One of the most abundant and best studied systems is the tRNA-like structure domain that is found in a number of plant viruses across eight genera. In this work, the folding dynamics of the tRNA-like structure domain of Brome Mosaic Virus are investigated using single-molecule Fluorescence Resonance Energy Transfer techniques. In particular, Burst fluorescence is applied to observe metal-ion induced folding in freely diffusing RNA constructs resembling the 3'-terminal 169nt of BMV RNA3. Histograms of EFRET probabilities reveal a complex equilibrium of three distinct populations. A step-wise kinetic model for TLS folding is developed in accord with the evolution of conformational populations and structural information in the literature. In this mechanism, formation of functional TLS domains from unfolded RNAs requires two consecutive steps; 1) hybridization of a long-range stem interaction followed by 2) formation of a 3' pseudoknot. This three-state equilibrium is well described by step-wise dissociation constants K1(328(30) μM) and K2(1092(183) μM) for [Mg2+] and K1(74(6) mM) and K2(243(52) mM) for [Na+]-induced folding. The kinetic model is validated by oligo competition with the STEM interaction. Implications of this conformational folding mechanism are discussed in regards to regulation of virus replication.
Modeling of autocatalytic hydrolysis of adefovir dipivoxil in solid formulations.

PubMed

Dong, Ying; Zhang, Yan; Xiang, Bingren; Deng, Haishan; Wu, Jingfang

2011-04-01

The stability and hydrolysis kinetics of a phosphate prodrug, adefovir dipivoxil, in solid formulations were studied. The stability relationship between five solid formulations was explored. An autocatalytic mechanism for hydrolysis could be proposed according to the kinetic behavior which fits the Prout-Tompkins model well. For the classical kinetic models could hardly describe and predict the hydrolysis kinetics of adefovir dipivoxil in solid formulations accurately when the temperature is high, a feedforward multilayer perceptron (MLP) neural network was constructed to model the hydrolysis kinetics. The build-in approaches in Weka, such as lazy classifiers and rule-based learners (IBk, KStar, DecisionTable and M5Rules), were used to verify the performance of MLP. The predictability of the models was evaluated by 10-fold cross-validation and an external test set. It reveals that MLP should be of general applicability proposing an alternative efficient way to model and predict autocatalytic hydrolysis kinetics for phosphate prodrugs.
Application of Time-Resolved Tryptophan Phosphorescence Spectroscopy to Protein Folding Studies.

NASA Astrophysics Data System (ADS)

Subramaniam, Vinod

This thesis presents studies of the protein folding problem, one of the most significant questions in contemporary biophysics. Sensitive biophysical techniques, including room temperature tryptophan phosphorescence, which reports on the local environment of the residue, and the lability of proteins to denaturation, a global parameter, were used to assess the validity of the traditional assumption that the biologically active state of a protein is the 'native' state, and to determine whether the pathways of folding in vitro lead to the folded state achieved in vivo. Phosphorescence techniques have also been extended to study, for the first time, emission from tryptophan residues engineered into specific positions as reporters of protein structure. During in vitro refolding of E. coli alkaline phosphatase and bovine 13-lactoglobulin, significant differences were found between the refolded proteins and the native conformations, which have no apparent effect on the biological functions. Slow conformational transitions, termed 'annealing,' that occur long after the return of enzyme activity of alkaline phosphatase are manifested in the retarded recovery of phosphorescence intensity, lifetime, and protein lability. While 'annealing' is not observed for beta -lactoglobulin, both phosphorescence and lability experiments reveal changes in the structure of the refolded protein, even though its biological activity, retinol binding, is fully recovered. This result suggests that the pathways of folding in vitro need not lead to the structure formed in vivo. We have used phosphorescence techniques to study the refolding of ribonuclease T1, which exhibits slow kinetics characteristic of proline isomerization. Furthermore, the ability to extract structural information from phosphorescent tryptophan probes engineered into selected regions represents an important advance in studying protein structure; we have reported the first such results from a mutant staphylococcal nuclease. The refolding data have been interpreted in the context of recent theoretical work on rugged energy landscape models of protein folding. Our results suggest that the barriers to folding can be as large as ~ 20 kcal-mol^{-1}, and imply that the conventional definition of the 'native' state as the biologically active conformation may need revision to acknowledge that the active state may represent a long-lived intermediate on the pathway to the native structure.
Ab initio RNA folding by discrete molecular dynamics: From structure prediction to folding mechanisms

PubMed Central

Ding, Feng; Sharma, Shantanu; Chalasani, Poornima; Demidov, Vadim V.; Broude, Natalia E.; Dokholyan, Nikolay V.

2008-01-01

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 Å deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNAPhe, pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting non-hierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses. PMID:18456842
The initial value problem in Lagrangian drift kinetic theory

NASA Astrophysics Data System (ADS)

Burby, J. W.

2016-06-01

> Existing high-order variational drift kinetic theories contain unphysical rapidly varying modes that are not seen at low orders. These unphysical modes, which may be rapidly oscillating, damped or growing, are ushered in by a failure of conventional high-order drift kinetic theory to preserve the structure of its parent model's initial value problem. In short, the (infinite dimensional) system phase space is unphysically enlarged in conventional high-order variational drift kinetic theory. I present an alternative, `renormalized' variational approach to drift kinetic theory that manifestly respects the parent model's initial value problem. The basic philosophy underlying this alternate approach is that high-order drift kinetic theory ought to be derived by truncating the all-orders system phase-space Lagrangian instead of the usual `field particle' Lagrangian. For the sake of clarity, this story is told first through the lens of a finite-dimensional toy model of high-order variational drift kinetics; the analogous full-on drift kinetic story is discussed subsequently. The renormalized drift kinetic system, while variational and just as formally accurate as conventional formulations, does not support the troublesome rapidly varying modes.
When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches

PubMed Central

Muñoz, Victor; Cerminara, Michele

2016-01-01

Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico. All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. PMID:27574021
Determination of thermodynamics and kinetics of RNA reactions by force

PubMed Central

Tinoco, Ignacio; Li, Pan T. X.; Bustamante, Carlos

2008-01-01

Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force. PMID:17040613
Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold*

PubMed Central

Vogt, Austin D.; Chakraborty, Pradipta; Di Cera, Enrico

2015-01-01

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold. PMID:26216877

Proteome-level interplay between folding and aggregation propensities of proteins.

PubMed

Tartaglia, Gian Gaetano; Vendruscolo, Michele

2010-10-08

With the advent of proteomics, there is an increasing need of tools for predicting the properties of large numbers of proteins by using the information provided by their amino acid sequences, even in the absence of the knowledge of their structures. One of the most important types of predictions concerns whether proteins will fold or aggregate. Here, we study the competition between these two processes by analyzing the relationship between the folding and aggregation propensity profiles for the human and Escherichia coli proteomes. These profiles are calculated, respectively, using the CamFold method, which we introduce in this work, and the Zyggregator method. Our results indicate that the kinetic behavior of proteins is, to a large extent, determined by the interplay between regions of low folding and high aggregation propensities. Copyright © 2010. Published by Elsevier Ltd.
Competition between protein folding and aggregation: A three-dimensional lattice-model simulation

NASA Astrophysics Data System (ADS)

Bratko, D.; Blanch, H. W.

2001-01-01

Aggregation of protein molecules resulting in the loss of biological activity and the formation of insoluble deposits represents a serious problem for the biotechnology and pharmaceutical industries and in medicine. Considerable experimental and theoretical efforts are being made in order to improve our understanding of, and ability to control, the process. In the present work, we describe a Monte Carlo study of a multichain system of coarse-grained model proteins akin to lattice models developed for simulations of protein folding. The model is designed to examine the competition between intramolecular interactions leading to the native protein structure, and intermolecular association, resulting in the formation of aggregates of misfolded chains. Interactions between the segments are described by a variation of the Go potential [N. Go and H. Abe, Biopolymers 20, 1013 (1981)] that extends the recognition between attracting types of segments to pairs on distinct chains. For the particular model we adopt, the global free energy minimum of a pair of protein molecules corresponds to a dimer of native proteins. When three or more molecules interact, clusters of misfolded chains can be more stable than aggregates of native folds. A considerable fraction of native structure, however, is preserved in these cases. Rates of conformational changes rapidly decrease with the size of the protein cluster. Within the timescale accessible to computer simulations, the folding-aggregation balance is strongly affected by kinetic considerations. Both the native form and aggregates can persist in metastable states, even if conditions such as temperature or concentration favor a transition to an alternative form. Refolding yield can be affected by the presence of an additional polymer species mimicking the function of a molecular chaperone.
Probing the Non-Native H Helix Translocation in Apomyoglobin Folding Intermediates

PubMed Central

2015-01-01

Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C–L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state. PMID:24857522
Interactions with the Bifunctional Interface of the Transcriptional Coactivator DCoH1 Are Kinetically Regulated

DOE PAGES

Wang, Dongli; Coco, Matthew W.; Rose, Robert B.

2014-12-23

Pterin-4a-carbinolamine dehydratase (PCD) is a highly conserved enzyme that evolved a second, unrelated function in mammals, as a transcriptional coactivator. As a coactivator, PCD is known as DCoH or dimerization cofactor of the transcription factor HNF-1. These two activities are associated with a change in oligomeric state: from two dimers interacting as an enzyme in the cytoplasm to a dimer interacting with a dimer of HNF-1 in the nucleus. The same interface of DCoH forms both complexes. To determine how DCoH partitions between its two functions, we studied in this paper the folding and stability of the DCoH homotetramer. Wemore » show that the DCoH1 homotetramer is kinetically trapped, meaning once it forms it will not dissociate to interact with HNF-1. In contrast, DCoH2, a paralog of DCoH1, unfolds within hours. A simple mutation in the interface of DCoH2 from Ser-51 to Thr, as found in DCoH1, increases the kinetic stability by 9 orders of magnitude, to τ½ ~2 million years. This suggests that the DCoH1·HNF-1 complex must co-fold to interact. We conclude that simple mutations can dramatically affect the dissociation kinetics of a complex. Residue 51 represents a “kinetic hot spot” instead of a “thermodynamic hot spot.” Kinetic regulation allows PCD to adopt two distinct functions. Finally, mutations in DCoH1 associated with diabetes affect both functions of DCoH1, perhaps by disrupting the balance between the two DCoH complexes.« less
Advancing age produces sex differences in vasomotor kinetics during and after skeletal muscle contraction.

PubMed

Bearden, Shawn E

2007-09-01

Little is known of the vasomotor responses of skeletal muscle arterioles during and following muscle contraction. We hypothesized that aging leads to impaired arteriolar responses to muscle contraction and recovery. Nitric oxide (NO) availability, which is age dependent, has been implicated in components of these kinetics. Therefore, we also hypothesized that changes in the kinetics of vascular responses are associated with the NO pathway. Groups were young (3 mo), old (24 mo), endothelial NO synthase knockout (eNOS-/-), and N(G)-nitro-L-arginine (L-NA)-treated male and female C57BL/6 mice. The kinetics of vasodilation during and following 1 min of contractions of the gluteus maximus muscle were recorded in second-order (regional distribution) and third-order (local control) arterioles. Baseline, peak (during contraction), and maximal diameters (pharmacological) were not affected by age or sex. The kinetics of dilation and recovery were not different between males and females at the young age. There was a significant slowing of vasodilation at the onset of contractions (approximately 2-fold; P < 0.05) and a significant speeding of recovery ( approximately 5-fold; P < 0.05) in old males vs. old females and vs. young eNOS-/-, and L-NA did not affect the kinetics at the onset of muscle contraction. eNOS-/- mimicked the rapid recovery of old males in second-order arterioles; acute NO production (L-NA) explained approximately 50% of this effect. These data demonstrate fundamental age-related differences between the sexes in the dynamic function of skeletal muscle arterioles. Understanding how youthful function persists in females but not males may provide therapeutic insight into clinical interventions to maintain dynamic microvascular control of nutrient supply with age.
Kinetic Parameters of Efflux of Penicillins by the Multidrug Efflux Transporter AcrAB-TolC of Escherichia coli▿

PubMed Central

Lim, Siew Ping; Nikaido, Hiroshi

2010-01-01

The multidrug efflux transporter AcrAB-TolC is known to pump out a diverse range of antibiotics, including β-lactams. However, the kinetic constants of the efflux process, needed for the quantitative understanding of resistance, were not available until those accompanying the efflux of some cephalosporins were recently determined by combining efflux with the hydrolysis of drugs by the periplasmic β-lactamase. In the present study we extended this approach to the study of a wide range of penicillins, from ampicillin and penicillin V to ureidopenicillins and isoxazolylpenicillins, by combining efflux with hydrolysis with the OXA-7 penicillinase. We found that the penicillins had a much stronger apparent affinity to AcrB and higher maximum rates of efflux than the cephalosporins. All penicillins showed strong positive cooperativity kinetics for export. The kinetic constants obtained were validated, as the MICs theoretically predicted on the basis of efflux and hydrolysis kinetics were remarkably similar to the observed MICs (except for the isoxazolylpenicillins). Surprisingly, however, the efflux kinetics of cloxacillin, for example, whose MIC decreased 512-fold in Escherichia coli upon the genetic deletion of the acrB gene, were quite similar to those of ampicillin, whose MIC decreased only 2-fold with the same treatment. Analysis of this phenomenon showed that the extensive decrease in the MIC for the acrB mutant is primarily due to the low permeation of the drug and that comparison of the MICs between the parent and the acrB strains is a very poor measure of the ability of AcrB to pump a drug out. PMID:20160052
LSENS: A General Chemical Kinetics and Sensitivity Analysis Code for homogeneous gas-phase reactions. Part 3: Illustrative test problems

NASA Technical Reports Server (NTRS)

Bittker, David A.; Radhakrishnan, Krishnan

1994-01-01

LSENS, the Lewis General Chemical Kinetics and Sensitivity Analysis Code, has been developed for solving complex, homogeneous, gas-phase chemical kinetics problems and contains sensitivity analysis for a variety of problems, including nonisothermal situations. This report is part 3 of a series of three reference publications that describe LSENS, provide a detailed guide to its usage, and present many example problems. Part 3 explains the kinetics and kinetics-plus-sensitivity analysis problems supplied with LSENS and presents sample results. These problems illustrate the various capabilities of, and reaction models that can be solved by, the code and may provide a convenient starting point for the user to construct the problem data file required to execute LSENS. LSENS is a flexible, convenient, accurate, and efficient solver for chemical reaction problems such as static system; steady, one-dimensional, inviscid flow; reaction behind incident shock wave, including boundary layer correction; and perfectly stirred (highly backmixed) reactor. In addition, the chemical equilibrium state can be computed for the following assigned states: temperature and pressure, enthalpy and pressure, temperature and volume, and internal energy and volume. For static problems the code computes the sensitivity coefficients of the dependent variables and their temporal derivatives with respect to the initial values of the dependent variables and/or the three rate coefficient parameters of the chemical reactions.
Probing the cytochrome c' folding landscape.

PubMed

Pletneva, Ekaterina V; Zhao, Ziqing; Kimura, Tetsunari; Petrova, Krastina V; Gray, Harry B; Winkler, Jay R

2007-11-01

The folding kinetics of R. palustris cytochrome c' (cyt c') have been monitored by heme absorption and native Trp72 fluorescence at pH 5. The Trp72 fluorescence burst signal suggests early compaction of the polypeptide ensemble. Analysis of heme transient absorption spectra reveals deviations from two-state behavior, including a prominent slow phase that is accelerated by the prolyl isomerase cyclophilin. A nonnative proline configuration (Pro21) likely interferes with the formation of the helical bundle surrounding the heme.
Inhibition of ligand exchange kinetics via active-site trapping with an antibody fragment.

PubMed

Oyen, David; Steyaert, Jan; Barlow, John N

2014-04-01

We describe the first example of an inhibitory antibody fragment (nanobody ca1697) that binds simultaneously to an enzyme (the enzyme dihydrofolate reductase from Escherichia coli) and its bound substrate (folate). Binding of the antibody to the substrate causes a 20-fold reduction in the rate of folate exchange kinetics. This work opens up the prospect of designing new types of antibody-based inhibitors of enzymes and receptors through suitable design of immunogens.
RNAslider: a faster engine for consecutive windows folding and its application to the analysis of genomic folding asymmetry.

PubMed

Horesh, Yair; Wexler, Ydo; Lebenthal, Ilana; Ziv-Ukelson, Michal; Unger, Ron

2009-03-04

Scanning large genomes with a sliding window in search of locally stable RNA structures is a well motivated problem in bioinformatics. Given a predefined window size L and an RNA sequence S of size N (L < N), the consecutive windows folding problem is to compute the minimal free energy (MFE) for the folding of each of the L-sized substrings of S. The consecutive windows folding problem can be naively solved in O(NL3) by applying any of the classical cubic-time RNA folding algorithms to each of the N-L windows of size L. Recently an O(NL2) solution for this problem has been described. Here, we describe and implement an O(NLpsi(L)) engine for the consecutive windows folding problem, where psi(L) is shown to converge to O(1) under the assumption of a standard probabilistic polymer folding model, yielding an O(L) speedup which is experimentally confirmed. Using this tool, we note an intriguing directionality (5'-3' vs. 3'-5') folding bias, i.e. that the minimal free energy (MFE) of folding is higher in the native direction of the DNA than in the reverse direction of various genomic regions in several organisms including regions of the genomes that do not encode proteins or ncRNA. This bias largely emerges from the genomic dinucleotide bias which affects the MFE, however we see some variations in the folding bias in the different genomic regions when normalized to the dinucleotide bias. We also present results from calculating the MFE landscape of a mouse chromosome 1, characterizing the MFE of the long ncRNA molecules that reside in this chromosome. The efficient consecutive windows folding engine described in this paper allows for genome wide scans for ncRNA molecules as well as large-scale statistics. This is implemented here as a software tool, called RNAslider, and applied to the scanning of long chromosomes, leading to the observation of features that are visible only on a large scale.
Kinetics and reaction coordinates of the reassembly of protein fragments via forward flux sampling.

PubMed

Borrero, Ernesto E; Contreras Martínez, Lydia M; DeLisa, Matthew P; Escobedo, Fernando A

2010-05-19

We studied the mechanism of the reassembly and folding process of two fragments of a split lattice protein by using forward flux sampling (FFS). Our results confirmed previous thermodynamics and kinetics analyses that suggested that the disruption of the critical core (of an unsplit protein that folds by a nucleation mechanism) plays a key role in the reassembly mechanism of the split system. For several split systems derived from a parent 48-mer model, we estimated the reaction coordinates in terms of collective variables by using the FFS least-square estimation method and found that the reassembly transition is best described by a combination of the total number of native contacts, the number of interchain native contacts, and the total conformational energy of the split system. We also analyzed the transition path ensemble obtained from FFS simulations using the estimated reaction coordinates as order parameters to identify the microscopic features that differentiate the reassembly of the different split systems studied. We found that in the fastest folding split system, a balanced distribution of the original-core amino acids (of the unsplit system) between protein fragments propitiates interchain interactions at early stages of the folding process. Only this system exhibits a different reassembly mechanism from that of the unsplit protein, involving the formation of a different folding nucleus. In the slowest folding system, the concentration of the folding nucleus in one fragment causes its early prefolding, whereas the second fragment tends to remain as a detached random coil. We also show that the reassembly rate can be either increased or decreased by tuning interchain cooperativeness via the introduction of a single point mutation that either strengthens or weakens one of the native interchain contacts (prevalent in the transition state ensemble). Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.
The Complexity of Folding Self-Folding Origami

NASA Astrophysics Data System (ADS)

Stern, Menachem; Pinson, Matthew B.; Murugan, Arvind

2017-10-01

Why is it difficult to refold a previously folded sheet of paper? We show that even crease patterns with only one designed folding motion inevitably contain an exponential number of "distractor" folding branches accessible from a bifurcation at the flat state. Consequently, refolding a sheet requires finding the ground state in a glassy energy landscape with an exponential number of other attractors of higher energy, much like in models of protein folding (Levinthal's paradox) and other NP-hard satisfiability (SAT) problems. As in these problems, we find that refolding a sheet requires actuation at multiple carefully chosen creases. We show that seeding successful folding in this way can be understood in terms of subpatterns that fold when cut out ("folding islands"). Besides providing guidelines for the placement of active hinges in origami applications, our results point to fundamental limits on the programmability of energy landscapes in sheets.
Predicting protein folding rate change upon point mutation using residue-level coevolutionary information.

PubMed

Mallik, Saurav; Das, Smita; Kundu, Sudip

2016-01-01

Change in folding kinetics of globular proteins upon point mutation is crucial to a wide spectrum of biological research, such as protein misfolding, toxicity, and aggregations. Here we seek to address whether residue-level coevolutionary information of globular proteins can be informative to folding rate changes upon point mutations. Generating residue-level coevolutionary networks of globular proteins, we analyze three parameters: relative coevolution order (rCEO), network density (ND), and characteristic path length (CPL). A point mutation is considered to be equivalent to a node deletion of this network and respective percentage changes in rCEO, ND, CPL are found linearly correlated (0.84, 0.73, and -0.61, respectively) with experimental folding rate changes. The three parameters predict the folding rate change upon a point mutation with 0.031, 0.045, and 0.059 standard errors, respectively. © 2015 Wiley Periodicals, Inc.
When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.

PubMed

Muñoz, Victor; Cerminara, Michele

2016-09-01

Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. © 2016 The Author(s).
Ultrafast Unzipping of a Beta-Hairpin Peptide

NASA Astrophysics Data System (ADS)

Zinth, W.; Schrader, T. E.; Schreier, W. J.; Koller, F. O.; Cordes, T.; Babitzki, G.; Denschlag, R.; Tavan, P.; Löweneck, M.; Dong, Shou-Liang; Moroder, L.; Renner, C.

Light induced switching of a beta-hairpin structure is investigated by femtosecond IR spectroscopy. While the unzipping process comprises ultrafast kinetics and is finished within 1 ns, the folding into the hairpin structure is a much slower process.
Unfolding and melting of DNA (RNA) hairpins: the concept of structure-specific 2D dynamic landscapes.

PubMed

Lin, Milo M; Meinhold, Lars; Shorokhov, Dmitry; Zewail, Ahmed H

2008-08-07

A 2D free-energy landscape model is presented to describe the (un)folding transition of DNA/RNA hairpins, together with molecular dynamics simulations and experimental findings. The dependence of the (un)folding transition on the stem sequence and the loop length is shown in the enthalpic and entropic contributions to the free energy. Intermediate structures are well defined by the two coordinates of the landscape during (un)zipping. Both the free-energy landscape model and the extensive molecular dynamics simulations totaling over 10 mus predict the existence of temperature-dependent kinetic intermediate states during hairpin (un)zipping and provide the theoretical description of recent ultrafast temperature-jump studies which indicate that hairpin (un)zipping is, in general, not a two-state process. The model allows for lucid prediction of the collapsed state(s) in simple 2D space and we term it the kinetic intermediate structure (KIS) model.
Rapid hybridization of nucleic acids using isotachophoresis

PubMed Central

Bercovici, Moran; Han, Crystal M.; Liao, Joseph C.; Santiago, Juan G.

2012-01-01

We use isotachophoresis (ITP) to control and increase the rate of nucleic acid hybridization reactions in free solution. We present a new physical model, validation experiments, and demonstrations of this assay. We studied the coupled physicochemical processes of preconcentration, mixing, and chemical reaction kinetics under ITP. Our experimentally validated model enables a closed form solution for ITP-aided reaction kinetics, and reveals a new characteristic time scale which correctly predicts order 10,000-fold speed-up of chemical reaction rate for order 100 pM reactants, and greater enhancement at lower concentrations. At 500 pM concentration, we measured a reaction time which is 14,000-fold lower than that predicted for standard second-order hybridization. The model and method are generally applicable to acceleration of reactions involving nucleic acids, and may be applicable to a wide range of reactions involving ionic reactants. PMID:22733732
Improvements in Mixing Time and Mixing Uniformity in Devices Designed for Studies of Protein Folding Kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Shuhuai; Bakajin, Olgica

2007-08-01

Using a microfluidic laminar flow mixer designed for studies of protein folding kinetics, we demonstrate a mixing time of 1 +/- 1 micros with sample consumption on the order of femtomoles. We recognize two limitations of previously proposed designs: (1) size and shape of the mixing region, which limits mixing uniformity and (2) the formation of Dean vortices at high flow rates, which limits the mixing time. We address these limitations by using a narrow shape-optimized nozzle and by reducing the bend of the side channel streamlines. The final design, which combines both of these features, achieves the best performance.more » We quantified the mixing performance of the different designs by numerical simulation of coupled Navier-Stokes and convection-diffusion equations and experiments using fluorescence resonance energy-transfer (FRET)-labeled DNA.« less
Communication: Role of explicit water models in the helix folding/unfolding processes

NASA Astrophysics Data System (ADS)

Palazzesi, Ferruccio; Salvalaglio, Matteo; Barducci, Alessandro; Parrinello, Michele

2016-09-01

In the last years, it has become evident that computer simulations can assume a relevant role in modelling protein dynamical motions for their ability to provide a full atomistic image of the processes under investigation. The ability of the current protein force-fields in reproducing the correct thermodynamics and kinetics systems behaviour is thus an essential ingredient to improve our understanding of many relevant biological functionalities. In this work, employing the last developments of the metadynamics framework, we compare the ability of state-of-the-art all-atom empirical functions and water models to consistently reproduce the folding and unfolding of a helix turn motif in a model peptide. This theoretical study puts in evidence that the choice of the water models can influence the thermodynamic and the kinetics of the system under investigation, and for this reason cannot be considered trivial.
On the role of conformational geometry in protein folding

NASA Astrophysics Data System (ADS)

Du, Rose; Pande, Vijay S.; Grosberg, Alexander Yu.; Tanaka, Toyoichi; Shakhnovich, Eugene

1999-12-01

Using a lattice model of protein folding, we find that once certain native contacts have been formed, folding to the native state is inevitable, even if the only energetic bias in the system is nonspecific, homopolymeric attraction to a collapsed state. These conformations can be quite geometrically unrelated to the native state (with as low as only 53% of the native contacts formed). We demonstrate these results by examining the Monte Carlo kinetics of both heteropolymers under Go interactions and homopolymers, with the folding of both types of polymers to the native state of the heteropolymer. Although we only consider a 48-mer lattice model, our findings shed light on the effects of geometrical restrictions, including those of chain connectivity and steric excluded volume, on protein folding. These effects play a complementary role to that of the rugged energy landscape. In addition, the results of this work can aid in the interpretation of experiments and computer simulations of protein folding performed at elevated temperatures.

Kinetic: A system code for analyzing nuclear thermal propulsion rocket engine transients

NASA Astrophysics Data System (ADS)

Schmidt, Eldon; Lazareth, Otto; Ludewig, Hans

The topics are presented in viewgraph form and include the following: outline of kinetic code; a kinetic information flow diagram; kinetic neutronic equations; turbopump/nozzle algorithm; kinetic heat transfer equations per node; and test problem diagram.
Self-catalyzed growth of S layers via an amorphous-to-crystalline transition limited by folding kinetics.

PubMed

Chung, Sungwook; Shin, Seong-Ho; Bertozzi, Carolyn R; De Yoreo, James J

2010-09-21

The importance of nonclassical, multistage crystallization pathways is increasingly evident from theoretical studies on colloidal systems and experimental investigations of proteins and biomineral phases. Although theoretical predictions suggest that proteins follow these pathways as a result of fluctuations that create unstable dense-liquid states, microscopic studies indicate these states are long-lived. Using in situ atomic force microscopy to follow 2D assembly of S-layer proteins on supported lipid bilayers, we have obtained a molecular-scale picture of multistage protein crystallization that reveals the importance of conformational transformations in directing the pathway of assembly. We find that monomers with an extended conformation first form a mobile adsorbed phase, from which they condense into amorphous clusters. These clusters undergo a phase transition through S-layer folding into crystalline clusters composed of compact tetramers. Growth then proceeds by formation of new tetramers exclusively at cluster edges, implying tetramer formation is autocatalytic. Analysis of the growth kinetics leads to a quantitative model in which tetramer creation is rate limiting. However, the estimated barrier is much smaller than expected for folding of isolated S-layer proteins, suggesting an energetic rationale for this multistage pathway.
In vitro kinetics of soybean lipoxygenase with combinatorial fatty substrates and its functional significance in off flavour development.

PubMed

Mandal, Somnath; Dahuja, Anil; Kar, Abhijit; Santha, I M

2014-03-01

Lipoxygenase (Lox) mediated oxidation of polyunsaturated fatty acids (PUFA) in mature soya seeds results in objectionable flavour. In the present study, Lox isozymes were purified to near homogeneity (107-fold). Lox-2 and 3 displayed remarkable kinetic preference (1.7 and 1.5-fold, respectively) for low PUFA ratios (LA/LeA) (PRs) among the selected PUFA combinations. Lox-1 displayed no specific preference. Pure Lox-1 displayed unbiased response towards substrates with marginal preference (1.2-fold) for linoleic acid at its optimum pH. Volatile compounds profiling showed a direct relationship between PRs and hexanal to trans-2-hexenal (1.47, 2.24 and 18.90 for 2, 7 and 15 PRs, respectively) ratio. The off-flavour determining parameters like TBA value, carbonyl value and lipid hydroperoxides (LHPODs) exhibited significant negative correlation (0.76, 0.74, 0.72; p<0.0001) in selected soya genotypes displaying varied PRs and significant positive correlation (0.89, 0.81. 0.89; p<0.0001) with ratio of PI (polyene index) to PRs - suggesting the plausible significance of PUFA ratios in biological lipid peroxidation. Copyright © 2013 Elsevier Ltd. All rights reserved.
The E. coli thioredoxin folding mechanism: the key role of the C-terminal helix.

PubMed

Vazquez, Diego S; Sánchez, Ignacio E; Garrote, Ana; Sica, Mauricio P; Santos, Javier

2015-02-01

In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability. Copyright © 2014 Elsevier B.V. All rights reserved.
Stability and kinetics of G-quadruplex structures

PubMed Central

Lane, Andrew N.; Chaires, J. Brad; Gray, Robert D.; Trent, John O.

2008-01-01

In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function. The unifying theme in this review is energetics. The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides. We provide an overview of the principles of stability and where available the experimental data that report on these principles. Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes. Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations. Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium. The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence. PMID:18718931
Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yechun; Yi, Hankuil; Wang, Melissa

2012-10-24

To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the firstmore » molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.« less
Integration of Inhibition Kinetics and Molecular Dynamics Simulations: A Urea-Mediated Folding Study on Acetaldehyde Dehydrogenase 1.

PubMed

Xu, Yingying; Lee, Jinhyuk; Lü, Zhi-Rong; Mu, Hang; Zhang, Qian; Park, Yong-Doo

2016-07-01

Understanding the mechanism of acetaldehyde dehydrogenase 1 (ALDH1) folding is important because this enzyme is directly involved in several types of cancers and other diseases. We investigated the urea-mediated unfolding of ALDH1 by integrating kinetic inhibition studies with computational molecular dynamics (MD) simulations. Conformational changes in the enzyme structure were also analyzed using intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence measurements. Kinetic studies revealed that the direct binding of urea to ALDH1 induces inactivation of ALDH1 in a manner of mixed-type inhibition. Tertiary structural changes associated with regional hydrophobic exposure of the active site were observed. The urea binding regions on ALDH1 were predicted by docking simulations and were partly shared with active site residues of ALDH1 and with interface residues of the oligomerization domain for tetramer formation. The docking results suggest that urea prevents formation of the ALDH1 normal shape for the tetramer state as well as entrance of the substrate into the active site. Our study provides insight into the structural changes that accompany urea-mediated unfolding of ALDH1 and the catalytic role associated with conformational changes.
Variation of iron redox kinetics and its relation with molecular composition of standard humic substances at circumneutral pH.

PubMed

Lee, Ying Ping; Fujii, Manabu; Kikuchi, Tetsuro; Terao, Koumei; Yoshimura, Chihiro

2017-01-01

Oxidation and reduction kinetics of iron (Fe) and proportion of steady-state Fe(II) concentration relative to total dissolved Fe (steady-state Fe(II) fraction) were investigated in the presence of various types of standard humic substances (HS) with particular emphasis on the photochemical and thermal reduction of Fe(III) and oxidation of Fe(II) by dissolved oxygen (O2) and hydrogen peroxide (H2O2) at circumneutral pH (pH 7-8). Rates of Fe(III) reduction were spectrophotometrically determined by a ferrozine method under the simulated sunlight and dark conditions, whereas rates of Fe(II) oxidation were examined in air-saturated solution using luminol chemiluminescence technique. The reduction and oxidation rate constants were determined to substantially vary depending on the type of HS. For example, the first-order rate constants varied by up to 10-fold for photochemical reduction and 7-fold for thermal reduction. The degree of variation in Fe(II) oxidation was larger for the H2O2-mediated reaction compared to the O2-mediated reaction (e.g., 15- and 3-fold changes for the former and latter reactions, respectively, at pH 8). The steady-state Fe(II) fraction under the simulated sunlight indicated that the Fe(II) fraction varies by up to 12-fold. The correlation analysis indicated that variation of Fe(II) oxidation is significantly associated with aliphatic content of HS, suggesting that Fe(II) complexation by aliphatic components accelerates Fe(II) oxidation. The reduction rate constant and steady-state Fe(II) fractions in the presence of sunlight had relatively strong positive relations with free radical content of HS, possibly due to the reductive property of radical semiquinone in HS. Overall, the findings in this study indicated that the Fe reduction and oxidation kinetics and resultant Fe(II) formation are substantially influenced by chemical properties of HS.
Kinetic resolution of drug intermediates catalyzed by lipase B from Candida antarctica immobilized on immobead-350.

PubMed

Pinheiro, Maísa Pessoa; Rios, Nathalia Saraiva; Fonseca, Thiago de S; Bezerra, Francisco de Aquino; Rodríguez-Castellón, Enrique; Fernandez-Lafuente, Roberto; Carlos de Mattos, Marcos; Dos Santos, José C S; Gonçalves, Luciana R B

2018-03-14

Novozyme 435, which is a commercial immobilized lipase B from Candida antarctica (CALB), has been proven to be inadequate for the kinetic resolution of rac-indanyl acetate. As it has been previously described that different immobilization protocols may greatly alter lipase features, in this work, CALB was covalently immobilized on epoxy Immobead-350 (IB-350) and on glyoxyl-agarose to ascertain if better kinetic resolution would result. Afterwards, all CALB biocatalysts were utilized in the hydrolytic resolution of rac-indanyl acetate and rac-(chloromethyl)-2-(o-methoxyphenoxy) ethyl acetate. After optimization of the immobilization protocol on IB-350, its loading capacity was 150 mg protein/g dried support. Furthermore, the CALB-IB-350 thermal and solvent stabilities were higher than that of the soluble enzyme (e.g., by a 14-fold factor at pH 5-70°C and by a 11-fold factor in dioxane 30%-65°C) and that of the glyoxyl-agarose-CALB (e.g., by a 12-fold factor at pH 10-50°C and by a 21-fold factor in dioxane 30%-65°C). The CALB-IB-350 preparation (with 98% immobilization yield and activity versus p-nitrophenyl butyrate of 6.26 ± 0.2 U/g) was used in the hydrolysis of rac-indanyl acetate using a biocatalyst/substrate ratio of 2:1 and a pH value of 7.0 at 30°C for 24 h. The conversion obtained was 48% and the enantiomeric excess of the product (e.e. p ) was 97%. These values were much higher than the ones obtained with Novozyme 435, 13% and 26% of conversion and e.e.p, respectively. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.
Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis.

PubMed Central

Witmer, M. R.; Palmieri-Young, D.; Villafranca, J. J.

1994-01-01

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS. PMID:7849593
LSENS, a general chemical kinetics and sensitivity analysis code for homogeneous gas-phase reactions. 2: Code description and usage

NASA Technical Reports Server (NTRS)

Radhakrishnan, Krishnan; Bittker, David A.

1994-01-01

LSENS, the Lewis General Chemical Kinetics Analysis Code, has been developed for solving complex, homogeneous, gas-phase chemical kinetics problems and contains sensitivity analysis for a variety of problems, including nonisothermal situations. This report is part 2 of a series of three reference publications that describe LSENS, provide a detailed guide to its usage, and present many example problems. Part 2 describes the code, how to modify it, and its usage, including preparation of the problem data file required to execute LSENS. Code usage is illustrated by several example problems, which further explain preparation of the problem data file and show how to obtain desired accuracy in the computed results. LSENS is a flexible, convenient, accurate, and efficient solver for chemical reaction problems such as static system; steady, one-dimensional, inviscid flow; reaction behind incident shock wave, including boundary layer correction; and perfectly stirred (highly backmixed) reactor. In addition, the chemical equilibrium state can be computed for the following assigned states: temperature and pressure, enthalpy and pressure, temperature and volume, and internal energy and volume. For static problems the code computes the sensitivity coefficients of the dependent variables and their temporal derivatives with respect to the initial values of the dependent variables and/or the three rate coefficient parameters of the chemical reactions. Part 1 (NASA RP-1328) derives the governing equations describes the numerical solution procedures for the types of problems that can be solved by lSENS. Part 3 (NASA RP-1330) explains the kinetics and kinetics-plus-sensitivity-analysis problems supplied with LSENS and presents sample results.
How Four Scientists Integrate Thermodynamic and Kinetic Theory, Context, Analogies, and Methods in Protein-Folding and Dynamics Research: Implications for Biochemistry Instruction.

PubMed

Jeffery, Kathleen A; Pelaez, Nancy; Anderson, Trevor R

2018-01-01

To keep biochemistry instruction current and relevant, it is crucial to expose students to cutting-edge scientific research and how experts reason about processes governed by thermodynamics and kinetics such as protein folding and dynamics. This study focuses on how experts explain their research into this topic with the intention of informing instruction. Previous research has modeled how expert biologists incorporate research methods, social or biological context, and analogies when they talk about their research on mechanisms. We used this model as a guiding framework to collect and analyze interview data from four experts. The similarities and differences that emerged from analysis indicate that all experts integrated theoretical knowledge with their research context, methods, and analogies when they explained how phenomena operate, in particular by mapping phenomena to mathematical models; they explored different processes depending on their explanatory aims, but readily transitioned between different perspectives and explanatory models; and they explained thermodynamic and kinetic concepts of relevance to protein folding in different ways that aligned with their particular research methods. We discuss how these findings have important implications for teaching and future educational research. © 2018 K. A. Jeffery et al. CBE—Life Sciences Education © 2018 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.

PubMed

Eichmann, Cédric; Preissler, Steffen; Riek, Roland; Deuerling, Elke

2010-05-18

The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from alpha-spectrin at sequential stages of elongation via in vivo ribosome-arrested (15)N,(13)C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like beta-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding.
Kinetics and thermodynamics of DNA polymerases with exonuclease proofreading

NASA Astrophysics Data System (ADS)

Gaspard, Pierre

2016-04-01

Kinetic theory and thermodynamics are applied to DNA polymerases with exonuclease activity, taking into account the dependence of the rates on the previously incorporated nucleotide. The replication fidelity is shown to increase significantly thanks to this dependence at the basis of the mechanism of exonuclease proofreading. In particular, this dependence can provide up to a 100-fold lowering of the error probability under physiological conditions. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.
Influence of architecture on the kinetic stability of molecular assemblies.

PubMed

Patel, Amesh B; Allen, Stephanie; Davies, Martyn C; Roberts, Clive J; Tendler, Saul J B; Williams, Philip M

2004-02-11

The strength of a multimolecular system depends on the number of interactions that hold it together. Using dynamic force spectroscopy, we show how the kinetic stability of a system decreases as the number of molecular bonds is increased, as predicted by theory. The data raise important considerations for experimental tests of bond strength and, as a paradigm, suggest both routes to and pitfalls in methods for computational simulation of molecular transitions, such as ligand binding and protein folding.
New insights into the folding of a β-sheet miniprotein in a reduced space of collective hydrogen bond variables: application to a hydrodynamic analysis of the folding flow.

PubMed

Kalgin, Igor V; Caflisch, Amedeo; Chekmarev, Sergei F; Karplus, Martin

2013-05-23

A new analysis of the 20 μs equilibrium folding/unfolding molecular dynamics simulations of the three-stranded antiparallel β-sheet miniprotein (beta3s) in implicit solvent is presented. The conformation space is reduced in dimensionality by introduction of linear combinations of hydrogen bond distances as the collective variables making use of a specially adapted principal component analysis (PCA); i.e., to make structured conformations more pronounced, only the formed bonds are included in determining the principal components. It is shown that a three-dimensional (3D) subspace gives a meaningful representation of the folding behavior. The first component, to which eight native hydrogen bonds make the major contribution (four in each beta hairpin), is found to play the role of the reaction coordinate for the overall folding process, while the second and third components distinguish the structured conformations. The representative points of the trajectory in the 3D space are grouped into conformational clusters that correspond to locally stable conformations of beta3s identified in earlier work. A simplified kinetic network based on the three components is constructed, and it is complemented by a hydrodynamic analysis. The latter, making use of "passive tracers" in 3D space, indicates that the folding flow is much more complex than suggested by the kinetic network. A 2D representation of streamlines shows there are vortices which correspond to repeated local rearrangement, not only around minima of the free energy surface but also in flat regions between minima. The vortices revealed by the hydrodynamic analysis are apparently not evident in folding pathways generated by transition-path sampling. Making use of the fact that the values of the collective hydrogen bond variables are linearly related to the Cartesian coordinate space, the RMSD between clusters is determined. Interestingly, the transition rates show an approximate exponential correlation with distance in the hydrogen bond subspace. Comparison with the many published studies shows good agreement with the present analysis for the parts that can be compared, supporting the robust character of our understanding of this "hydrogen atom" of protein folding.
Evolution, Energy Landscapes and the Paradoxes of Protein Folding

PubMed Central

Wolynes, Peter G.

2014-01-01

Protein folding has been viewed as a difficult problem of molecular self-organization. The search problem involved in folding however has been simplified through the evolution of folding energy landscapes that are funneled. The funnel hypothesis can be quantified using energy landscape theory based on the minimal frustration principle. Strong quantitative predictions that follow from energy landscape theory have been widely confirmed both through laboratory folding experiments and from detailed simulations. Energy landscape ideas also have allowed successful protein structure prediction algorithms to be developed. The selection constraint of having funneled folding landscapes has left its imprint on the sequences of existing protein structural families. Quantitative analysis of co-evolution patterns allows us to infer the statistical characteristics of the folding landscape. These turn out to be consistent with what has been obtained from laboratory physicochemical folding experiments signalling a beautiful confluence of genomics and chemical physics. PMID:25530262
Inclusion bodies and purification of proteins in biologically active forms.

PubMed

Mukhopadhyay, A

1997-01-01

Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved.
Maximizing neotissue growth kinetics in a perfusion bioreactor: An in silico strategy using model reduction and Bayesian optimization.

PubMed

Mehrian, Mohammad; Guyot, Yann; Papantoniou, Ioannis; Olofsson, Simon; Sonnaert, Maarten; Misener, Ruth; Geris, Liesbet

2018-03-01

In regenerative medicine, computer models describing bioreactor processes can assist in designing optimal process conditions leading to robust and economically viable products. In this study, we started from a (3D) mechanistic model describing the growth of neotissue, comprised of cells, and extracellular matrix, in a perfusion bioreactor set-up influenced by the scaffold geometry, flow-induced shear stress, and a number of metabolic factors. Subsequently, we applied model reduction by reformulating the problem from a set of partial differential equations into a set of ordinary differential equations. Comparing the reduced model results to the mechanistic model results and to dedicated experimental results assesses the reduction step quality. The obtained homogenized model is 10 5 fold faster than the 3D version, allowing the application of rigorous optimization techniques. Bayesian optimization was applied to find the medium refreshment regime in terms of frequency and percentage of medium replaced that would maximize neotissue growth kinetics during 21 days of culture. The simulation results indicated that maximum neotissue growth will occur for a high frequency and medium replacement percentage, a finding that is corroborated by reports in the literature. This study demonstrates an in silico strategy for bioprocess optimization paying particular attention to the reduction of the associated computational cost. © 2017 Wiley Periodicals, Inc.
SVM-Fold: a tool for discriminative multi-class protein fold and superfamily recognition

PubMed Central

Melvin, Iain; Ie, Eugene; Kuang, Rui; Weston, Jason; Stafford, William Noble; Leslie, Christina

2007-01-01

Background Predicting a protein's structural class from its amino acid sequence is a fundamental problem in computational biology. Much recent work has focused on developing new representations for protein sequences, called string kernels, for use with support vector machine (SVM) classifiers. However, while some of these approaches exhibit state-of-the-art performance at the binary protein classification problem, i.e. discriminating between a particular protein class and all other classes, few of these studies have addressed the real problem of multi-class superfamily or fold recognition. Moreover, there are only limited software tools and systems for SVM-based protein classification available to the bioinformatics community. Results We present a new multi-class SVM-based protein fold and superfamily recognition system and web server called SVM-Fold, which can be found at . Our system uses an efficient implementation of a state-of-the-art string kernel for sequence profiles, called the profile kernel, where the underlying feature representation is a histogram of inexact matching k-mer frequencies. We also employ a novel machine learning approach to solve the difficult multi-class problem of classifying a sequence of amino acids into one of many known protein structural classes. Binary one-vs-the-rest SVM classifiers that are trained to recognize individual structural classes yield prediction scores that are not comparable, so that standard "one-vs-all" classification fails to perform well. Moreover, SVMs for classes at different levels of the protein structural hierarchy may make useful predictions, but one-vs-all does not try to combine these multiple predictions. To deal with these problems, our method learns relative weights between one-vs-the-rest classifiers and encodes information about the protein structural hierarchy for multi-class prediction. In large-scale benchmark results based on the SCOP database, our code weighting approach significantly improves on the standard one-vs-all method for both the superfamily and fold prediction in the remote homology setting and on the fold recognition problem. Moreover, our code weight learning algorithm strongly outperforms nearest-neighbor methods based on PSI-BLAST in terms of prediction accuracy on every structure classification problem we consider. Conclusion By combining state-of-the-art SVM kernel methods with a novel multi-class algorithm, the SVM-Fold system delivers efficient and accurate protein fold and superfamily recognition. PMID:17570145

A kMC-MD method with generalized move-sets for the simulation of folding of α-helical and β-stranded peptides.

PubMed

Peter, Emanuel K; Pivkin, Igor V; Shea, Joan-Emma

2015-04-14

In Monte-Carlo simulations of protein folding, pathways and folding times depend on the appropriate choice of the Monte-Carlo move or process path. We developed a generalized set of process paths for a hybrid kinetic Monte Carlo-Molecular dynamics algorithm, which makes use of a novel constant time-update and allows formation of α-helical and β-stranded secondary structures. We apply our new algorithm to the folding of 3 different proteins: TrpCage, GB1, and TrpZip4. All three systems are seen to fold within the range of the experimental folding times. For the β-hairpins, we observe that loop formation is the rate-determining process followed by collapse and formation of the native core. Cluster analysis of both peptides reveals that GB1 folds with equal likelihood along a zipper or a hydrophobic collapse mechanism, while TrpZip4 follows primarily a zipper pathway. The difference observed in the folding behavior of the two proteins can be attributed to the different arrangements of their hydrophobic core, strongly packed, and dry in case of TrpZip4, and partially hydrated in the case of GB1.
Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy

PubMed Central

Snow, Christopher D.; Qiu, Linlin; Du, Deguo; Gai, Feng; Hagen, Stephen J.; Pande, Vijay S.

2004-01-01

We studied the microsecond folding dynamics of three β hairpins (Trp zippers 1–3, TZ1–TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLSaa) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1–TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLSaa TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations. PMID:15020773
Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy

NASA Astrophysics Data System (ADS)

Snow, Christopher D.; Qiu, Linlin; Du, Deguo; Gai, Feng; Hagen, Stephen J.; Pande, Vijay S.

2004-03-01

We studied the microsecond folding dynamics of three hairpins (Trp zippers 1-3, TZ1-TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLSaa) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1-TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLSaa TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations.
Design Principles of DNA Enzyme-Based Walkers: Translocation Kinetics and Photoregulation.

PubMed

Cha, Tae-Gon; Pan, Jing; Chen, Haorong; Robinson, Heather N; Li, Xiang; Mao, Chengde; Choi, Jong Hyun

2015-07-29

Dynamic DNA enzyme-based walkers complete their stepwise movements along the prescribed track through a series of reactions, including hybridization, enzymatic cleavage, and strand displacement; however, their overall translocation kinetics is not well understood. Here, we perform mechanistic studies to elucidate several key parameters that govern the kinetics and processivity of DNA enzyme-based walkers. These parameters include DNA enzyme core type and structure, upper and lower recognition arm lengths, and divalent metal cation species and concentration. A theoretical model is developed within the framework of single-molecule kinetics to describe overall translocation kinetics as well as each reaction step. A better understanding of kinetics and design parameters enables us to demonstrate a walker movement near 5 μm at an average speed of ∼1 nm s(-1). We also show that the translocation kinetics of DNA walkers can be effectively controlled by external light stimuli using photoisomerizable azobenzene moieties. A 2-fold increase in the cleavage reaction is observed when the hairpin stems of enzyme catalytic cores are open under UV irradiation. This study provides general design guidelines to construct highly processive, autonomous DNA walker systems and to regulate their translocation kinetics, which would facilitate the development of functional DNA walkers.
A prospectus on kinetic heliophysics

NASA Astrophysics Data System (ADS)

Howes, Gregory G.

2017-05-01

Under the low density and high temperature conditions typical of heliospheric plasmas, the macroscopic evolution of the heliosphere is strongly affected by the kinetic plasma physics governing fundamental microphysical mechanisms. Kinetic turbulence, collisionless magnetic reconnection, particle acceleration, and kinetic instabilities are four poorly understood, grand-challenge problems that lie at the new frontier of kinetic heliophysics. The increasing availability of high cadence and high phase-space resolution measurements of particle velocity distributions by current and upcoming spacecraft missions and of massively parallel nonlinear kinetic simulations of weakly collisional heliospheric plasmas provides the opportunity to transform our understanding of these kinetic mechanisms through the full utilization of the information contained in the particle velocity distributions. Several major considerations for future investigations of kinetic heliophysics are examined. Turbulent dissipation followed by particle heating is highlighted as an inherently two-step process in weakly collisional plasmas, distinct from the more familiar case in fluid theory. Concerted efforts must be made to tackle the big-data challenge of visualizing the high-dimensional (3D-3V) phase space of kinetic plasma theory through physics-based reductions. Furthermore, the development of innovative analysis methods that utilize full velocity-space measurements, such as the field-particle correlation technique, will enable us to gain deeper insight into these four grand-challenge problems of kinetic heliophysics. A systems approach to tackle the multi-scale problem of heliophysics through a rigorous connection between the kinetic physics at microscales and the self-consistent evolution of the heliosphere at macroscales will propel the field of kinetic heliophysics into the future.
A prospectus on kinetic heliophysics

PubMed Central

2017-01-01

Under the low density and high temperature conditions typical of heliospheric plasmas, the macroscopic evolution of the heliosphere is strongly affected by the kinetic plasma physics governing fundamental microphysical mechanisms. Kinetic turbulence, collisionless magnetic reconnection, particle acceleration, and kinetic instabilities are four poorly understood, grand-challenge problems that lie at the new frontier of kinetic heliophysics. The increasing availability of high cadence and high phase-space resolution measurements of particle velocity distributions by current and upcoming spacecraft missions and of massively parallel nonlinear kinetic simulations of weakly collisional heliospheric plasmas provides the opportunity to transform our understanding of these kinetic mechanisms through the full utilization of the information contained in the particle velocity distributions. Several major considerations for future investigations of kinetic heliophysics are examined. Turbulent dissipation followed by particle heating is highlighted as an inherently two-step process in weakly collisional plasmas, distinct from the more familiar case in fluid theory. Concerted efforts must be made to tackle the big-data challenge of visualizing the high-dimensional (3D-3V) phase space of kinetic plasma theory through physics-based reductions. Furthermore, the development of innovative analysis methods that utilize full velocity-space measurements, such as the field-particle correlation technique, will enable us to gain deeper insight into these four grand-challenge problems of kinetic heliophysics. A systems approach to tackle the multi-scale problem of heliophysics through a rigorous connection between the kinetic physics at microscales and the self-consistent evolution of the heliosphere at macroscales will propel the field of kinetic heliophysics into the future. PMID:29104421
LSENS, A General Chemical Kinetics and Sensitivity Analysis Code for Homogeneous Gas-Phase Reactions. Part 2; Code Description and Usage

NASA Technical Reports Server (NTRS)

Radhakrishnan, Krishnan; Bittker, David A.

1994-01-01

LSENS, the Lewis General Chemical Kinetics and Sensitivity Analysis Code, has been developed for solving complex, homogeneous, gas-phase chemical kinetics problems and contains sensitivity analysis for a variety of problems, including nonisothermal situations. This report is part II of a series of three reference publications that describe LSENS, provide a detailed guide to its usage, and present many example problems. Part II describes the code, how to modify it, and its usage, including preparation of the problem data file required to execute LSENS. Code usage is illustrated by several example problems, which further explain preparation of the problem data file and show how to obtain desired accuracy in the computed results. LSENS is a flexible, convenient, accurate, and efficient solver for chemical reaction problems such as static system; steady, one-dimensional, inviscid flow; reaction behind incident shock wave, including boundary layer correction; and perfectly stirred (highly backmixed) reactor. In addition, the chemical equilibrium state can be computed for the following assigned states: temperature and pressure, enthalpy and pressure, temperature and volume, and internal energy and volume. For static problems the code computes the sensitivity coefficients of the dependent variables and their temporal derivatives with respect to the initial values of the dependent variables and/or the three rate coefficient parameters of the chemical reactions. Part I (NASA RP-1328) derives the governing equations and describes the numerical solution procedures for the types of problems that can be solved by LSENS. Part III (NASA RP-1330) explains the kinetics and kinetics-plus-sensitivity-analysis problems supplied with LSENS and presents sample results.
Effects of Polymer Hydrophobicity on Protein Structure and Aggregation Kinetics in Crowded Milieu.

PubMed

Breydo, Leonid; Sales, Amanda E; Frege, Telma; Howell, Mark C; Zaslavsky, Boris Y; Uversky, Vladimir N

2015-05-19

We examined the effects of water-soluble polymers of various degrees of hydrophobicity on the folding and aggregation of proteins. The polymers we chose were polyethylene glycol (PEG) and UCON (1:1 copolymer of ethylene glycol and propylene glycol). The presence of additional methyl groups in UCON makes it more hydrophobic than PEG. Our earlier analysis revealed that similarly sized PEG and UCON produced different changes in the solvent properties of water in their solutions and induced morphologically different α-synuclein aggregates [Ferreira, L. A., et al. (2015) Role of solvent properties of aqueous media in macromolecular crowding effects. J. Biomol. Struct. Dyn., in press]. To improve our understanding of molecular mechanisms defining behavior of proteins in a crowded environment, we tested the effects of these polymers on secondary and tertiary structure and aromatic residue solvent accessibility of 10 proteins [five folded proteins, two hybrid proteins; i.e., protein containing ordered and disordered domains, and three intrinsically disordered proteins (IDPs)] and on the aggregation kinetics of insulin and α-synuclein. We found that effects of both polymers on secondary and tertiary structures of folded and hybrid proteins were rather limited with slight unfolding observed in some cases. Solvent accessibility of aromatic residues was significantly increased for the majority of the studied proteins in the presence of UCON but not PEG. PEG also accelerated the aggregation of protein into amyloid fibrils, whereas UCON promoted aggregation to amyloid oligomers instead. These results indicate that even a relatively small change in polymer structure leads to a significant change in the effect of this polymer on protein folding and aggregation. This is an indication that protein folding and especially aggregation are highly sensitive to the presence of other macromolecules, and an excluded volume effect is insufficient to describe their effect.
In vitro-in vivo extrapolation of CYP2D6 inactivation by paroxetine: prediction of nonstationary pharmacokinetics and drug interaction magnitude.

PubMed

Venkatakrishnan, Karthik; Obach, R Scott

2005-06-01

Attempts at predicting drug-drug interactions perpetrated by paroxetine from in vitro data have utilized reversible enzyme inhibition models and have been unsuccessful to date, grossly underpredicting interaction magnitude. Recent data have provided evidence for mechanism-based inactivation of CYP2D6 by paroxetine. We have predicted the pharmacokinetic consequences of CYP2D6 inactivation by paroxetine from in vitro inactivation kinetics (kinact 0.17 min(-1), unbound KI 0.315 microM), in vivo inhibitor concentrations, and an estimated CYP2D6 degradation half-life of 51 h, using a mathematical model of mechanism-based inhibition. The model-predicted accumulation ratio of paroxetine was 5 times that expected from single-dose kinetics and in excellent agreement with the observed 5- to 6-fold greater accumulation. Magnitudes of interactions produced by paroxetine (20-30 mg/day) with desipramine, risperidone, perphenazine, atomoxetine, (S)-metoprolol, and (R)-metoprolol were predicted, considering the contribution of CYP2D6 to their oral clearance. Predicted fold-increases in victim drug AUC were 5-, 6-, 5-, 6-, 4-, and 6-fold, respectively, and are in reasonable agreement with observed values of 5-, 6-, >7-, 7-, 5-, and 8-fold, respectively. Failure to consider microsomal binding in vitro adversely affected predictive accuracy. Simulation of the sensitivities of these predictions to model inputs suggests a 2-fold underprediction of interaction magnitude when a CYP2D6 degradation half-life of 14 h (reported for rat CYP3A) is used. In summary, the scaling model for mechanism-based inactivation successfully predicted the pharmacokinetic consequences of CYP2D6 inactivation by paroxetine from in vitro data.
Enhanced Recyclable Magnetized Palm Shell Waste-Based Powdered Activated Carbon for the Removal of Ibuprofen: Insights for Kinetics and Mechanisms

PubMed Central

Wong, Kien Tiek; Yoon, Yeomin; Jang, Min

2015-01-01

A novel preparation method of magnetized palm shell waste-based powdered activated carbon (MPPAC, avg. size 112 μm) was developed. The prepared MPPAC was assessed by several physicochemical analyses, and batch tests were performed for ibuprofen (IBP) removal. Field emission scanning electron microscopy (FESEM) and N2 gas isotherms revealed that magnetite and maghemite were homogeneous and deposited mostly on the surface of PPAC without a significant clogging effect on the micropores. Isotherm results showed that 3.8% Fe (w/w) impregnated PPAC [MPPAC-Fe(3.8%)] had about 2.2-fold higher maximum sorption capacity (157.3 mg g-1) and a 2.5-fold higher sorption density (0.23 mg m-2) than pristine PPAC. Both Fourier-transform infrared spectroscopy (FTIR) and isotherm data indicated that the high sorption capacity and density of IBP by MPPAC was primarily attributable to donor-acceptor complexes with the C = O group and dispersive π-π interactions with the carbon surface. Based on kinetic and repeated adsorption tests, pore diffusion was the rate-limiting step, and MPPAC-Fe(3.8%) had about 1.9~2.8- and 9.1~15.8-fold higher rate constants than MPPAC-Fe(8.6%) and palm shell-waste granular activated carbon (PGAC, avg. size 621 μm), respectively. MPPAC showed almost eight fold greater re-adsorption capacity than PPAC due to a thermal catalytic effect of magnetite/maghemite. PMID:26496196
Postharvest stilbene-enrichment of red and white table grape varieties using UV-C irradiation pulses.

PubMed

Cantos, Emma; Espín, Juan Carlos; Tomás-Barberán, Francisco A

2002-10-23

The red table grape varieties Flame, Red Globe, Crimson, and Napoleon, as well as the white varieties Superior, Dominga, and Moscatel Italica, were irradiated with a previously optimized UV-C postharvest irradiation protocol (510 W, 40 cm, 60 s). The induction kinetics of the stilbenes trans-resveratrol, trans-piceid, trans-piceatannol, trans-astringin, and viniferins was followed by using HPLC-DAD/MS/MS. The most inducible stilbenes were trans-resveratrol, trans-piceatannol, and viniferins. Both quantitative and qualitative differences were observed in both the stilbene induction kinetics and stilbene content in the seven table grapes analyzed here. The total resveratrol content ranged from 0.69 mg/100 g fw (Dominga) to 2.3 mg/100 g fw (Red Globe). The net resveratrol induction ranged from 3.4-fold (Flame) to 2315-fold (Red Globe). The highest viniferins content was observed in the variety Flame (0.73 mg/100 g fw), although the variety Red Globe presented the highest viniferins induction (175-fold). The highest content and induction of piceatannol (0.17 mg/100 g fw and 173-fold, respectively) was observed in the variety Flame. It should be stressed that taking into account the health-beneficial effects claimed for stilbenes, the UV-C irradiated table grapes can be considered as new functional fruits that can supply (a serving of unpeeled 200 g table grapes) the resveratrol content (depending on the variety) equivalent to more than seven glasses of red wine ( approximately 1.5 L) with high resveratrol content.
Enhanced Recyclable Magnetized Palm Shell Waste-Based Powdered Activated Carbon for the Removal of Ibuprofen: Insights for Kinetics and Mechanisms.

PubMed

Wong, Kien Tiek; Yoon, Yeomin; Jang, Min

2015-01-01

A novel preparation method of magnetized palm shell waste-based powdered activated carbon (MPPAC, avg. size 112 μm) was developed. The prepared MPPAC was assessed by several physicochemical analyses, and batch tests were performed for ibuprofen (IBP) removal. Field emission scanning electron microscopy (FESEM) and N2 gas isotherms revealed that magnetite and maghemite were homogeneous and deposited mostly on the surface of PPAC without a significant clogging effect on the micropores. Isotherm results showed that 3.8% Fe (w/w) impregnated PPAC [MPPAC-Fe(3.8%)] had about 2.2-fold higher maximum sorption capacity (157.3 mg g-1) and a 2.5-fold higher sorption density (0.23 mg m-2) than pristine PPAC. Both Fourier-transform infrared spectroscopy (FTIR) and isotherm data indicated that the high sorption capacity and density of IBP by MPPAC was primarily attributable to donor-acceptor complexes with the C = O group and dispersive π-π interactions with the carbon surface. Based on kinetic and repeated adsorption tests, pore diffusion was the rate-limiting step, and MPPAC-Fe(3.8%) had about 1.9~2.8- and 9.1~15.8-fold higher rate constants than MPPAC-Fe(8.6%) and palm shell-waste granular activated carbon (PGAC, avg. size 621 μm), respectively. MPPAC showed almost eight fold greater re-adsorption capacity than PPAC due to a thermal catalytic effect of magnetite/maghemite.
Specificity and Affinity Quantification of Flexible Recognition from Underlying Energy Landscape Topography

PubMed Central

Chu, Xiakun; Wang, Jin

2014-01-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition. PMID:25144525
Iron translocation into and out of Listeria innocua Dps and size distribution of the protein-enclosed nanomineral are modulated by the electrostatic gradient at the 3-fold "ferritin-like" pores.

PubMed

Bellapadrona, Giuliano; Stefanini, Simonetta; Zamparelli, Carlotta; Theil, Elizabeth C; Chiancone, Emilia

2009-07-10

Elucidating pore function at the 3-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management of iron/hydrogen peroxide. The Dps pores are called "ferritin-like" because of the structural resemblance to the 3-fold channels of 24-subunit ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the pores generate a negative electrostatic gradient that guides iron ions toward the ferroxidase centers for catalysis with oxidant and destined for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in Listeria innocua Dps act in a similar fashion, D121N, D126N, D130N, and D121N/D126N/D130N proteins were produced; kinetics of iron uptake/release and the size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal growth in a confined space, indicate that iron uses the hydrophilic 3-fold pores to traverse the protein shell. For the first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.
Specificity and affinity quantification of flexible recognition from underlying energy landscape topography.

PubMed

Chu, Xiakun; Wang, Jin

2014-08-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition.
Pressure-jump small-angle x-ray scattering detected kinetics of staphylococcal nuclease folding.

PubMed Central

Woenckhaus, J; Köhling, R; Thiyagarajan, P; Littrell, K C; Seifert, S; Royer, C A; Winter, R

2001-01-01

The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, approximately 1 h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique. PMID:11222312
Subdoses of 17DD yellow fever vaccine elicit equivalent virological/immunological kinetics timeline.

PubMed

Campi-Azevedo, Ana Carolina; de Almeida Estevam, Paula; Coelho-Dos-Reis, Jordana Grazziela; Peruhype-Magalhães, Vanessa; Villela-Rezende, Gabriela; Quaresma, Patrícia Flávia; Maia, Maria de Lourdes Sousa; Farias, Roberto Henrique Guedes; Camacho, Luiz Antonio Bastos; Freire, Marcos da Silva; Galler, Ricardo; Yamamura, Anna Maya Yoshida; Almeida, Luiz Fernando Carvalho; Lima, Sheila Maria Barbosa; Nogueira, Rita Maria Ribeiro; Silva Sá, Gloria Regina; Hokama, Darcy Akemi; de Carvalho, Ricardo; Freire, Ricardo Aguiar Villanova; Filho, Edson Pereira; Leal, Maria da Luz Fernandes; Homma, Akira; Teixeira-Carvalho, Andréa; Martins, Reinaldo Menezes; Martins-Filho, Olindo Assis

2014-07-15

The live attenuated 17DD Yellow Fever vaccine is one of the most successful prophylactic interventions for controlling disease expansion ever designed and utilized in larger scale. However, increase on worldwide vaccine demands and manufacturing restrictions urge for more detailed dose sparing studies. The establishment of complementary biomarkers in addition to PRNT and Viremia could support a secure decision-making regarding the use of 17DD YF vaccine subdoses. The present work aimed at comparing the serum chemokine and cytokine kinetics triggered by five subdoses of 17DD YF Vaccine. Neutralizing antibody titers, viremia, cytokines and chemokines were tested on blood samples obtained from eligible primary vaccinees. The results demonstrated that a fifty-fold lower dose of 17DD-YF vaccine (587 IU) is able to trigger similar immunogenicity, as evidenced by significant titers of anti-YF PRNT. However, only subdoses as low as 3,013 IU elicit viremia kinetics with an early peak at five days after primary vaccination equivalent to the current dose (27,476 IU), while other subdoses show a distinct, lower in magnitude and later peak at day 6 post-vaccination. Although the subdose of 587 IU is able to trigger equivalent kinetics of IL-8/CXCL-8 and MCP-1/CCL-2, only the subdose of 3,013 IU is able to trigger similar kinetics of MIG/CXCL-9, pro-inflammatory (TNF, IFN-γ and IL-2) and modulatory cytokines (IL-5 and IL-10). The analysis of serum biomarkers IFN-γ and IL-10, in association to PRNT and viremia, support the recommendation of use of a ten-fold lower subdose (3,013 IU) of 17DD-YF vaccine.
Revealing the global map of protein folding space by large-scale simulations

NASA Astrophysics Data System (ADS)

Sinner, Claude; Lutz, Benjamin; Verma, Abhinav; Schug, Alexander

2015-12-01

The full characterization of protein folding is a remarkable long-standing challenge both for experiment and simulation. Working towards a complete understanding of this process, one needs to cover the full diversity of existing folds and identify the general principles driving the process. Here, we want to understand and quantify the diversity in folding routes for a large and representative set of protein topologies covering the full range from all alpha helical topologies towards beta barrels guided by the key question: Does the majority of the observed routes contribute to the folding process or only a particular route? We identified a set of two-state folders among non-homologous proteins with a sequence length of 40-120 residues. For each of these proteins, we ran native-structure based simulations both with homogeneous and heterogeneous contact potentials. For each protein, we simulated dozens of folding transitions in continuous uninterrupted simulations and constructed a large database of kinetic parameters. We investigate folding routes by tracking the formation of tertiary structure interfaces and discuss whether a single specific route exists for a topology or if all routes are equiprobable. These results permit us to characterize the complete folding space for small proteins in terms of folding barrier ΔG‡, number of routes, and the route specificity RT.
Program Helps To Determine Chemical-Reaction Mechanisms

NASA Technical Reports Server (NTRS)

Bittker, D. A.; Radhakrishnan, K.

1995-01-01

General Chemical Kinetics and Sensitivity Analysis (LSENS) computer code developed for use in solving complex, homogeneous, gas-phase, chemical-kinetics problems. Provides for efficient and accurate chemical-kinetics computations and provides for sensitivity analysis for variety of problems, including problems involving honisothermal conditions. Incorporates mathematical models for static system, steady one-dimensional inviscid flow, reaction behind incident shock wave (with boundary-layer correction), and perfectly stirred reactor. Computations of equilibrium properties performed for following assigned states: enthalpy and pressure, temperature and pressure, internal energy and volume, and temperature and volume. Written in FORTRAN 77 with exception of NAMELIST extensions used for input.
Alternative kinetic energy metrics for Lagrangian systems

NASA Astrophysics Data System (ADS)

Sarlet, W.; Prince, G.

2010-11-01

We examine Lagrangian systems on \\ {R}^n with standard kinetic energy terms for the possibility of additional, alternative Lagrangians with kinetic energy metrics different to the Euclidean one. Using the techniques of the inverse problem in the calculus of variations we find necessary and sufficient conditions for the existence of such Lagrangians. We illustrate the problem in two and three dimensions with quadratic and cubic potentials. As an aside we show that the well-known anomalous Lagrangians for the Coulomb problem can be removed by switching on a magnetic field, providing an appealing resolution of the ambiguous quantizations of the hydrogen atom.

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

PubMed Central

An, Na; Fleming, Aaron M.; Middleton, Eric G.; Burrows, Cynthia J.

2014-01-01

Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′ that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structure-dependent electrical signatures through DNA–protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2′-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology. PMID:25225404
Importance of Diffuse Metal Ion Binding to RNA

PubMed Central

Tan, Zhi-Jie; Chen, Shi-Jie

2016-01-01

RNAs are highly charged polyanionic molecules. RNA structure and function are strongly correlated with the ionic condition of the solution. The primary focus of this article is on the role of diffusive ions in RNA folding. Due to the long-range nature of electrostatic interactions, the diffuse ions can contribute significantly to RNA structural stability and folding kinetics. We present an overview of the experimental findings as well as the theoretical developments on the diffuse ion effects in RNA folding. This review places heavy emphasis on the effect of magnesium ions. Magnesium ions play a highly efficient role in stabilizing RNA tertiary structures and promoting tertiary structural folding. The highly efficient role goes beyond the mean-field effect such as the ionic strength. In addition to the effects of specific ion binding and ion dehydration, ion-ion correlation for the diffuse ions can contribute to the efficient role of the multivalent ions such as the magnesium ions in RNA folding. PMID:22010269
Importance of diffuse metal ion binding to RNA.

PubMed

Tan, Zhi-Jie; Chen, Shi-Jie

2011-01-01

RNAs are highly charged polyanionic molecules. RNA structure and function are strongly correlated with the ionic condition of the solution. The primary focus of this article is on the role of diffusive ions in RNA folding. Due to the long-range nature of electrostatic interactions, the diffuse ions can contribute significantly to RNA structural stability and folding kinetics. We present an overview of the experimental findings as well as the theoretical developments on the diffuse ion effects in RNA folding. This review places heavy emphasis on the effect of magnesium ions. Magnesium ions play a highly efficient role in stabilizing RNA tertiary structures and promoting tertiary structural folding. The highly efficient role goes beyond the mean-field effect such as the ionic strength. In addition to the effects of specific ion binding and ion dehydration, ion-ion correlation for the diffuse ions can contribute to the efficient role of the multivalent ions such as the magnesium ions in RNA folding.
Acceleration through passive destabilization: protein folding in a weak hydrophobic environment

NASA Astrophysics Data System (ADS)

Jewett, Andrew; Baumketner, Andrij; Shea, Joan-Emma

2004-03-01

The GroEL chaperonin is a biomolecule which assists the folding of an extremely diverse range of proteins in Eubacteria. Some proteins undergo many rounds of ATP-regulated binding and dissociation from GroEL/ES before folding. It has been proposed that transient stress from ATP-regulated binding and release from GroEL/ES frees frustrated proteins from misfolded conformations. However recent evidence suggests that chaperonin-accelerated protein folding can take place entirely within a mutated GroEL+ES cavity that is unable to open and release the protein. Using molecular dynamics, we demonstrate that static confinement within a weakly hydrophobic (attractive) cavity (similar to the interior of the cavity formed by the GroEL+ES complex) is sufficient to significantly accelerate the folding of a highly frustrated protein-like heteropolymer. Our frustrated molecule benifits kinetically from a static hydrophobic environment that destabilizes misfolded conformations. This may shed light on the mechanisms used by other chaperones which do not depend on ATP.
A de novo designed 11 kDa polypeptide: model for amyloidogenic intrinsically disordered proteins.

PubMed

Topilina, Natalya I; Ermolenkov, Vladimir V; Sikirzhytski, Vitali; Higashiya, Seiichiro; Lednev, Igor K; Welch, John T

2010-07-01

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) has a significant number of identical weakly interacting beta-strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that beta-sheet folding of the 11-kDa amyloidogenic polypeptide is completely aggregation driven.
Intermediates and the folding of proteins L and G

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Scott; Head-Gordon, Teresa

We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G that are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted {beta}-1 and {beta}-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contactsmore » involving the third {beta}-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment.« less
Intermediates and the folding of proteins L and G

PubMed Central

Brown, Scott; Head-Gordon, Teresa

2004-01-01

We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted β-1 and β-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding, and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third β-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally, the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first-order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment. PMID:15044729
Elucidating quantitative stability/flexibility relationships within thioredoxin and its fragments using a distance constraint model.

PubMed

Jacobs, Donald J; Livesay, Dennis R; Hules, Jeremy; Tasayco, Maria Luisa

2006-05-05

Numerous quantitative stability/flexibility relationships, within Escherichia coli thioredoxin (Trx) and its fragments are determined using a minimal distance constraint model (DCM). A one-dimensional free energy landscape as a function of global flexibility reveals Trx to fold in a low-barrier two-state process, with a voluminous transition state. Near the folding transition temperature, the native free energy basin is markedly skewed to allow partial unfolded forms. Under native conditions the skewed shape is lost, and the protein forms a compact structure with some flexibility. Predictions on ten Trx fragments are generally consistent with experimental observations that they are disordered, and that complementary fragments reconstitute. A hierarchical unfolding pathway is uncovered using an exhaustive computational procedure of breaking interfacial cross-linking hydrogen bonds that span over a series of fragment dissociations. The unfolding pathway leads to a stable core structure (residues 22-90), predicted to act as a kinetic trap. Direct connection between degree of rigidity within molecular structure and non-additivity of free energy is demonstrated using a thermodynamic cycle involving fragments and their hierarchical unfolding pathway. Additionally, the model provides insight about molecular cooperativity within Trx in its native state, and about intermediate states populating the folding/unfolding pathways. Native state cooperativity correlation plots highlight several flexibly correlated regions, giving insight into the catalytic mechanism that facilitates access to the active site disulfide bond. Residual native cooperativity correlations are present in the core substructure, suggesting that Trx can function when it is partly unfolded. This natively disordered kinetic trap, interpreted as a molten globule, has a wide temperature range of metastability, and it is identified as the "slow intermediate state" observed in kinetic experiments. These computational results are found to be in overall agreement with a large array of experimental data.
Kinetics during the redox biotransformation of pollutants mediated by immobilized and soluble humic acids.

PubMed

Cervantes, Francisco J; Martínez, Claudia M; Gonzalez-Estrella, Jorge; Márquez, Arturo; Arriaga, Sonia

2013-03-01

The aim of this study was to elucidate the kinetic constraints during the redox biotransformation of the azo dye, Reactive Red 2 (RR2), and carbon tetrachloride (CT) mediated by soluble humic acids (HAs) and immobilized humic acids (HAi), as well as by the quinoid model compounds, anthraquinone-2,6-disulfonate (AQDS) and 1,2-naphthoquinone-4-sulfonate (NQS). The microbial reduction of both HAs and HAi by anaerobic granular sludge (AGS) was the rate-limiting step during decolorization of RR2 since the reduction of RR2 by reduced HAi proceeded at more than three orders of magnitute faster than the electron-transferring rate observed during the microbial reduction of HAi by AGS. Similarly, the reduction of RR2 by reduced AQDS proceeded 1.6- and 1.9-fold faster than the microbial reduction of AQDS by AGS when this redox mediator (RM) was supplied in soluble and immobilized form, respectively. In contrast, the reduction of NQS by AGS occurred 1.6- and 19.2-fold faster than the chemical reduction of RR2 by reduced NQS when this RM was supplied in soluble and immobilized form, respectively. The microbial reduction of HAs and HAi by a humus-reducing consortium proceeded 1,400- and 790-fold faster than the transfer of electrons from reduced HAs and HAi, respectively, to achieve the reductive dechlorination of CT to chloroform. Overall, the present study provides elucidation on the rate-limiting steps involved in the redox biotransformation of priority pollutants mediated by both HAs and HAi and offers technical suggestions to overcome the kinetic restrictions identified in the redox reactions evaluated.
Influence of thermodynamically unfavorable secondary structures on DNA hybridization kinetics

PubMed Central

Hata, Hiroaki; Kitajima, Tetsuro

2018-01-01

Abstract Nucleic acid secondary structure plays an important role in nucleic acid–nucleic acid recognition/hybridization processes, and is also a vital consideration in DNA nanotechnology. Although the influence of stable secondary structures on hybridization kinetics has been characterized, unstable secondary structures, which show positive ΔG° with self-folding, can also form, and their effects have not been systematically investigated. Such thermodynamically unfavorable secondary structures should not be ignored in DNA hybridization kinetics, especially under isothermal conditions. Here, we report that positive ΔG° secondary structures can change the hybridization rate by two-orders of magnitude, despite the fact that their hybridization obeyed second-order reaction kinetics. The temperature dependence of hybridization rates showed non-Arrhenius behavior; thus, their hybridization is considered to be nucleation limited. We derived a model describing how ΔG° positive secondary structures affect hybridization kinetics in stopped-flow experiments with 47 pairs of oligonucleotides. The calculated hybridization rates, which were based on the model, quantitatively agreed with the experimental rate constant. PMID:29220504
QCM-D and ToF-SIMS Investigation to Deconvolute the Relationship between Lipid Adsorption and Orientation on Lipase Activity.

PubMed

Joyce, Paul; Kempson, Ivan; Prestidge, Clive A

2015-09-22

Quartz crystal microbalance with dissipation (QCM-D) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to provide insights into the relationship between lipid adsorption kinetics and molecular behavior in porous silica particles of varying hydrophobicities on lipase activity. Lipase (an interfacial enzyme that cleaves ester bonds to break down lipids to fatty acids and monoglycerides) activity was controlled by loading triglycerides at different surface coverages in hydrophilic and hydrophobic porous silica particles. The rate of lipid adsorption increased 2-fold for the hydrophobic surface compared to the hydrophilic surface. However, for submonolayer lipid coverage, the hydrophilic surface enhanced lipase activity 4-fold, whereas the hydrophobic surface inhibited lipase activity 16-fold, compared to lipid droplets in water. A difference in lipid orientation for low surface coverage, evidenced by ToF-SIMS, indicated that lipid adsorbs to hydrophilic silica in a conformation promoting hydrolysis. Multilayer coverage on hydrophobic and hydrophilic surfaces was indistinguishable with ToF-SIMS analysis. Increased lipid adsorption for both substrates facilitated digestion kinetics comparable to a conventional emulsion. Improved understanding of the interfacial adsorption and orientation of lipid and its digestibility in porous silica has implications in improving the uptake of pharmaceuticals and nutrients from lipid-based delivery systems.
Kinetics of Internal-Loop Formation in Polypeptide Chains: A Simulation Study

PubMed Central

Doucet, Dana; Roitberg, Adrian; Hagen, Stephen J.

2007-01-01

The speed of simple diffusional motions, such as the formation of loops in the polypeptide chain, places one physical limit on the speed of protein folding. Many experimental studies have explored the kinetics of formation of end-to-end loops in polypeptide chains; however, protein folding more often requires the formation of contacts between interior points on the chain. One expects that, for loops of fixed contour length, interior loops will form more slowly than end-to-end loops, owing to the additional excluded volume associated with the “tails”. We estimate the magnitude of this effect by generating ensembles of randomly coiled, freely jointed chains, and then using the theory of Szabo, Schulten, and Schulten to calculate the corresponding contact formation rates for these ensembles. Adding just a few residues, to convert an end-to-end loop to an internal loop, sharply decreases the contact rate. Surprisingly, the relative change in rate increases for a longer loop; sufficiently long tails, however, actually reverse the effect and accelerate loop formation slightly. Our results show that excluded volume effects in real, full-length polypeptides may cause the rates of loop formation during folding to depart significantly from the values derived from recent loop-formation experiments on short peptides. PMID:17208979
Incorporating beta-turns and a turn mimetic out of context in loop 1 of the WW domain affords cooperatively folded beta-sheets.

PubMed

Kaul, R; Angeles, A R; Jäger, M; Powers, E T; Kelly, J W

2001-06-06

To probe the conformational requirements of loop 1 in the Pin1 WW domain, the residues at the i + 2 and i + 3 positions of a beta-turn within this loop were replaced by dPro-Gly and Asn-Gly, which are known to prefer the conformations required at the i + 1 and i + 2 positions of type II' and type I' beta-turns. Conformational specificity or lack thereof was further examined by incorporating into the i + 2 and i + 3 positions a non-alpha-amino acid-based beta-turn mimetic (4-(2'-aminoethyl)-6-dibenzofuran propionic acid residue, 1), which was designed to replace the i + 1 and i + 2 positions of beta-turns. All these Pin WW variants are monomeric and folded as discerned by analytical ultracentrifugation, NMR, and CD. They exhibit cooperative two-state transitions and display thermodynamic stability within 0.5 kcal/mol of the wild-type WW domain, demonstrating that the acquisition of native structure and stability does not require a specific sequence and, by extension, conformation within loop 1. However, it could be that these loop 1 mutations alter the kinetics of antiparallel beta-sheet folding, which will be addressed by subsequent kinetic studies.
Changes in cell-cycle kinetics responsible for limiting somatic growth in mice

PubMed Central

Chang, Maria; Parker, Elizabeth A.; Muller, Tessa J. M.; Haenen, Caroline; Mistry, Maanasi; Finkielstain, Gabriela P.; Murphy-Ryan, Maureen; Barnes, Kevin M.; Sundaram, Rajeshwari; Baron, Jeffrey

2009-01-01

In mammals, the rate of somatic growth is rapid in early postnatal life but then slows with age, approaching zero as the animal approaches adult body size. To investigate the underlying changes in cell-cycle kinetics, [methyl-3H]thymidine and 5’-bromo-2’deoxyuridine were used to double-label proliferating cells in 1-, 2-, and 3-week-old mice for four weeks. Proliferation of renal tubular epithelial cells and hepatocytes decreased with age. The average cell-cycle time did not increase in liver and increased only 1.7 fold in kidney. The fraction of cells in S-phase that will divide again declined approximately 10 fold with age. Concurrently, average cell area increased approximately 2 fold. The findings suggest that somatic growth deceleration primarily results not from an increase in cell-cycle time but from a decrease in growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cells enlarge to a greater volume, perhaps because they are relieved of the size constraint imposed by cell division. In conclusion, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size. PMID:18535488
Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

DTIC Science & Technology

2014-01-01

wild-type spores but ~15-fold higher deltaTrelease values; v ) germination kinetics of wild-type spores given a ? 30 sec 140 MPa HP pulse followed by...15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average Tlag values; and ( v ) the germination of wild-type...committed spores, as it does for nutrient-committed spores (14)? ( v ) Can these HP-com- mitted spores be isolated under conditions that do not allow
Efficient Ligation of the Schistosoma Hammerhead Ribozyme †

PubMed Central

Canny, Marella D.; Jucker, Fiona M.; Pardi, Arthur

2011-01-01

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by a ~2,000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2–3 at physiological Mg2+ ion concentrations (0.1 –1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules, and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme. PMID:17319693
The Proteasomal ATPases Use a Slow but Highly Processive Strategy to Unfold Proteins

PubMed Central

Snoberger, Aaron; Anderson, Raymond T.; Smith, David M.

2017-01-01

All domains of life have ATP-dependent compartmentalized proteases that sequester their peptidase sites on their interior. ATPase complexes will often associate with these compartmentalized proteases in order to unfold and inject substrates into the protease for degradation. Significant effort has been put into understanding how ATP hydrolysis is used to apply force to proteins and cause them to unfold. The unfolding kinetics of the bacterial ATPase, ClpX, have been shown to resemble a fast motor that traps unfolded intermediates as a strategy to unfold proteins. In the present study, we sought to determine if the proteasomal ATPases from eukaryotes and archaea exhibit similar unfolding kinetics. We found that the proteasomal ATPases appear to use a different kinetic strategy for protein unfolding, behaving as a slower but more processive and efficient translocation motor, particularly when encountering a folded domain. We expect that these dissimilarities are due to differences in the ATP binding/exchange cycle, the presence of a trans-arginine finger, or the presence of a threading ring (i.e., the OB domain), which may be used as a rigid platform to pull folded domains against. We speculate that these differences may have evolved due to the differing client pools these machines are expected to encounter. PMID:28421184
Accelerated Simulation of Kinetic Transport Using Variational Principles and Sparsity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caflisch, Russel

This project is centered on the development and application of techniques of sparsity and compressed sensing for variational principles, PDEs and physics problems, in particular for kinetic transport. This included derivation of sparse modes for elliptic and parabolic problems coming from variational principles. The research results of this project are on methods for sparsity in differential equations and their applications and on application of sparsity ideas to kinetic transport of plasmas.
Advances in electron kinetics and theory of gas discharges

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kolobov, Vladimir I.; The University of Alabama in Huntsville, Huntsville, Alabama 35899

2013-10-15

“Electrons, like people, are fertile and infertile: high-energy electrons are fertile and able to reproduce.”—Lev TsendinModern physics of gas discharges increasingly uses physical kinetics for analysis of non-equilibrium plasmas. The description of underlying physics at the kinetic level appears to be important for plasma applications in modern technologies. In this paper, we attempt to grasp the legacy of Professor Lev Tsendin, who advocated the use of the kinetic approach for understanding fundamental problems of gas discharges. We outline the fundamentals of electron kinetics in low-temperature plasmas, describe elements of the modern kinetic theory of gas discharges, and show examples ofmore » the theoretical approach to gas discharge problems used by Lev Tsendin. Important connections between electron kinetics in gas discharges and semiconductors are also discussed. Using several examples, we illustrate how Tsendin's ideas and methods are currently being developed for the implementation of next generation computational tools for adaptive kinetic-fluid simulations of gas discharges used in modern technologies.« less
Deciphering the mechanisms of binding induced folding at nearly atomic resolution: The Φ value analysis applied to IDPs.

PubMed

Gianni, Stefano; Dogan, Jakob; Jemth, Per

2014-01-01

The Φ value analysis is a method to analyze the structure of metastable states in reaction pathways. Such a methodology is based on the quantitative analysis of the effect of point mutations on the kinetics and thermodynamics of the probed reaction. The Φ value analysis is routinely used in protein folding studies and is potentially an extremely powerful tool to analyze the mechanism of binding induced folding of intrinsically disordered proteins. In this review we recapitulate the key equations and experimental advices to perform the Φ value analysis in the perspective of the possible caveats arising in intrinsically disordered systems. Finally, we briefly discuss some few examples already available in the literature.

Chloride concentration affects Kv channel voltage-gating kinetics: Importance of experimental anion concentrations.

PubMed

Bekar, L K; Loewen, M E; Forsyth, G W; Walz, W

2005-09-30

Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539-7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657-670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016-11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207-216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142-150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary conformation as cytoskeletal interaction appears to mask the effect. Furthermore, as cytoskeletal disruption only impacts channel gating kinetics at low physiological intracellular chloride concentrations, these studies highlight the importance of paying close attention to anion concentrations used under experimental conditions.
Frustration Sculpts the Early Stages of Protein Folding.

PubMed

Di Silvio, Eva; Brunori, Maurizio; Gianni, Stefano

2015-09-07

The funneled energy landscape theory implies that protein structures are minimally frustrated. Yet, because of the divergent demands between folding and function, regions of frustrated patterns are present at the active site of proteins. To understand the effects of such local frustration in dictating the energy landscape of proteins, here we compare the folding mechanisms of the two alternative spliced forms of a PDZ domain (PDZ2 and PDZ2as) that share a nearly identical sequence and structure, while displaying different frustration patterns. The analysis, based on the kinetic characterization of a large number of site-directed mutants, reveals that although the late stages for folding are very robust and biased by native topology, the early stages are more malleable and dominated by local frustration. The results are briefly discussed in the context of the energy-landscape theory. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding.

PubMed

Pechmann, Sebastian; Frydman, Judith

2013-02-01

The choice of codons can influence local translation kinetics during protein synthesis. Whether codon preference is linked to cotranslational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale that incorporates the competition between tRNA supply and demand. Applying this scale to ten closely related yeast species, we uncover the evolutionary conservation of codon optimality in eukaryotes. This analysis reveals universal patterns of conserved optimal and nonoptimal codons, often in clusters, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Our analysis suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate cotranslational polypeptide folding, beyond its previously proposed role of adapting to the cost of expression. These findings establish how mRNA sequences are generally under selection to optimize the cotranslational folding of corresponding polypeptides.
In-cell thermodynamics and a new role for protein surfaces.

PubMed

Smith, Austin E; Zhou, Larry Z; Gorensek, Annelise H; Senske, Michael; Pielak, Gary J

2016-02-16

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.
Kinetically trapped metastable intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase.

PubMed

Sugimoto, Hayuki; Nakaura, Miho; Nishimura, Shigenori; Karita, Shuichi; Miyake, Hideo; Tanaka, Akiyoshi

2009-08-01

Refolding of a thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and (1)H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half-lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with beta-cyclodextrin as the native state, suggesting that the intermediate is highly-ordered and native-like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far-UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off-pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.
Nonminimal kinetic coupled gravity: Inflation on the warped DGP brane

NASA Astrophysics Data System (ADS)

Darabi, F.; Parsiya, A.; Atazadeh, K.

2016-03-01

We consider the nonminimally kinetic coupled version of DGP brane model, where the kinetic term of the scalar field is coupled to the metric and Einstein tensor on the brane by a coupling constant ζ. We obtain the corresponding field equations, using the Friedmann-Robertson-Walker metric and the perfect fluid, and study the inflationary scenario to confront the numerical analysis of six typical scalar field potentials with the current observational results. We find that among the suggested potentials and coupling constants, subject to the e-folding N = 60, the potentials V (ϕ) = σϕ, V (ϕ) = σϕ2 and V (ϕ) = σϕ3 provide the best fits with both Planck+WP+highL data and Planck+WP+highL+BICEP2 data.
Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II

NASA Astrophysics Data System (ADS)

Nemtseva, Elena V.; Lashchuk, Olesya O.; Gerasimova, Marina A.; Melnik, Tatiana N.; Nagibina, Galina S.; Melnik, Bogdan S.

2018-01-01

In most cases, intermediate states of multistage folding proteins are not ‘visible’ under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.
Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II.

PubMed

Nemtseva, Elena V; Lashchuk, Olesya O; Gerasimova, Marina A; Melnik, Tatiana N; Nagibina, Galina S; Melnik, Bogdan S

2017-12-21

In most cases, intermediate states of multistage folding proteins are not 'visible' under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.
The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization.

PubMed

Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

2016-11-01

Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified.
Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI).

PubMed

Esperante, Sebastián A; Covaleda, Giovanni; Trejo, Sebastián A; Bronsoms, Sílvia; Aviles, Francesc X; Ventura, Salvador

2017-07-14

Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the oxidative folding and reductive unfolding pathways of NvCI. Much debate has gone on whether protein conformational folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the folding of NvCI is under thermodynamic control and follows a "trial and error" mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both folding routes is similar. The present study illustrates thus a surprising plasticity in the folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications.
Oxidative Folding and N-terminal Cyclization of Onconase+

PubMed Central

Welker, Ervin; Hathaway, Laura; Xu, Guoqiang; Narayan, Mahesh; Pradeep, Lovy; Shin, Hang-Cheol; Scheraga, Harold A.

2008-01-01

Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally. PMID:17439243
Folding free-energy landscape of villin headpiece subdomain from molecular dynamics simulations.

PubMed

Lei, Hongxing; Wu, Chun; Liu, Haiguang; Duan, Yong

2007-03-20

High-accuracy ab initio folding has remained an elusive objective despite decades of effort. To explore the folding landscape of villin headpiece subdomain HP35, we conducted two sets of replica exchange molecular dynamics for 200 ns each and three sets of conventional microsecond-long molecular dynamics simulations, using AMBER FF03 force field and a generalized-Born solvation model. The protein folded consistently to the native state; the lowest C(alpha)-rmsd from the x-ray structure was 0.46 A, and the C(alpha)- rmsd of the center of the most populated cluster was 1.78 A at 300 K. ab initio simulations have previously not reached this level. The folding landscape of HP35 can be partitioned into the native, denatured, and two intermediate-state regions. The native state is separated from the major folding intermediate state by a small barrier, whereas a large barrier exists between the major folding intermediate and the denatured states. The melting temperature T(m) = 339 K extracted from the heat-capacity profile was in close agreement with the experimentally derived T(m) = 342 K. A comprehensive picture of the kinetics and thermodynamics of HP35 folding emerges when the results from replica exchange and conventional molecular dynamics simulations are combined.
High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.

PubMed

Zhang, Yi; Kitazawa, Soichiro; Peran, Ivan; Stenzoski, Natalie; McCallum, Scott A; Raleigh, Daniel P; Royer, Catherine A

2016-11-23

Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.
Characterization of protein folding by a Φ-value calculation with a statistical-mechanical model.

PubMed

Wako, Hiroshi; Abe, Haruo

2016-01-01

The Φ-value analysis approach provides information about transition-state structures along the folding pathway of a protein by measuring the effects of an amino acid mutation on folding kinetics. Here we compared the theoretically calculated Φ values of 27 proteins with their experimentally observed Φ values; the theoretical values were calculated using a simple statistical-mechanical model of protein folding. The theoretically calculated Φ values reflected the corresponding experimentally observed Φ values with reasonable accuracy for many of the proteins, but not for all. The correlation between the theoretically calculated and experimentally observed Φ values strongly depends on whether the protein-folding mechanism assumed in the model holds true in real proteins. In other words, the correlation coefficient can be expected to illuminate the folding mechanisms of proteins, providing the answer to the question of which model more accurately describes protein folding: the framework model or the nucleation-condensation model. In addition, we tried to characterize protein folding with respect to various properties of each protein apart from the size and fold class, such as the free-energy profile, contact-order profile, and sensitivity to the parameters used in the Φ-value calculation. The results showed that any one of these properties alone was not enough to explain protein folding, although each one played a significant role in it. We have confirmed the importance of characterizing protein folding from various perspectives. Our findings have also highlighted that protein folding is highly variable and unique across different proteins, and this should be considered while pursuing a unified theory of protein folding.
Characterization of protein folding by a Φ-value calculation with a statistical-mechanical model

PubMed Central

Wako, Hiroshi; Abe, Haruo

2016-01-01

The Φ-value analysis approach provides information about transition-state structures along the folding pathway of a protein by measuring the effects of an amino acid mutation on folding kinetics. Here we compared the theoretically calculated Φ values of 27 proteins with their experimentally observed Φ values; the theoretical values were calculated using a simple statistical-mechanical model of protein folding. The theoretically calculated Φ values reflected the corresponding experimentally observed Φ values with reasonable accuracy for many of the proteins, but not for all. The correlation between the theoretically calculated and experimentally observed Φ values strongly depends on whether the protein-folding mechanism assumed in the model holds true in real proteins. In other words, the correlation coefficient can be expected to illuminate the folding mechanisms of proteins, providing the answer to the question of which model more accurately describes protein folding: the framework model or the nucleation-condensation model. In addition, we tried to characterize protein folding with respect to various properties of each protein apart from the size and fold class, such as the free-energy profile, contact-order profile, and sensitivity to the parameters used in the Φ-value calculation. The results showed that any one of these properties alone was not enough to explain protein folding, although each one played a significant role in it. We have confirmed the importance of characterizing protein folding from various perspectives. Our findings have also highlighted that protein folding is highly variable and unique across different proteins, and this should be considered while pursuing a unified theory of protein folding. PMID:28409079
Isolation of a latent polyphenol oxidase from loquat fruit (Eriobotrya japonica Lindl.): kinetic characterization and comparison with the active form.

PubMed

Sellés-Marchart, Susana; Casado-Vela, Juan; Bru-Martínez, Roque

2006-02-15

Polyphenol oxidase (PPO) has been extracted from both soluble and particulate fractions of loquat fruit (Eriobotrya japonica Lindl. cv. Algerie). The soluble PPO (20% of total activity) was partially purified 3.3-fold after ammonium sulfate fractionation being in its active state. The particulate PPO fraction (80% of total activity) was purified to homogeneity in a latent form being activable by sodium dodecyl sulfate (SDS). The enzyme was purified 40.0-fold with a total yield of 15.3% after extraction by phase partitioning in Triton X-114 followed by three chromatographic steps. The molecular weight was estimated to be about 59.2 and 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, respectively, indicating that latent PPO is a monomer. Latent PPO catalyzed the oxidation of chlorogenic acid (CA) at a rate 50-fold faster than that of 4-tert-butylcatechol (TBC) but the soluble active counterpart only twice. Both PPOs exhibited similar Km values for TBC but Km for CA was 5-fold higher for the latent than for the active soluble PPO. Other kinetic characteristics, including sensitivity to inhibitors, substrate specificity, thermal stability, temperature, and pH profiles, were quite different between both PPOs. These results provide strong evidences that the soluble active and the particulate latent are different forms of PPO in loquat fruit flesh. The results suggest that the major PPO form for the oxidation of CA, leading to enzymatic browning under physiological conditions, is the latent one.
Modulation of frustration in folding by sequence permutation.

PubMed

Nobrega, R Paul; Arora, Karunesh; Kathuria, Sagar V; Graceffa, Rita; Barrea, Raul A; Guo, Liang; Chakravarthy, Srinivas; Bilsel, Osman; Irving, Thomas C; Brooks, Charles L; Matthews, C Robert

2014-07-22

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.
New Insights into the Folding of a β-Sheet Miniprotein in a Reduced Space of Collective Hydrogen Bond Variables: Application to a Hydrodynamic Analysis of the Folding Flow

PubMed Central

Kalgin, Igor V.; Caflisch, Amedeo; Chekmarev, Sergei F.; Karplus, Martin

2013-01-01

A new analysis of the 20 μs equilibrium folding/unfolding molecular dynamics simulations of the three-stranded antiparallel β-sheet miniprotein (beta3s) in implicit solvent is presented. The conformation space is reduced in dimensionality by introduction of linear combinations of hydrogen bond distances as the collective variables making use of a specially adapted Principal Component Analysis (PCA); i.e., to make structured conformations more pronounced, only the formed bonds are included in determining the principal components. It is shown that a three-dimensional (3D) subspace gives a meaningful representation of the folding behavior. The first component, to which eight native hydrogen bonds make the major contribution (four in each beta hairpin), is found to play the role of the reaction coordinate for the overall folding process, while the second and third components distinguish the structured conformations. The representative points of the trajectory in the 3D space are grouped into conformational clusters that correspond to locally stable conformations of beta3s identified in earlier work. A simplified kinetic network based on the three components is constructed and it is complemented by a hydrodynamic analysis. The latter, making use of “passive tracers” in 3D space, indicates that the folding flow is much more complex than suggested by the kinetic network. A 2D representation of streamlines shows there are vortices which correspond to repeated local rearrangement, not only around minima of the free energy surface, but also in flat regions between minima. The vortices revealed by the hydrodynamic analysis are apparently not evident in folding pathways generated by transition-path sampling. Making use of the fact that the values of the collective hydrogen bond variables are linearly related to the Cartesian coordinate space, the RMSD between clusters is determined. Interestingly, the transition rates show an approximate exponential correlation with distance in the hydrogen bond subspace. Comparison with the many published studies shows good agreement with the present analysis for the parts that can be compared, supporting the robust character of our understanding of this “hydrogen atom” of protein folding. PMID:23621790
The effect of medium viscosity on kinetics of ATP hydrolysis by the chloroplast coupling factor CF1.

PubMed

Malyan, Alexander N

2016-05-01

The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca(2+)- and Mg(2+)-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca(2+)-dependent ATPase without changing the apparent Michaelis constant (К m), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg(2+)-dependent reaction, the presence of sucrose increased К m without changing the maximal rate that is many-fold as high as that of Ca(2+)-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg(2+)-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water.
Conceptual Transformation and Cognitive Processes in Origami Paper Folding

ERIC Educational Resources Information Center

Tenbrink, Thora; Taylor, Holly A.

2015-01-01

Research on problem solving typically does not address tasks that involve following detailed and/or illustrated step-by-step instructions. Such tasks are not seen as cognitively challenging problems to be solved. In this paper, we challenge this assumption by analyzing verbal protocols collected during an Origami folding task. Participants…

The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations.

PubMed

Wang, Moye; Hu, Jie; Zhang, Zhuqing

2016-04-26

As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5-10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.
The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations

PubMed Central

Wang, Moye; Hu, Jie; Zhang, Zhuqing

2016-01-01

As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5–10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design. PMID:27128902
A resource facility for kinetic analysis: modeling using the SAAM computer programs.

PubMed

Foster, D M; Boston, R C; Jacquez, J A; Zech, L

1989-01-01

Kinetic analysis and integrated system modeling have contributed significantly to understanding the physiology and pathophysiology of metabolic systems in humans and animals. Many experimental biologists are aware of the usefulness of these techniques and recognize that kinetic modeling requires special expertise. The Resource Facility for Kinetic Analysis (RFKA) provides this expertise through: (1) development and application of modeling technology for biomedical problems, and (2) development of computer-based kinetic modeling methodologies concentrating on the computer program Simulation, Analysis, and Modeling (SAAM) and its conversational version, CONversational SAAM (CONSAM). The RFKA offers consultation to the biomedical community in the use of modeling to analyze kinetic data and trains individuals in using this technology for biomedical research. Early versions of SAAM were widely applied in solving dosimetry problems; many users, however, are not familiar with recent improvements to the software. The purpose of this paper is to acquaint biomedical researchers in the dosimetry field with RFKA, which, together with the joint National Cancer Institute-National Heart, Lung and Blood Institute project, is overseeing SAAM development and applications. In addition, RFKA provides many service activities to the SAAM user community that are relevant to solving dosimetry problems.
Free energy profile of RNA hairpins: a molecular dynamics simulation study.

PubMed

Deng, Nan-Jie; Cieplak, Piotr

2010-02-17

RNA hairpin loops are one of the most abundant secondary structure elements and participate in RNA folding and protein-RNA recognition. To characterize the free energy surface of RNA hairpin folding at an atomic level, we calculated the potential of mean force (PMF) as a function of the end-to-end distance, by using umbrella sampling simulations in explicit solvent. Two RNA hairpins containing tetraloop cUUCGg and cUUUUg are studied with AMBER ff99 and CHARMM27 force fields. Experimentally, the UUCG hairpin is known to be significantly more stable than UUUU. In this study, the calculations using AMBER force field give a qualitatively correct description for the folding of two RNA hairpins, as the calculated PMF confirms the global stability of the folded structures and the resulting relative folding free energy is in quantitative agreement with the experimental result. The hairpin stabilities are also correctly differentiated by the more rapid molecular mechanics-Poisson Boltzmann-surface area approach, but the relative free energy estimated from this method is overestimated. The free energy profile shows that the native state basin and the unfolded state plateau are separated by a wide shoulder region, which samples a variety of native-like structures with frayed terminal basepair. The calculated PMF lacks major barriers that are expected near the transition regions, and this is attributed to the limitation of the 1-D reaction coordinate. The PMF results are compared with other studies of small RNA hairpins using kinetics method and coarse grained models. The two RNA hairpins described by CHARMM27 are significantly more deformable than those represented by AMBER. Compared with the AMBER results, the CHARMM27 calculated DeltaG(fold) for the UUUU tetraloop is in better agreement with the experimental results. However, the CHARMM27 calculation does not confirm the global stability of the experimental UUCG structure; instead, the extended conformations are predicted to be thermodynamically stable in solution. This finding is further supported by separate unrestrained CHARMM27 simulations, in which the UUCG hairpin unfolds spontaneously within 10 ns. The instability of the UUCG hairpin originates from the loop region, and propagates to the stem. The results of this study provide a molecular picture of RNA hairpin unfolding and reveal problems in the force field descriptions for the conformational energy of certain RNA hairpin. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Characterization of Folding Mechanisms of Trp-cage and WW-domain by Network Analysis of Simulations with a Hybrid-resolution Model

PubMed Central

Han, Wei; Schulten, Klaus

2013-01-01

In this study, we apply a hybrid-resolution model, namely PACE, to characterize the free energy surfaces (FESs) of trp-cage and a WW domain variant along with the respective folding mechanisms. Unbiased, independent simulations with PACE are found to achieve together multiple folding and unfolding events for both proteins, allowing us to perform network analysis of the FESs to identify folding pathways. PACE reproduces for both proteins expected complexity hidden in the folding FESs, in particular, meta-stable non-native intermediates. Pathway analysis shows that some of these intermediates are, actually, on-pathway folding intermediates and that intermediates kinetically closest to the native states can be either critical on-pathway or off-pathway intermediates, depending on the protein. Apart from general insights into folding, specific folding mechanisms of the proteins are resolved. We find that trp-cage folds via a dominant pathway in which hydrophobic collapse occurs before the N-terminal helix forms; full incorporation of Trp6 into the hydrophobic core takes place as the last step of folding, which, however, may not be the rate-limiting step. For the WW domain variant studied we observe two main folding pathways with opposite orders of formation of the two hairpins involved in the structure; for either pathway, formation of hairpin 1 is more likely to be the rate-limiting step. Altogether, our results suggest that PACE combined with network analysis is a computationally efficient and valuable tool for the study of protein folding. PMID:23915394
Towards fast, rigorous and efficient conformational sampling of biomolecules: Advances in accelerated molecular dynamics.

PubMed

Doshi, Urmi; Hamelberg, Donald

2015-05-01

Accelerated molecular dynamics (aMD) has been proven to be a powerful biasing method for enhanced sampling of biomolecular conformations on general-purpose computational platforms. Biologically important long timescale events that are beyond the reach of standard molecular dynamics can be accessed without losing the detailed atomistic description of the system in aMD. Over other biasing methods, aMD offers the advantages of tuning the level of acceleration to access the desired timescale without any advance knowledge of the reaction coordinate. Recent advances in the implementation of aMD and its applications to small peptides and biological macromolecules are reviewed here along with a brief account of all the aMD variants introduced in the last decade. In comparison to the original implementation of aMD, the recent variant in which all the rotatable dihedral angles are accelerated (RaMD) exhibits faster convergence rates and significant improvement in statistical accuracy of retrieved thermodynamic properties. RaMD in conjunction with accelerating diffusive degrees of freedom, i.e. dual boosting, has been rigorously tested for the most difficult conformational sampling problem, protein folding. It has been shown that RaMD with dual boosting is capable of efficiently sampling multiple folding and unfolding events in small fast folding proteins. RaMD with the dual boost approach opens exciting possibilities for sampling multiple timescales in biomolecules. While equilibrium properties can be recovered satisfactorily from aMD-based methods, directly obtaining dynamics and kinetic rates for larger systems presents a future challenge. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014 Elsevier B.V. All rights reserved.
FROM FOLDING THEORIES TO FOLDING PROTEINS: A Review and Assessment of Simulation Studies of Protein Folding and Unfolding

NASA Astrophysics Data System (ADS)

Shea, Joan-Emma; Brooks, Charles L., III

2001-10-01

Beginning with simplified lattice and continuum "minimalist" models and progressing to detailed atomic models, simulation studies have augmented and directed development of the modern landscape perspective of protein folding. In this review we discuss aspects of detailed atomic simulation methods applied to studies of protein folding free energy surfaces, using biased-sampling free energy methods and temperature-induced protein unfolding. We review studies from each on systems of particular experimental interest and assess the strengths and weaknesses of each approach in the context of "exact" results for both free energies and kinetics of a minimalist model for a beta-barrel protein. We illustrate in detail how each approach is implemented and discuss analysis methods that have been developed as components of these studies. We describe key insights into the relationship between protein topology and the folding mechanism emerging from folding free energy surface calculations. We further describe the determination of detailed "pathways" and models of folding transition states that have resulted from unfolding studies. Our assessment of the two methods suggests that both can provide, often complementary, details of folding mechanism and thermodynamics, but this success relies on (a) adequate sampling of diverse conformational regions for the biased-sampling free energy approach and (b) many trajectories at multiple temperatures for unfolding studies. Furthermore, we find that temperature-induced unfolding provides representatives of folding trajectories only when the topology and sequence (energy) provide a relatively funneled landscape and "off-pathway" intermediates do not exist.
Folding of multidomain proteins: biophysical consequences of tethering even in apparently independent folding.

PubMed

Arviv, Oshrit; Levy, Yaakov

2012-12-01

Most eukaryotic and a substantial fraction of prokaryotic proteins are composed of more than one domain. The tethering of these evolutionary, structural, and functional units raises, among others, questions regarding the folding process of conjugated domains. Studying the folding of multidomain proteins in silico enables one to identify and isolate the tethering-induced biophysical determinants that govern crosstalks generated between neighboring domains. For this purpose, we carried out coarse-grained and atomistic molecular dynamics simulations of two two-domain constructs from the immunoglobulin-like β-sandwich fold. Each of these was experimentally shown to behave as the "sum of its parts," that is, the thermodynamic and kinetic folding behavior of the constituent domains of these constructs seems to occur independently, with the folding of each domain uncoupled from the folding of its partner in the two-domain construct. We show that the properties of the individual domains can be significantly affected by conjugation to another domain. The tethering may be accompanied by stabilizing as well as destabilizing factors whose magnitude depends on the size of the interface, the length, and the flexibility of the linker, and the relative stability of the domains. Accordingly, the folding of a multidomain protein should not be viewed as the sum of the folding patterns of each of its parts, but rather, it involves abrogating several effects that lead to this outcome. An imbalance between these effects may result in either stabilization or destabilization owing to the tethering. Copyright © 2012 Wiley Periodicals, Inc.
New techniques for positron emission tomography in the study of human neurological disorders: Progress report, 15 June 1992--31 October 1992

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1992-01-01

During the past six months, we have continued work on the fronts of kinetic modeling of radioligands for studying neurotransmitter/receptor systems, iterative reconstruction techniques, and methodology for PET cerebral blood flow activation studies. Initial human PET studies have been performed and analyzed with many different kinetic model formulations to determine the quantitative potential of the neuronwsmitter/receptor ligand, ({sup 11}C)N-methyl piperidyl benzilate (NMPB), a muscarinic cholinergic antagonist. In addition, initial human studies using ({sup 11}C)tetrabenazine (TBZ), a marker for monoantine nerve terminal density. Results of the NWB studies have indicated that this new agent yields better estimates of receptor density thanmore » previous muscarinic ligands developed at our facility, ({sup 11}C)-TRB and ({sup 11}C)scopolamine. TRB and scopolamine have previously been shown to be only partially successful ligands due to sub-optimal values of the individual rate constants, causing varying degrees of flow limitation. This is found to be much less of a problem for NMPB due to the 2.0--2.5 fold increase in ligand transport observed in the human studies ({approximately}60% first pass extraction). A 2-parameter 2-compartment simplification had previously been implemented for the benzodiazepine ligand, (C-11)FMZ, and a similar model appears to be suitable for TBZ based on the preliminary human data.« less
New techniques for positron emission tomography in the study of human neurological disorders: Progress report, 15 June 1992--31 October 1992

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1992-10-01

During the past six months, we have continued work on the fronts of kinetic modeling of radioligands for studying neurotransmitter/receptor systems, iterative reconstruction techniques, and methodology for PET cerebral blood flow activation studies. Initial human PET studies have been performed and analyzed with many different kinetic model formulations to determine the quantitative potential of the neuronwsmitter/receptor ligand, [{sup 11}C]N-methyl piperidyl benzilate (NMPB), a muscarinic cholinergic antagonist. In addition, initial human studies using [{sup 11}C]tetrabenazine (TBZ), a marker for monoantine nerve terminal density. Results of the NWB studies have indicated that this new agent yields better estimates of receptor density thanmore » previous muscarinic ligands developed at our facility, [{sup 11}C]-TRB and [{sup 11}C]scopolamine. TRB and scopolamine have previously been shown to be only partially successful ligands due to sub-optimal values of the individual rate constants, causing varying degrees of flow limitation. This is found to be much less of a problem for NMPB due to the 2.0--2.5 fold increase in ligand transport observed in the human studies ({approximately}60% first pass extraction). A 2-parameter 2-compartment simplification had previously been implemented for the benzodiazepine ligand, [C-11]FMZ, and a similar model appears to be suitable for TBZ based on the preliminary human data.« less
Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

PubMed

Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K; Nidetzky, Bernd

2016-06-15

L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release.
Effects of electrostatic interactions on ligand dissociation kinetics

NASA Astrophysics Data System (ADS)

Erbaş, Aykut; de la Cruz, Monica Olvera; Marko, John F.

2018-02-01

We study unbinding of multivalent cationic ligands from oppositely charged polymeric binding sites sparsely grafted on a flat neutral substrate. Our molecular dynamics simulations are suggested by single-molecule studies of protein-DNA interactions. We consider univalent salt concentrations spanning roughly a 1000-fold range, together with various concentrations of excess ligands in solution. To reveal the ionic effects on unbinding kinetics of spontaneous and facilitated dissociation mechanisms, we treat electrostatic interactions both at a Debye-Hückel (DH) (or implicit ions, i.e., use of an electrostatic potential with a prescribed decay length) level and by the more precise approach of considering all ionic species explicitly in the simulations. We find that the DH approach systematically overestimates unbinding rates, relative to the calculations where all ion pairs are present explicitly in solution, although many aspects of the two types of calculation are qualitatively similar. For facilitated dissociation (FD) (acceleration of unbinding by free ligands in solution) explicit-ion simulations lead to unbinding at lower free-ligand concentrations. Our simulations predict a variety of FD regimes as a function of free-ligand and ion concentrations; a particularly interesting regime is at intermediate concentrations of ligands where nonelectrostatic binding strength controls FD. We conclude that explicit-ion electrostatic modeling is an essential component to quantitatively tackle problems in molecular ligand dissociation, including nucleic-acid-binding proteins.
Facilitated release of substrate protein from prefoldin by chaperonin.

PubMed

Zako, Tamotsu; Iizuka, Ryo; Okochi, Mina; Nomura, Tomoko; Ueno, Taro; Tadakuma, Hisashi; Yohda, Masafumi; Funatsu, Takashi

2005-07-04

Prefoldin is a chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. However, kinetics of interactions between prefoldin and substrate proteins have not been investigated. In this study, dissociation constants and dissociation rate constants of unfolded proteins with prefoldin were firstly measured using fluorescence microscopy. Our results suggest that binding and release of prefoldin from hyperthermophilic archaea with substrate proteins were in a dynamic equilibrium. Interestingly, the release of substrate proteins from prefoldin was facilitated when chaperonin was present, supporting a handoff mechanism of substrate proteins from prefoldin to the chaperonin.
STRONG ORACLE OPTIMALITY OF FOLDED CONCAVE PENALIZED ESTIMATION.

PubMed

Fan, Jianqing; Xue, Lingzhou; Zou, Hui

2014-06-01

Folded concave penalization methods have been shown to enjoy the strong oracle property for high-dimensional sparse estimation. However, a folded concave penalization problem usually has multiple local solutions and the oracle property is established only for one of the unknown local solutions. A challenging fundamental issue still remains that it is not clear whether the local optimum computed by a given optimization algorithm possesses those nice theoretical properties. To close this important theoretical gap in over a decade, we provide a unified theory to show explicitly how to obtain the oracle solution via the local linear approximation algorithm. For a folded concave penalized estimation problem, we show that as long as the problem is localizable and the oracle estimator is well behaved, we can obtain the oracle estimator by using the one-step local linear approximation. In addition, once the oracle estimator is obtained, the local linear approximation algorithm converges, namely it produces the same estimator in the next iteration. The general theory is demonstrated by using four classical sparse estimation problems, i.e., sparse linear regression, sparse logistic regression, sparse precision matrix estimation and sparse quantile regression.
STRONG ORACLE OPTIMALITY OF FOLDED CONCAVE PENALIZED ESTIMATION

PubMed Central

Fan, Jianqing; Xue, Lingzhou; Zou, Hui

2014-01-01

Folded concave penalization methods have been shown to enjoy the strong oracle property for high-dimensional sparse estimation. However, a folded concave penalization problem usually has multiple local solutions and the oracle property is established only for one of the unknown local solutions. A challenging fundamental issue still remains that it is not clear whether the local optimum computed by a given optimization algorithm possesses those nice theoretical properties. To close this important theoretical gap in over a decade, we provide a unified theory to show explicitly how to obtain the oracle solution via the local linear approximation algorithm. For a folded concave penalized estimation problem, we show that as long as the problem is localizable and the oracle estimator is well behaved, we can obtain the oracle estimator by using the one-step local linear approximation. In addition, once the oracle estimator is obtained, the local linear approximation algorithm converges, namely it produces the same estimator in the next iteration. The general theory is demonstrated by using four classical sparse estimation problems, i.e., sparse linear regression, sparse logistic regression, sparse precision matrix estimation and sparse quantile regression. PMID:25598560
Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule

PubMed Central

Rosen, Laura E.; Connell, Katelyn B.; Marqusee, Susan

2014-01-01

The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates. PMID:25258414
Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule.

PubMed

Rosen, Laura E; Connell, Katelyn B; Marqusee, Susan

2014-10-14

The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.
Deceleration of arginine kinase refolding by induced helical structures.

PubMed

Li, Hai-Long; Zhou, Sheng-Mei; Park, Daeui; Jeong, Hyoung Oh; Chung, Hae Young; Yang, Jun-Mo; Meng, Fan-Guo; Hu, Wei-Jiang

2012-04-01

Arginine kinase (AK) is a key metabolic enzyme for keeping energy balance in invertebrates. Therefore, regulation of the enzymatic activity and the folding studies of AK from the various invertebrates have been the focus of investigation. We studied the effects of helical structures by using hexafluoroisopropanol (HFIP) on AK folding. Folding kinetic studies showed that the folding rates of the urea-denatured AKs were significantly decelerated after being induced in various concentrations of HFIP. AK lost its activity completely at concentrations greater than 60%. The results indicated that the HFIP-induced helical structures in the denatured state play a negative role in protein folding, and the helical structures induced in 5% (v/v) HFIP act as the most effective barrier against AK taking its native structure. The computational docking simulations (binding energies for -2.19 kcal/mol for AutoDock4.2 and -20.47 kcal/mol for Dock6.3) suggested that HFIP interacts with the several important residues that are predicted by both programs. The excessively pre-organized helical structures not only hampered the folding process, but also ultimately brought about changes in the three-dimensional conformation and biological function of AK.
Helix formation via conformation diffusion search

PubMed Central

Huang, Cheng-Yen; Getahun, Zelleka; Zhu, Yongjin; Klemke, Jason W.; DeGrado, William F.; Gai, Feng

2002-01-01

The helix-coil transition kinetics of an α-helical peptide were investigated by time-resolved infrared spectroscopy coupled with laser-induced temperature-jump initiation method. Specific isotope labeling of the amide carbonyl groups with 13C at selected residues was used to obtain site-specific information. The relaxation kinetics following a temperature jump, obtained by probing the amide I′ band of the peptide backbone, exhibit nonexponential behavior and are sensitive to both initial and final temperatures. These data are consistent with a conformation diffusion process on the folding energy landscape, in accord with a recent molecular dynamics simulation study. PMID:11867741
Landscapes with megabasins: Polyamorphism in liquids and biopolymers and the role of nucleation in folding and folding diseases

NASA Astrophysics Data System (ADS)

Angell, C. A.

1997-02-01

We show how energy landscape concepts can rationalize the observations on glassforming liquids over the whole range of behavior, strong to fragile. In particular, we show how the existence of landscapes with both strong and fragile megabasins can provide a basis for understanding the nature of quasi-first-order transitions between amorphous states such as those observed to occur in the glassy states of “strong” glassformers. We show how this propensity originates in the liquid state and then emphasize the analogy provided, at the mesoscopic level, by the folding transition in proteins. Recognition that the folding transition is an equilibrium first-order transition between polyamorphic forms of a complex system implies recognition of the need for a nucleation step in the process. When nucleated phase transitions are kinetically retarded, their probability can be influenced by time-temperature history and by the presence of nucleating agents. Nucleation events are statistically rare in mesoscopic systems, hence the ability to fold rapidly should require special features in the folding molecular structure or the presence of nucleating agents. We propose that the unwanted folding events leading to pathogenic forms of certain proteins (prions) can be stimulated by nucleating agents, which thus may be the unidentified infectious agents in “mad cow” disease and related maladies.

Universality and diversity of folding mechanics for three-helix bundle proteins.

PubMed

Yang, Jae Shick; Wallin, Stefan; Shakhnovich, Eugene I

2008-01-22

In this study we evaluate, at full atomic detail, the folding processes of two small helical proteins, the B domain of protein A and the Villin headpiece. Folding kinetics are studied by performing a large number of ab initio Monte Carlo folding simulations using a single transferable all-atom potential. Using these trajectories, we examine the relaxation behavior, secondary structure formation, and transition-state ensembles (TSEs) of the two proteins and compare our results with experimental data and previous computational studies. To obtain a detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Moreover, rigorous p(fold) analysis is used to obtain representative samples of the TSEs and a good quantitative agreement between experimental and simulated Phi values is obtained for protein A. Phi values for Villin also are obtained and left as predictions to be tested by future experiments. Our analysis shows that the two-helix hairpin is a common partially stable structural motif that gets formed before entering the TSE in the studied proteins. These results together with our earlier study of Engrailed Homeodomain and recent experimental studies provide a comprehensive, atomic-level picture of folding mechanics of three-helix bundle proteins.
Deciphering the Hidden Informational Content of Protein Sequences

PubMed Central

Liu, Ming; Hua, Qing-xin; Hu, Shi-Quan; Jia, Wenhua; Yang, Yanwu; Saith, Sunil Evan; Whittaker, Jonathan; Arvan, Peter; Weiss, Michael A.

2010-01-01

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (PheB1 in placental mammals) is variably positioned within crystal structures and exhibits 1H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1–B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus. PMID:20663888
Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

PubMed Central

Warepam, Marina; Sharma, Gurumayum Suraj; Dar, Tanveer Ali; Khan, Md. Khurshid Alam; Singh, Laishram Rajendrakumar

2014-01-01

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K m and k cat), thermodynamic stability (T m and ΔH m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes. PMID:25313668
Four residues of propeptide are essential for precursor folding of nattokinase.

PubMed

Jia, Yan; Cao, Xinhua; Deng, Yu; Bao, Wei; Tang, Changyan; Ding, Hanjing; Zheng, Zhongliang; Zou, Guolin

2014-11-01

Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies confirmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage (i) and (ii) of precursor folding, Tyr10 and Gly13 would form the part of interface with subtilisin domain. While Gly34 and Gly35 connected with an α-helix that would stabilize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Modeling the mechanism of CLN025 beta-hairpin formation

NASA Astrophysics Data System (ADS)

McKiernan, Keri A.; Husic, Brooke E.; Pande, Vijay S.

2017-09-01

Beta-hairpins are substructures found in proteins that can lend insight into more complex systems. Furthermore, the folding of beta-hairpins is a valuable test case for benchmarking experimental and theoretical methods. Here, we simulate the folding of CLN025, a miniprotein with a beta-hairpin structure, at its experimental melting temperature using a range of state-of-the-art protein force fields. We construct Markov state models in order to examine the thermodynamics, kinetics, mechanism, and rate-determining step of folding. Mechanistically, we find the folding process is rate-limited by the formation of the turn region hydrogen bonds, which occurs following the downhill hydrophobic collapse of the extended denatured protein. These results are presented in the context of established and contradictory theories of the beta-hairpin folding process. Furthermore, our analysis suggests that the AMBER-FB15 force field, at this temperature, best describes the characteristics of the full experimental CLN025 conformational ensemble, while the AMBER ff99SB-ILDN and CHARMM22* force fields display a tendency to overstabilize the native state.
New theoretical framework for designing nonionic surfactant mixtures that exhibit a desired adsorption kinetics behavior.

PubMed

Moorkanikkara, Srinivas Nageswaran; Blankschtein, Daniel

2010-12-21

How does one design a surfactant mixture using a set of available surfactants such that it exhibits a desired adsorption kinetics behavior? The traditional approach used to address this design problem involves conducting trial-and-error experiments with specific surfactant mixtures. This approach is typically time-consuming and resource-intensive and becomes increasingly challenging when the number of surfactants that can be mixed increases. In this article, we propose a new theoretical framework to identify a surfactant mixture that most closely meets a desired adsorption kinetics behavior. Specifically, the new theoretical framework involves (a) formulating the surfactant mixture design problem as an optimization problem using an adsorption kinetics model and (b) solving the optimization problem using a commercial optimization package. The proposed framework aims to identify the surfactant mixture that most closely satisfies the desired adsorption kinetics behavior subject to the predictive capabilities of the chosen adsorption kinetics model. Experiments can then be conducted at the identified surfactant mixture condition to validate the predictions. We demonstrate the reliability and effectiveness of the proposed theoretical framework through a realistic case study by identifying a nonionic surfactant mixture consisting of up to four alkyl poly(ethylene oxide) surfactants (C(10)E(4), C(12)E(5), C(12)E(6), and C(10)E(8)) such that it most closely exhibits a desired dynamic surface tension (DST) profile. Specifically, we use the Mulqueen-Stebe-Blankschtein (MSB) adsorption kinetics model (Mulqueen, M.; Stebe, K. J.; Blankschtein, D. Langmuir 2001, 17, 5196-5207) to formulate the optimization problem as well as the SNOPT commercial optimization solver to identify a surfactant mixture consisting of these four surfactants that most closely exhibits the desired DST profile. Finally, we compare the experimental DST profile measured at the surfactant mixture condition identified by the new theoretical framework with the desired DST profile and find good agreement between the two profiles.
Kinetic study of hydroxytyrosol oxidation and its related compounds by Red Globe grape polyphenol oxidase.

PubMed

García-García, María Inmaculada; Hernández-García, Samanta; Sánchez-Ferrer, Álvaro; García-Carmona, Francisco

2013-06-26

Red Globe grape polyphenol oxidase, partially purified using phase partitioning with Triton-X114, was used to study the oxidation of hydroxytytosol (HT) and its related compounds tyrosol (TS), tyrosol acetate (TSA), and hydroxytyrosol acetate (HTA). The enzyme showed activity toward both monophenols (monophenolase activity) and o-diphenols (diphenolase activity) with a pH optimum (pH 6.5) that was independent of the phenol used. However, the optimal temperature for diphenolase activity was substrate-dependent, with a broad optimum of 25-65 °C for HT, compared with the maximum obtained for HTA (40 °C). Monophenolase activity showed the typical lag period, which was modulated by pH, substrate and enzyme concentrations, and the presence of catalytic amounts of o-diphenols. When the catalytic power (Vmax/K(M)) was determined for both activities, higher values were observed for o-diphenols than for monophenols: 9-fold higher for the HT/TS pair and 4-fold higher for HTA/TSA pair. Surprisingly, this ratio was equally higher for TSA (2.2-fold) compared with that of TS, whereas no such effect was observed for o-diphenols. This higher efficiency of TSA could be related to its greater hydrophobicity. Acetyl modification of these phenols not only changes the kinetic parameters of the enzyme but also affects their antioxidant activity (ORAC-FL assays), which is lower in HTA than in HT.
A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics.

PubMed

Subach, Oksana M; Entenberg, David; Condeelis, John S; Verkhusha, Vladislav V

2012-09-12

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via Förster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.
Determination of antibody response to influenza virus surface glycoproteins by kinetic enzyme-linked immunosorbent assay.

PubMed Central

Snyder, M H; Banks, S; Murphy, B R

1988-01-01

We modified an existing enzyme-linked immunosorbent assay (ELISA) to be able to use new spectrophotometers which can measure the rate of color development in microtiter wells. This new kinetic-based ELISA (KELISA) required only a single dilution of specimen rather than the multiple dilutions required with endpoint ELISA. In addition, 10- to 100-fold-less specimen was required to perform the KELISA than the ELISA. The level of serum or nasal wash antibody against surface glycoproteins of influenza A or influenza B viruses determined by KELISA was reproducible and correlated highly with the results of endpoint ELISA or hemagglutination inhibition tests. The difference between the KELISA rates, which indicated than an antibody response to infection had occurred, was defined and was analogous to a 2.2-fold rise in titer for serum and a 3.4-fold rise in titer for nasal wash determined by endpoint ELISA. The KELISA was similar to endpoint ELISAs in its ability to detect rises in antibody level in paired serum or nasal wash specimens obtained from volunteers who received live attenuated influenza A reassortant virus vaccines. By eliminating the need for multiple dilutions, the use of KELISA offers the advantage of increasing the number of assays that can be performed by the same personnel compared with endpoint ELISA, while it maintains sensitivity and specificity. PMID:3182992
Designed protein reveals structural determinants of extreme kinetic stability

PubMed Central

Broom, Aron; Ma, S. Martha; Xia, Ke; Rafalia, Hitesh; Trainor, Kyle; Colón, Wilfredo; Gosavi, Shachi; Meiering, Elizabeth M.

2015-01-01

The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge. PMID:26554002
Protein folding by NMR.

PubMed

Zhuravleva, Anastasia; Korzhnev, Dmitry M

2017-05-01

Protein folding is a highly complex process proceeding through a number of disordered and partially folded nonnative states with various degrees of structural organization. These transiently and sparsely populated species on the protein folding energy landscape play crucial roles in driving folding toward the native conformation, yet some of these nonnative states may also serve as precursors for protein misfolding and aggregation associated with a range of devastating diseases, including neuro-degeneration, diabetes and cancer. Therefore, in vivo protein folding is often reshaped co- and post-translationally through interactions with the ribosome, molecular chaperones and/or other cellular components. Owing to developments in instrumentation and methodology, solution NMR spectroscopy has emerged as the central experimental approach for the detailed characterization of the complex protein folding processes in vitro and in vivo. NMR relaxation dispersion and saturation transfer methods provide the means for a detailed characterization of protein folding kinetics and thermodynamics under native-like conditions, as well as modeling high-resolution structures of weakly populated short-lived conformational states on the protein folding energy landscape. Continuing development of isotope labeling strategies and NMR methods to probe high molecular weight protein assemblies, along with advances of in-cell NMR, have recently allowed protein folding to be studied in the context of ribosome-nascent chain complexes and molecular chaperones, and even inside living cells. Here we review solution NMR approaches to investigate the protein folding energy landscape, and discuss selected applications of NMR methodology to studying protein folding in vitro and in vivo. Together, these examples highlight a vast potential of solution NMR in providing atomistic insights into molecular mechanisms of protein folding and homeostasis in health and disease. Copyright © 2016 Elsevier B.V. All rights reserved.
Kinetic Modeling of Next-Generation High-Energy, High-Intensity Laser-Ion Accelerators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Albright, Brian James; Yin, Lin; Stark, David James

One of the long-standing problems in the community is the question of how we can model “next-generation” laser-ion acceleration in a computationally tractable way. A new particle tracking capability in the LANL VPIC kinetic plasma modeling code has enabled us to solve this long-standing problem
Human telomere sequence DNA in water-free and high-viscosity solvents: G-quadruplex folding governed by Kramers rate theory.

PubMed

Lannan, Ford M; Mamajanov, Irena; Hud, Nicholas V

2012-09-19

Structures formed by human telomere sequence (HTS) DNA are of interest due to the implication of telomeres in the aging process and cancer. We present studies of HTS DNA folding in an anhydrous, high viscosity deep eutectic solvent (DES) comprised of choline choride and urea. In this solvent, the HTS DNA forms a G-quadruplex with the parallel-stranded ("propeller") fold, consistent with observations that reduced water activity favors the parallel fold, whereas alternative folds are favored at high water activity. Surprisingly, adoption of the parallel structure by HTS DNA in the DES, after thermal denaturation and quick cooling to room temperature, requires several months, as opposed to less than 2 min in an aqueous solution. This extended folding time in the DES is, in part, due to HTS DNA becoming kinetically trapped in a folded state that is apparently not accessed in lower viscosity solvents. A comparison of times required for the G-quadruplex to convert from its aqueous-preferred folded state to its parallel fold also reveals a dependence on solvent viscosity that is consistent with Kramers rate theory, which predicts that diffusion-controlled transitions will slow proportionally with solvent friction. These results provide an enhanced view of a G-quadruplex folding funnel and highlight the necessity to consider solvent viscosity in studies of G-quadruplex formation in vitro and in vivo. Additionally, the solvents and analyses presented here should prove valuable for understanding the folding of many other nucleic acids and potentially have applications in DNA-based nanotechnology where time-dependent structures are desired.
Direct Observation of Parallel Folding Pathways Revealed Using a Symmetric Repeat Protein System

PubMed Central

Aksel, Tural; Barrick, Doug

2014-01-01

Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule. PMID:24988356
Bioavailability of House Dust Mite Allergens in Sublingual Allergy Tablets Is Highly Dependent on the Formulation.

PubMed

Ohashi-Doi, Katsuyo; Kito, Hirokazu; Du, Weibin; Nakazawa, Hiroshi; Ipsen, Henrik; Gudmann, Pernille; Lund, Kaare

2017-01-01

In sublingual immunotherapy (SLIT), the immune system is addressed by solubilized allergen that interacts with immunocompetent cells of the oral mucosa, the efficiency of which is governed by 2 main factors of SLIT allergen bioavailability: the allergen concentration and the mucosal contact time. Recently, 3 house dust mite (HDM) SLIT tablets were developed that differ with regard to allergen content, nominal strength (maintenance doses: 6 SQ-HDM/10,000 Japanese Allergen Units [JAU], 12 SQ-HDM/ 20,000 JAU, and 300 IR/57,000 JAU), and formulation (freeze-dried/compressed). Here, the importance of the SLIT tablet formulation for HDM major allergen bioavailability is examined. The HDM major allergen content, tablet disintegration times, and allergen release kinetics were determined. Dissolution kinetics (allergen concentration vs. time) of Der f 1, Der p 1, and Der 2 were measured. Area under the curve (AUC) was used as a surrogate parameter for allergen bioavailability. The release of HDM major allergens from the freeze-dried tablets was complete after 30 s, while only partial release was achieved with the compressed tablets, even after prolonged dissolution. At 1 min, i.e., the recommended sublingual holding time for the freeze-dried tablets, the allergen bioavailability (AUC) of the compressed 300 IR/57,000 JAU tablet was 4.7-fold (Der f 1), 10.8-fold (Der p 1), and 23.6-fold (Der 2) lower than that of the freeze-dried 12 SQ-HDM/20,000 JAU tablet and similar to (Der f 1) and 5.3-fold (Der p 1) and 12.5-fold (Der 2) lower than that of the freeze-dried 6 SQ-HDM/10,000 JAU tablet. SLIT tablet allergen bioavailability depends highly on the tablet formulation. Only the fast-dissolving freeze-dried tablets provide maximal delivery of soluble allergens and achieve allergen concentrations that reflect the nominal tablet strengths within the recommended sublingual holding time. © 2017 S. Karger AG, Basel.
Increased robustness of single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone versus real-time kinetic measurements.

PubMed

Selck, David A; Karymov, Mikhail A; Sun, Bing; Ismagilov, Rustem F

2013-11-19

Quantitative bioanalytical measurements are commonly performed in a kinetic format and are known to not be robust to perturbation that affects the kinetics itself or the measurement of kinetics. We hypothesized that the same measurements performed in a "digital" (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a 2-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7 × 10(-7)), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20 min change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R(2) = 0.9997 vs true counts over a 100-fold dynamic range). Fluorescent output of multiplexed PCR amplification could also be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification schemes based on organic, inorganic, and biochemical reactions have been developed, but their robustness is not well understood. This work implies that these chemistries may be significantly more robust in the digital, rather than kinetic, format. It also calls for theoretical studies to predict robustness of these chemistries and, more generally, to design robust reaction architectures. The SlipChip that we used here and other digital microfluidic technologies already exist to enable testing of these predictions. Such work may lead to identification or creation of robust amplification chemistries that enable rapid and precise quantitative molecular measurements under LRS. Furthermore, it may provide more general principles describing robustness of chemical and biological networks in digital formats.
Comparison of detailed and reduced kinetics mechanisms of silane oxidation in the basis of detonation wave structure problem

NASA Astrophysics Data System (ADS)

Fedorov, A. V.; Tropin, D. A.; Fomin, P. A.

2018-03-01

The paper deals with the problem of the structure of detonation waves in the silane-air mixture within the framework of mathematical model of a nonequilibrium gas dynamics. Detailed kinetic scheme of silane oxidation as well as the newly developed reduced kinetic model of detonation combustion of silane are used. On its basis the detonation wave (DW) structure in stoichiometric silane - air mixture and dependences of Chapman-Jouguet parameters of mixture on stoichiometric ratio between the fuel (silane) and an oxidizer (air) were obtained.
Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

PubMed Central

Charlier, Cyril; Alderson, T. Reid; Courtney, Joseph M.; Ying, Jinfa; Anfinrud, Philip

2018-01-01

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state. PMID:29666248
Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes

NASA Astrophysics Data System (ADS)

Habchi, Johnny; Chia, Sean; Galvagnion, Céline; Michaels, Thomas C. T.; Bellaiche, Mathias M. J.; Ruggeri, Francesco Simone; Sanguanini, Michele; Idini, Ilaria; Kumita, Janet R.; Sparr, Emma; Linse, Sara; Dobson, Christopher M.; Knowles, Tuomas P. J.; Vendruscolo, Michele

2018-06-01

Alzheimer's disease is a neurodegenerative disorder associated with the aberrant aggregation of the amyloid-β peptide. Although increasing evidence implicates cholesterol in the pathogenesis of Alzheimer's disease, the detailed mechanistic link between this lipid molecule and the disease process remains to be fully established. To address this problem, we adopt a kinetics-based strategy that reveals a specific catalytic role of cholesterol in the aggregation of Aβ42 (the 42-residue form of the amyloid-β peptide). More specifically, we demonstrate that lipid membranes containing cholesterol promote Aβ42 aggregation by enhancing its primary nucleation rate by up to 20-fold through a heterogeneous nucleation pathway. We further show that this process occurs as a result of cooperativity in the interaction of multiple cholesterol molecules with Aβ42. These results identify a specific microscopic pathway by which cholesterol dramatically enhances the onset of Aβ42 aggregation, thereby helping rationalize the link between Alzheimer's disease and the impairment of cholesterol homeostasis.
Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells†

PubMed Central

Das, Lipsa; Anderson, Todd A.; Gard, Jaime M.C.; Sroka, Isis C.; Strautman, Stephanie R.; Nagle, Raymond B.; Morrissey, Colm; Knudsen, Beatrice S.; Cress, Anne E.

2017-01-01

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modelling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual) of 3.25min−1, 3-fold faster than α3 integrin (1.0 min−1), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min−1), and significantly slower than the unrelated transferrin receptor (CD71) (15 min−1). Silencing of α3 integrin protein expression in DU145, PC3 and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8 fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. This article is protected by copyright. All rights reserved PMID:27509031

Amyloidogenesis of Natively Unfolded Proteins

PubMed Central

Uversky, Vladimir N.

2009-01-01

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation, which shares many structural properties with the pre-molten globule state, a partially folded intermediate first found during the equilibrium and kinetic (un)folding studies of several globular proteins and later described as one of the structural forms of natively unfolded proteins. The flexibility of this structural form is essential for the conformational rearrangements driving the formation of the core cross-beta structure of the amyloid fibril. Obviously, molecular mechanisms describing amyloidogenesis of ordered and natively unfolded proteins are different. For ordered protein to fibrillate, its unique and rigid structure has to be destabilized and partially unfolded. On the other hand, fibrillogenesis of a natively unfolded protein involves the formation of partially folded conformation; i.e., partial folding rather than unfolding. In this review recent findings are surveyed to illustrate some unique features of the natively unfolded proteins amyloidogenesis. PMID:18537543
The force-dependent mechanism of DnaK-mediated mechanical folding

PubMed Central

Perales-Calvo, Judit; Giganti, David; Stirnemann, Guillaume; Garcia-Manyes, Sergi

2018-01-01

It is well established that chaperones modulate the protein folding free-energy landscape. However, the molecular determinants underlying chaperone-mediated mechanical folding remain largely elusive, primarily because the force-extended unfolded conformation fundamentally differs from that characterized in biochemistry experiments. We use single-molecule force-clamp spectroscopy, combined with molecular dynamics simulations, to study the effect that the Hsp70 system has on the mechanical folding of three mechanically stiff model proteins. Our results demonstrate that, when working independently, DnaJ (Hsp40) and DnaK (Hsp70) work as holdases, blocking refolding by binding to distinct substrate conformations. Whereas DnaK binds to molten globule–like forms, DnaJ recognizes a cryptic sequence in the extended state in an unanticipated force-dependent manner. By contrast, the synergetic coupling of the Hsp70 system exhibits a marked foldase behavior. Our results offer unprecedented molecular and kinetic insights into the mechanisms by which mechanical force finely regulates chaperone binding, directly affecting protein elasticity. PMID:29487911
A desensitization-selective potentiator of AMPA-type glutamate receptors

PubMed Central

Sekiguchi, Masayuki; Nishikawa, Kaori; Aoki, Shunsuke; Wada, Keiji

2002-01-01

We examined the effects of PEPA, an allosteric potentiator of AMPA receptors, on AMPA receptor kinetics. PEPA did not affect the deactivation of glutamate responses but potently attenuated the extent of receptor desensitization without slowing the onset of desensitization in most of the recombinant AMPA receptors (GluR1-flip, GluR1-flop, GluR3-flip, GluR3-flip + GluR2-flip, and GluR3-flop + GluR2-flop) expressed in Xenopus oocytes. For the GluR3-flop subunit, PEPA attenuated the extent of desensitization and only weakly prolonged deactivation (1.3 fold). PEPA did not significantly affect recovery from desensitization in oocytes expressing GluR3-flip, GluR1-flop, and GluR1-flop, but weakly accelerated (2.6 fold) recovery from desensitization in oocytes expressing GluR3-flop. PEPA's effect on desensitization of GluR3-flop-containing receptors is unique in that onset is very slow. Simulation studies using simplified kinetic models for AMPA receptors are utilized to explore the differential effects of PEPA on GluR3-flip and -flop. It is possible to simulate the action on GluR3-flip by modulating two rate constants in a 12-state kinetic model. For simulation of the action on GluR3-flop, the 12-state kinetic model is not enough, and it is necessary to invoke a 13th state, a PEPA-bound receptor to which glutamate cannot bind. These results suggest that attenuation of extent of desensitization represents the principal mechanism underlying the potentiation of AMPA receptors by PEPA, and that PEPA exhibits different mechanisms with respect to GluR3-flip and GluR3-flop. PMID:12145103
Kinetic, mechanistic, and structural modeling studies of truncated wild-type leucine-rich repeat kinase 2 and the G2019S mutant.

PubMed

Liu, Min; Kang, Stephanie; Ray, Soumya; Jackson, Justin; Zaitsev, Alexandra D; Gerber, Scott A; Cuny, Gregory D; Glicksman, Marcie A

2011-11-01

Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity.
Transgenic mouse α- and β-cardiac myosins containing the R403Q mutation show isoform-dependent transient kinetic differences.

PubMed

Lowey, Susan; Bretton, Vera; Gulick, James; Robbins, Jeffrey; Trybus, Kathleen M

2013-05-24

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.
Cation-limited kinetic model for microbial extracellular electron transport via an outer membrane cytochrome C complex

PubMed Central

Okamoto, Akihiro; Tokunou, Yoshihide; Saito, Junki

2016-01-01

Outer-membrane c-type cytochrome (OM c-Cyt) complexes in several genera of iron-reducing bacteria, such as Shewanella and Geobacter, are capable of transporting electrons from the cell interior to extracellular solids as a terminal step of anaerobic respiration. The kinetics of this electron transport has implications for controlling the rate of microbial electron transport during bioenergy or biochemical production, iron corrosion, and natural mineral cycling. Herein, we review the findings from in-vivo and in-vitro studies examining electron transport kinetics through single OM c-Cyt complexes in Shewanella oneidensis MR-1. In-vitro electron flux via a purified OM c-Cyt complex, comprised of MtrA, B, and C proteins from S. oneidensis MR-1, embedded in a proteoliposome system is reported to be 10- to 100-fold faster compared with in-vivo estimates based on measurements of electron flux per cell and OM c-Cyts density. As the proteoliposome system is estimated to have 10-fold higher cation flux via potassium channels than electrons, we speculate that the slower rate of electron-coupled cation transport across the OM is responsible for the significantly lower electron transport rate that is observed in-vivo. As most studies to date have primarily focused on the energetics or kinetics of interheme electron hopping in OM c-Cyts in this microbial electron transport mechanism, the proposed model involving cation transport provides new insight into the rate detemining step of EET, as well as the role of self-secreted flavin molecules bound to OM c-Cyt and proton management for energy conservation and production in S. oneidensis MR-1. PMID:27924259
Studies of G-quadruplexes formed within self-assembled DNA mini-circles.

PubMed

Klejevskaja, Beata; Pyne, Alice L B; Reynolds, Matthew; Shivalingam, Arun; Thorogate, Richard; Hoogenboom, Bart W; Ying, Liming; Vilar, Ramon

2016-10-13

We have developed self-assembled DNA mini-circles that contain a G-quadruplex-forming sequence from the c-Myc oncogene promoter and demonstrate by FRET that the G-quadruplex unfolding kinetics are 10-fold slower than for the simpler 24-mer G-quadruplex that is commonly used for FRET experiments.
Soft Computing Techniques for the Protein Folding Problem on High Performance Computing Architectures.

PubMed

Llanes, Antonio; Muñoz, Andrés; Bueno-Crespo, Andrés; García-Valverde, Teresa; Sánchez, Antonia; Arcas-Túnez, Francisco; Pérez-Sánchez, Horacio; Cecilia, José M

2016-01-01

The protein-folding problem has been extensively studied during the last fifty years. The understanding of the dynamics of global shape of a protein and the influence on its biological function can help us to discover new and more effective drugs to deal with diseases of pharmacological relevance. Different computational approaches have been developed by different researchers in order to foresee the threedimensional arrangement of atoms of proteins from their sequences. However, the computational complexity of this problem makes mandatory the search for new models, novel algorithmic strategies and hardware platforms that provide solutions in a reasonable time frame. We present in this revision work the past and last tendencies regarding protein folding simulations from both perspectives; hardware and software. Of particular interest to us are both the use of inexact solutions to this computationally hard problem as well as which hardware platforms have been used for running this kind of Soft Computing techniques.
Development and evaluation of granule and emulsifiable concentrate formulations containing Derris elliptica extract for crop pest control.

PubMed

Wiwattanapatapee, Ruedeekorn; Sae-Yun, Attawadee; Petcharat, Jiraporn; Ovatlarnporn, Chitchamai; Itharat, Arunporn

2009-12-09

Derris elliptica Benth. extracts containing rotenone have long been used as natural insecticides, but time-consuming preparation processes and the short shelf life of the extract limit their use in pest control. In this study, stable water-dispersible granules and emulsifiable concentrate liquids containing Derris extract (equivalent to 5% w/w of rotenone) were developed with simple techniques. Accelerated degradation kinetics of rotenone in the Derris extract, and in both formulations, indicated that its degradation followed first-order kinetics. The predicted half-life (t(1/2)) and shelf life (t(90%)) at 30 degrees C of rotenone in Derris extract were 520 and 79 days, respectively. Derris granules and emulsifiable concentrate clearly prolong the stability of rotenone 8-fold (t(90%) = 633 days) and 1.4-fold (t(90%) = 110 days), respectively. The study of rotenone degradation after application onto plants indicated that both formulations would be effective for up to 3 days after spraying. Preliminary efficacy testing indicated that the Derris emulsifiable concentrate was clearly more effective than Derris water-dispersible granules in controlling Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae).
A new mode of regulation of N-type inactivation in a Caenorhabditis elegans voltage-gated potassium channel.

PubMed

Cai, Shi-Qing; Sesti, Federico

2007-06-22

N-type inactivation in voltage-gated K+ (Kv) channels is a widespread means to modulate neuronal excitability and signaling. Here we have shown a novel mechanism of N-type inactivation in a Caenorhabditis elegans Kv channel. The N-terminal sequence of KVS-1 contains a domain of 22 amino acids that resembles the inactivation ball in A-type channels, which is preceded by a domain of eighteen amino acids. Wild type KVS-1 currents can be described as A-type; however, their kinetics are significantly (approximately 5-fold) slower. When the putative inactivation ball is deleted, the current becomes non-inactivating. Inactivation is restored in non-inactivating channels by diffusion of the missing inactivation domain in the cytoplasm. Deletion of the domain in front of the ball speeds inactivation kinetics approximately 5-fold. We conclude that KVS-1 is the first example of a novel type of Kv channel simultaneously possessing an N-inactivating ball preceded by an N inactivation regulatory domain (NIRD) that acts to slow down inactivation through steric mechanisms.
Hydrothermal Atomic Force Microscopy Investigation of Barite Growth: Role of Spectator Ions in Elementary Step Edge Growth Kinetics and Hillock Morphology [Supporting Information Only

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jindra, Sarah A.; Bertagni, Angela L.; Bracco, Jacquelyn N.

Here, to better understand the role of spectator ions in barite growth, the kinetics of step edge growth on barite (001) surfaces were studied under various salt solutions. Hydrothermal atomic force microscopy (HAFM) was used to investigate the effect of background electrolytes (NaCl, NaBr, and NaNO 3) as a function of saturation index and ionic strength ( I) on barite growth sourced at dislocations at 108 °C. Results demonstrate that hillock morphology is affected by I, as well as type of anion, where the prevalence of steps aligned on the [010] direction is highest under Cl –. There is amore » modest increase in kinetic coefficient of 55–130% with a 10-fold increase in I for each salt. In comparing the kinetic coefficients of the salts at low ionic strength (0.01 M), there is a moderate difference, suggesting that the anion may play a role in barium attachment.« less
Extraction of anthocyanins from red cabbage using high pressure CO2.

PubMed

Xu, Zhenzhen; Wu, Jihong; Zhang, Yan; Hu, Xiaosong; Liao, Xiaojun; Wang, Zhengfu

2010-09-01

The extraction kinetics of anthocyanins from red cabbage using high pressure CO(2) (HPCD) against conventional acidified water (CAW) was investigated. The HPCD time, temperature, pressure and volume ratio of solid-liquid mixture vs. pressurized CO(2) (R((S+L)/G)) exhibited important roles on the extraction kinetics of anthocyanins. The extraction kinetics showed two phases, the yield increased with increasing the time in the first phase, the yield defined as steady-state yield (y(*)) was constant in the second phase. The y(*) of anthocyanins using HPCD increased with higher temperature, higher pressure and lower R((S+L)/G). The general mass transfer model with higher regression coefficients (R(2)>0.97) fitted the kinetic data better than the Fick's second law diffusion model. As compared with CAW, the time (t(*)) to reach the y(*) of anthocyanins using HPCD was reduced by half while its corresponding overall volumetric mass transfer coefficients k(L)xa from the general mass transfer model increased by two folds. Copyright 2010 Elsevier Ltd. All rights reserved.
First-Order or Second-Order Kinetics? A Monte Carlo Answer

ERIC Educational Resources Information Center

Tellinghuisen, Joel

2005-01-01

Monte Carlo computational experiments reveal that the ability to discriminate between first- and second-order kinetics from least-squares analysis of time-dependent concentration data is better than implied in earlier discussions of the problem. The problem is rendered as simple as possible by assuming that the order must be either 1 or 2 and that…
Microbially Mediated Kinetic Sulfur Isotope Fractionation: Reactive Transport Modeling Benchmark

NASA Astrophysics Data System (ADS)

Wanner, C.; Druhan, J. L.; Cheng, Y.; Amos, R. T.; Steefel, C. I.; Ajo Franklin, J. B.

2014-12-01

Microbially mediated sulfate reduction is a ubiquitous process in many subsurface systems. Isotopic fractionation is characteristic of this anaerobic process, since sulfate reducing bacteria (SRB) favor the reduction of the lighter sulfate isotopologue (S32O42-) over the heavier isotopologue (S34O42-). Detection of isotopic shifts have been utilized as a proxy for the onset of sulfate reduction in subsurface systems such as oil reservoirs and aquifers undergoing uranium bioremediation. Reactive transport modeling (RTM) of kinetic sulfur isotope fractionation has been applied to field and laboratory studies. These RTM approaches employ different mathematical formulations in the representation of kinetic sulfur isotope fractionation. In order to test the various formulations, we propose a benchmark problem set for the simulation of kinetic sulfur isotope fractionation during microbially mediated sulfate reduction. The benchmark problem set is comprised of four problem levels and is based on a recent laboratory column experimental study of sulfur isotope fractionation. Pertinent processes impacting sulfur isotopic composition such as microbial sulfate reduction and dispersion are included in the problem set. To date, participating RTM codes are: CRUNCHTOPE, TOUGHREACT, MIN3P and THE GEOCHEMIST'S WORKBENCH. Preliminary results from various codes show reasonable agreement for the problem levels simulating sulfur isotope fractionation in 1D.
Common diagnoses and treatments in professional voice users.

PubMed

Franco, Ramon A; Andrus, Jennifer G

2007-10-01

Common problems among all patients seen by the laryngologist are also common among professional voice users. These include laryngopharyngeal reflux, muscle tension dysphonia, fibrovascular vocal fold lesions (eg, nodules and polyps), cysts, vocal fold scarring, changes in vocal fold mobility, and age-related changes. Microvascular lesions and their associated sequelae of vocal fold hemorrhage and laryngitis due to voice overuse are more common among professional voice users. Much more common among professional voice users is the negative impact that voice problems have on their ability to work, on their overall sense of well-being, and sometimes on their very sense of self. This article reviews the diagnosis and treatment options for these and other problems among professional voice users, describing the relevant roles of medical treatment, voice therapy, and surgery. The common scenario of multiple concomitant entities contributing to a symptom complex is underscored. Emphasis is placed on gaining insight into the "whole" patient so that individualized management plans can be developed. Videos of select diagnoses accompany this content online.
Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

PubMed Central

Huang, Beijing K.; Sikes, Hadley D.

2014-01-01

Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS) in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies. PMID:25460730
Sodium hydroxide catalyzed monodispersed high surface area silica nanoparticles.

PubMed

Bhakta, Snehasis; Dixit, Chandra K; Bist, Itti; Jalil, Karim Abdel; Suib, Steven L; Rusling, James F

2016-07-01

Understanding of the synthesis kinetics and our ability to modulate medium conditions allowed us to generate nanoparticles via an ultra-fast process. The synthesis medium is kept quite simple with tetraethyl orthosilicate (TEOS) as precursor and 50% ethanol and sodium hydroxide catalyst. Synthesis is performed under gentle conditions at 20 °C for 20 min Long synthesis time and catalyst-associated drawbacks are most crucial in silica nanoparticle synthesis. We have addressed both these bottlenecks by replacing the conventional Stober catalyst, ammonium hydroxide, with sodium hydroxide. We have reduced the overall synthesis time from 20 to 1/3 h, ~60-fold decrease, and obtained highly monodispersed nanoparticles with 5-fold higher surface area than Stober particles. We have demonstrated that the developed NPs with ~3-fold higher silane can be used as efficient probes for biosensor applications.
Measuring internal friction of an ultrafast-folding protein.

PubMed

Cellmer, Troy; Henry, Eric R; Hofrichter, James; Eaton, William A

2008-11-25

Nanosecond laser T-jump was used to measure the viscosity dependence of the folding kinetics of the villin subdomain under conditions where the viscogen has no effect on its equilibrium properties. The dependence of the unfolding/refolding relaxation time on solvent viscosity indicates a major contribution to the dynamics from internal friction. The internal friction increases with increasing temperature, suggesting a shift in the transition state along the reaction coordinate toward the native state with more compact structures, and therefore, a smaller diffusion coefficient due to increased landscape roughness. Fitting the data with an Ising-like model yields a relatively small position dependence for the diffusion coefficient. This finding is consistent with the excellent correlation found between experimental and calculated folding rates based on free energy barrier heights using the same diffusion coefficient for every protein.
Extreme Folding

NASA Astrophysics Data System (ADS)

Demaine, Erik

2012-02-01

Our understanding of the mathematics and algorithms behind paper folding, and geometric folding in general, has increased dramatically over the past several years. These developments have found a surprisingly broad range of applications. In the art of origami, it has helped spur the technical origami revolution. In engineering and science, it has helped solve problems in areas such as manufacturing, robotics, graphics, and protein folding. On the recreational side, it has led to new kinds of folding puzzles and magic. I will give an overview of the mathematics and algorithms of folding, with a focus on new mathematics and sculpture.
Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

PubMed Central

Dupuis, Nicholas F.; Holmstrom, Erik D.; Nesbitt, David J.

2013-01-01

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′on = 2.1(1) × 106 M−1 s−1) and 6 bp (k′on = 5.0(1) × 106 M−1 s−1) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s−1) to 6 bp (koff = 14 s−1) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, konincreases by >40-fold (kon = 0.10(1) s−1 to 4.0(4) s−1 between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s−1 to 0.17(7) s−1) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation. PMID:23931323

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

PubMed Central

Almaqwashi, Ali A.; Andersson, Johanna; Lincoln, Per; Rouzina, Ioulia; Westerlund, Fredrik; Williams, Mark C.

2016-01-01

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)4Ru2]4+ (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)2dppz) and the distal subunit (Ru(phen)2ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δxeq, and the dynamic DNA structural deformations during ligand association xon and dissociation xoff. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a Kd of 27 ± 3 nM for (Δ,Λ)-Piz to a Kd of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δxeq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and xon from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited xoff changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics. PMID:27028636
Protein folding, protein structure and the origin of life: Theoretical methods and solutions of dynamical problems

NASA Technical Reports Server (NTRS)

Weaver, D. L.

1982-01-01

Theoretical methods and solutions of the dynamics of protein folding, protein aggregation, protein structure, and the origin of life are discussed. The elements of a dynamic model representing the initial stages of protein folding are presented. The calculation and experimental determination of the model parameters are discussed. The use of computer simulation for modeling protein folding is considered.
Computational Modeling of Proteins based on Cellular Automata: A Method of HP Folding Approximation.

PubMed

Madain, Alia; Abu Dalhoum, Abdel Latif; Sleit, Azzam

2018-06-01

The design of a protein folding approximation algorithm is not straightforward even when a simplified model is used. The folding problem is a combinatorial problem, where approximation and heuristic algorithms are usually used to find near optimal folds of proteins primary structures. Approximation algorithms provide guarantees on the distance to the optimal solution. The folding approximation approach proposed here depends on two-dimensional cellular automata to fold proteins presented in a well-studied simplified model called the hydrophobic-hydrophilic model. Cellular automata are discrete computational models that rely on local rules to produce some overall global behavior. One-third and one-fourth approximation algorithms choose a subset of the hydrophobic amino acids to form H-H contacts. Those algorithms start with finding a point to fold the protein sequence into two sides where one side ignores H's at even positions and the other side ignores H's at odd positions. In addition, blocks or groups of amino acids fold the same way according to a predefined normal form. We intend to improve approximation algorithms by considering all hydrophobic amino acids and folding based on the local neighborhood instead of using normal forms. The CA does not assume a fixed folding point. The proposed approach guarantees one half approximation minus the H-H endpoints. This lower bound guaranteed applies to short sequences only. This is proved as the core and the folds of the protein will have two identical sides for all short sequences.
Rotational Energy.

ERIC Educational Resources Information Center

Lockett, Keith

1988-01-01

Demonstrates several objects rolling down a slope to explain the energy transition among potential energy, translational kinetic energy, and rotational kinetic energy. Contains a problem from Galileo's rolling ball experiment. (YP)
Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

PubMed Central

Strulson, Christopher A.; Boyer, Joshua A.; Whitman, Elisabeth E.; Bevilacqua, Philip C.

2014-01-01

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg2+ ion concentrations are low, K+ concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo–like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg2+ (0.5–2 mM) and K+ (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution. PMID:24442612
[Temperament of children with vocal fold nodules].

PubMed

Wei, Youhua; Wang, Zhinan; Xu, Zhongqiang; Chen, Ping; Hao, Lili

2009-11-01

To examine the temperament of children with vocal fold nodules. To compare the temperament dimension and temperamental types of 42 children with vocal fold nodules with 46 vocally normal children, using Chinese children's Temperament Problem Screening system (CCTPSs). The children with vocal fold nodules differed significantly from the comparison group in their temperament dimension's adaptability, intensity of reaction, mood value, persistency and temperamental types. There are more difficult and slow-to-warm-up children in patients with vocal fold nodules than vocally normal children.
Exploring the Universe of Protein Structures beyond the Protein Data Bank

PubMed Central

Cossio, Pilar; Trovato, Antonio; Pietrucci, Fabio; Seno, Flavio; Maritan, Amos; Laio, Alessandro

2010-01-01

It is currently believed that the atlas of existing protein structures is faithfully represented in the Protein Data Bank. However, whether this atlas covers the full universe of all possible protein structures is still a highly debated issue. By using a sophisticated numerical approach, we performed an exhaustive exploration of the conformational space of a 60 amino acid polypeptide chain described with an accurate all-atom interaction potential. We generated a database of around 30,000 compact folds with at least of secondary structure corresponding to local minima of the potential energy. This ensemble plausibly represents the universe of protein folds of similar length; indeed, all the known folds are represented in the set with good accuracy. However, we discover that the known folds form a rather small subset, which cannot be reproduced by choosing random structures in the database. Rather, natural and possible folds differ by the contact order, on average significantly smaller in the former. This suggests the presence of an evolutionary bias, possibly related to kinetic accessibility, towards structures with shorter loops between contacting residues. Beside their conceptual relevance, the new structures open a range of practical applications such as the development of accurate structure prediction strategies, the optimization of force fields, and the identification and design of novel folds. PMID:21079678
Analyzing the effect of homogeneous frustration in protein folding.

PubMed

Contessoto, Vinícius G; Lima, Debora T; Oliveira, Ronaldo J; Bruni, Aline T; Chahine, Jorge; Leite, Vitor B P

2013-10-01

The energy landscape theory has been an invaluable theoretical framework in the understanding of biological processes such as protein folding, oligomerization, and functional transitions. According to the theory, the energy landscape of protein folding is funneled toward the native state, a conformational state that is consistent with the principle of minimal frustration. It has been accepted that real proteins are selected through natural evolution, satisfying the minimum frustration criterion. However, there is evidence that a low degree of frustration accelerates folding. We examined the interplay between topological and energetic protein frustration. We employed a Cα structure-based model for simulations with a controlled nonspecific energetic frustration added to the potential energy function. Thermodynamics and kinetics of a group of 19 proteins are completely characterized as a function of increasing level of energetic frustration. We observed two well-separated groups of proteins: one group where a little frustration enhances folding rates to an optimal value and another where any energetic frustration slows down folding. Protein energetic frustration regimes and their mechanisms are explained by the role of non-native contact interactions in different folding scenarios. These findings strongly correlate with the protein free-energy folding barrier and the absolute contact order parameters. These computational results are corroborated by principal component analysis and partial least square techniques. One simple theoretical model is proposed as a useful tool for experimentalists to predict the limits of improvements in real proteins. Copyright © 2013 Wiley Periodicals, Inc.
Kinetics of Exocytosis Is Faster in Cones Than in Rods

PubMed Central

Rabl, Katalin; Cadetti, Lucia; Thoreson, Wallace B.

2006-01-01

Cone-driven responses of second-order retinal neurons are considerably faster than rod-driven responses. We examined whether differences in the kinetics of synaptic transmitter release from rods and cones may contribute to differences in postsynaptic response kinetics. Exocytosis from rods and cones was triggered by membrane depolarization and monitored in two ways: (1) by measuring EPSCs evoked in second-order neurons by depolarizing steps applied to presynaptic rods or cones during simultaneous paired whole-cell recordings or (2) by direct measurements of exocytotic increases in membrane capacitance. The kinetics of release was assessed by varying the length of the depolarizing test step. Both measures of release revealed two kinetic components to the increase in exocytosis as a function of the duration of a step depolarization. In addition to slow sustained components in both cell types, the initial fast component of exocytosis had a time constant of <5 ms in cones, >10-fold faster than that of rods. Rod/cone differences in the kinetics of release were substantiated by a linear correlation between depolarization-evoked capacitance increases and EPSC charge transfer. Experiments on isolated rods indicate that the slower kinetics of exocytosis from rods was not a result of rod–rod coupling. The initial rapid release of vesicles from cones can shape the postsynaptic response and may contribute to the faster responses of cone-driven cells observed at light offset. PMID:15872111
Rapid, bilateral changes in growth rate and curvature during gravitropism of cucumber hypocotyls: implications for mechanism of growth control

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.

1990-01-01

The growth response of etiolated cucumber (Cucumis sativus L.) hypocotyls to gravitropic stimulation was examined by means of time-lapse photography and high-resolution analysis of surface expansion and curvature. In comparison with video analysis, the technique described here has five- to 20-fold better resolution; moreover, the mathematical fitting method (cubic splines) allows direct estimation of local and integrated curvature. After switching seedlings from a vertical to horizontal position, both upper and lower surfaces of the stem reacted after a lag of about 11 min with a two- to three-fold increase in surface expansion rate on the lower side and a cessation of expansion, or slight compression, on the upper surface. This growth asymmetry was initiated simultaneously along the length of the hypocotyl, on both upper and lower surfaces, and did not migrate basipetally from the apex. Later stages in the gravitropic response involved a complex reversal of the growth asymmetry, with the net result being a basipetal migration of the curved region. This secondary growth reversal may reflect oscillatory and/or self-regulatory behaviour of growing cells. With some qualifications, the kinetics and pattern of growth response are consistent with a mechanism involving hormone redistribution, although they do not prove such a mechanism. The growth kinetics require a growth mechanism which can be stimulated by two- to three-fold or completely inhibited within a few minutes.
Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

PubMed

Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

2004-06-29

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.
Two states or not two states: Single-molecule folding studies of protein L

NASA Astrophysics Data System (ADS)

Aviram, Haim Yuval; Pirchi, Menahem; Barak, Yoav; Riven, Inbal; Haran, Gilad

2018-03-01

Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ˜7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.
A hybrid computer program for rapidly solving flowing or static chemical kinetic problems involving many chemical species

NASA Technical Reports Server (NTRS)

Mclain, A. G.; Rao, C. S. R.

1976-01-01

A hybrid chemical kinetic computer program was assembled which provides a rapid solution to problems involving flowing or static, chemically reacting, gas mixtures. The computer program uses existing subroutines for problem setup, initialization, and preliminary calculations and incorporates a stiff ordinary differential equation solution technique. A number of check cases were recomputed with the hybrid program and the results were almost identical to those previously obtained. The computational time saving was demonstrated with a propane-oxygen-argon shock tube combustion problem involving 31 chemical species and 64 reactions. Information is presented to enable potential users to prepare an input data deck for the calculation of a problem.
Folding of viscous sheets and filaments

NASA Astrophysics Data System (ADS)

Skorobogatiy, M.; Mahadevan, L.

2000-12-01

We consider the nonlinear folding behavior of a viscous filament or a sheet under the influence of an external force such as gravity. Everyday examples of this phenomenon are provided by the periodic folding of a sheet of honey as it impinges on toast, or the folding of a stream of shampoo as it falls on one's hand. To understand the evolution of a fold, we formulate and solve a free-boundary problem for the phenomenon, give scaling laws for the size of the folds and the frequency with which they are laid out, and verify these experimentally.
Recent developments in the theory of protein folding: searching for the global energy minimum.

PubMed

Scheraga, H A

1996-04-16

Statistical mechanical theories and computer simulation are being used to gain an understanding of the fundamental features of protein folding. A major obstacle in the computation of protein structures is the multiple-minima problem arising from the existence of many local minima in the multidimensional energy landscape of the protein. This problem has been surmounted for small open-chain and cyclic peptides, and for regular-repeating sequences of models of fibrous proteins. Progress is being made in resolving this problem for globular proteins.
The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

NASA Astrophysics Data System (ADS)

Beedle, Amy E. M.; Lezamiz, Ainhoa; Stirnemann, Guillaume; Garcia-Manyes, Sergi

2015-08-01

Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.
Unravelling Origami Metamaterial Behavior

NASA Astrophysics Data System (ADS)

Eidini, Maryam; Paulino, Glaucio

2015-03-01

Origami has shown to be a substantial source of inspiration for innovative design of mechanical metamaterials for which the material properties arise from their geometry and structural layout. Most research on origami-inspired materials relies on known patterns, especially on classic Miura-ori pattern. In the present research, we have created origami-inspired metamaterials and we have shown that the folded materials possess properties as remarkable as those of Miura-ori on which there is a lot of recent research. We have also introduced and placed emphasis on several important concepts that are confused or overlooked in the literature, e.g. concept of planar Poisson's ratio for folded materials from different conceptual viewpoints, and we have clarified the importance of such concepts by applying them to the folded sheet metamaterials introduced in our research. The new patterns are appropriate for a broad range of applications, from mechanical metamaterials to deployable and kinetic structures, at both small and large scales.
Protein Quality Control Acts on Folding Intermediates to Shape the Effects of Mutations on Organismal Fitness

PubMed Central

Bershtein, Shimon; Mu, Wanmeng; Serohijos, Adrian W. R.; Zhou, Jingwen; Shakhnovich, Eugene I.

2012-01-01

Summary What are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli’s gene encoding dihydrofolate reductase (DHFR), and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: Over-expression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains. Kinetic steady-state modeling predicts and experimentation verifies that mutations affect fitness by shifting the flux balance in cellular milieu between protein production, folding and degradation orchestrated by PQC through the interaction with folding intermediates. PMID:23219534
The energy landscape of adenylate kinase during catalysis

PubMed Central

Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; Pontiggia, Francesco; Otten, Renee; Pachov, Dimitar V.; Kutter, Steffen; Phung, Lien A.; Murphy, Padraig N.; Thai, Vu; Alber, Tom; Hagan, Michael F.; Kern, Dorothee

2014-01-01

Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, MD simulations, and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events, phosphoryl transfer (>105-fold) and lid-opening (103-fold). In contrast, mutation of an essential active-site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid-opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a pre-organized active site. PMID:25580578
Designing cooperatively folded abiotic uni- and multimolecular helix bundles

NASA Astrophysics Data System (ADS)

de, Soumen; Chi, Bo; Granier, Thierry; Qi, Ting; Maurizot, Victor; Huc, Ivan

2018-01-01

Abiotic foldamers, that is foldamers that have backbones chemically remote from peptidic and nucleotidic skeletons, may give access to shapes and functions different to those of peptides and nucleotides. However, design methodologies towards abiotic tertiary and quaternary structures are yet to be developed. Here we report rationally designed interactional patterns to guide the folding and assembly of abiotic helix bundles. Computational design facilitated the introduction of hydrogen-bonding functionalities at defined locations on the aromatic amide backbones that promote cooperative folding into helix-turn-helix motifs in organic solvents. The hydrogen-bond-directed aggregation of helices not linked by a turn unit produced several thermodynamically and kinetically stable homochiral dimeric and trimeric bundles with structures that are distinct from the designed helix-turn-helix. Relative helix orientation within the bundles may be changed from parallel to tilted on subtle solvent variations. Altogether, these results prefigure the richness and uniqueness of abiotic tertiary structure behaviour.

Separating the effects of internal friction and transition state energy to explain the slow, frustrated folding of spectrin domains

PubMed Central

Wensley, Beth G.; Kwa, Lee Gyan; Shammas, Sarah L.; Rogers, Joseph M.; Browning, Stuart; Yang, Ziqi; Clarke, Jane

2012-01-01

The elongated three-helix bundle domains spectrin R16 and R17 fold some two to three orders of magnitude more slowly than their homologue R15. We have shown that this slow folding is due, at least in part, to roughness in the free-energy landscape of R16 and R17. We have proposed that this roughness is due to a frustrated search for the correct docking of partly preformed helices. However, this accounts for only a small part of the slowing of folding and unfolding. Five residues on the A helix of R15, when inserted together into R16 or R17, increase the folding rate constants, reduce landscape roughness, and alter the folding mechanism to one resembling R15. The effect of each of these mutations individually is investigated here. No one mutation causes the behavior seen for the five in combination. However, two mutations, E18F and K25V, significantly increase the folding and unfolding rates of both R16 and R17 but without a concomitant loss in landscape roughness. E18F has the greatest effect on the kinetics, and a Φ-value analysis of the C helix reveals that the folding mechanism is unchanged. For both E18F and K25V the removal of the charge and resultant transition state stabilization is the main origin of the faster folding. Consequently, the major cause of the unusually slow folding of R16 and R17 is the non-native burial of the two charged residues in the transition state. The slowing due to landscape roughness is only about fivefold. PMID:22711800
Separating the effects of internal friction and transition state energy to explain the slow, frustrated folding of spectrin domains.

PubMed

Wensley, Beth G; Kwa, Lee Gyan; Shammas, Sarah L; Rogers, Joseph M; Browning, Stuart; Yang, Ziqi; Clarke, Jane

2012-10-30

The elongated three-helix bundle domains spectrin R16 and R17 fold some two to three orders of magnitude more slowly than their homologue R15. We have shown that this slow folding is due, at least in part, to roughness in the free-energy landscape of R16 and R17. We have proposed that this roughness is due to a frustrated search for the correct docking of partly preformed helices. However, this accounts for only a small part of the slowing of folding and unfolding. Five residues on the A helix of R15, when inserted together into R16 or R17, increase the folding rate constants, reduce landscape roughness, and alter the folding mechanism to one resembling R15. The effect of each of these mutations individually is investigated here. No one mutation causes the behavior seen for the five in combination. However, two mutations, E18F and K25V, significantly increase the folding and unfolding rates of both R16 and R17 but without a concomitant loss in landscape roughness. E18F has the greatest effect on the kinetics, and a Φ-value analysis of the C helix reveals that the folding mechanism is unchanged. For both E18F and K25V the removal of the charge and resultant transition state stabilization is the main origin of the faster folding. Consequently, the major cause of the unusually slow folding of R16 and R17 is the non-native burial of the two charged residues in the transition state. The slowing due to landscape roughness is only about fivefold.
First passage analysis of the folding of a β-sheet miniprotein: is it more realistic than the standard equilibrium approach?

PubMed

Kalgin, Igor V; Chekmarev, Sergei F; Karplus, Martin

2014-04-24

Simulations of first-passage folding of the antiparallel β-sheet miniprotein beta3s, which has been intensively studied under equilibrium conditions by A. Caflisch and co-workers, show that the kinetics and dynamics are significantly different from those for equilibrium folding. Because the folding of a protein in a living system generally corresponds to the former (i.e., the folded protein is stable and unfolding is a rare event), the difference is of interest. In contrast to equilibrium folding, the Ch-curl conformations become very rare because they contain unfavorable parallel β-strand arrangements, which are difficult to form dynamically due to the distant N- and C-terminal strands. At the same time, the formation of helical conformations becomes much easier (particularly in the early stage of folding) due to short-range contacts. The hydrodynamic descriptions of the folding reaction have also revealed that while the equilibrium flow field presented a collection of local vortices with closed "streamlines", the first-passage folding is characterized by a pronounced overall flow from the unfolded states to the native state. The flows through the locally stable structures Cs-or and Ns-or, which are conformationally close to the native state, are negligible due to detailed balance established between these structures and the native state. Although there are significant differences in the general picture of the folding process from the equilibrium and first-passage folding simulations, some aspects of the two are in agreement. The rate of transitions between the clusters of characteristic protein conformations in both cases decreases approximately exponentially with the distance between the clusters in the hydrogen bond distance space of collective variables, and the folding time distribution in the first-passage segments of the equilibrium trajectory is in good agreement with that for the first-passage folding simulations.
First Passage Analysis of the Folding of a β-Sheet Miniprotein: Is it More Realistic Than the Standard Equilibrium Approach?

PubMed Central

2015-01-01

Simulations of first-passage folding of the antiparallel β-sheet miniprotein beta3s, which has been intensively studied under equilibrium conditions by A. Caflisch and co-workers, show that the kinetics and dynamics are significantly different from those for equilibrium folding. Because the folding of a protein in a living system generally corresponds to the former (i.e., the folded protein is stable and unfolding is a rare event), the difference is of interest. In contrast to equilibrium folding, the Ch-curl conformations become very rare because they contain unfavorable parallel β-strand arrangements, which are difficult to form dynamically due to the distant N- and C-terminal strands. At the same time, the formation of helical conformations becomes much easier (particularly in the early stage of folding) due to short-range contacts. The hydrodynamic descriptions of the folding reaction have also revealed that while the equilibrium flow field presented a collection of local vortices with closed ”streamlines”, the first-passage folding is characterized by a pronounced overall flow from the unfolded states to the native state. The flows through the locally stable structures Cs-or and Ns-or, which are conformationally close to the native state, are negligible due to detailed balance established between these structures and the native state. Although there are significant differences in the general picture of the folding process from the equilibrium and first-passage folding simulations, some aspects of the two are in agreement. The rate of transitions between the clusters of characteristic protein conformations in both cases decreases approximately exponentially with the distance between the clusters in the hydrogen bond distance space of collective variables, and the folding time distribution in the first-passage segments of the equilibrium trajectory is in good agreement with that for the first-passage folding simulations. PMID:24669953
Fission fragment mass and total kinetic energy distributions of spontaneously fissioning plutonium isotopes

NASA Astrophysics Data System (ADS)

Pomorski, K.; Nerlo-Pomorska, B.; Bartel, J.; Schmitt, C.

2018-03-01

The fission-fragment mass and total kinetic energy (TKE) distributions are evaluated in a quantum mechanical framework using elongation, mass asymmetry, neck degree of freedom as the relevant collective parameters in the Fourier shape parametrization recently developed by us. The potential energy surfaces (PES) are calculated within the macroscopic-microscopic model based on the Lublin-Strasbourg Drop (LSD), the Yukawa-folded (YF) single-particle potential and a monopole pairing force. The PES are presented and analysed in detail for even-even Plutonium isotopes with A = 236-246. They reveal deep asymmetric valleys. The fission-fragment mass and TKE distributions are obtained from the ground state of a collective Hamiltonian computed within the Born-Oppenheimer approximation, in the WKB approach by introducing a neck-dependent fission probability. The calculated mass and total kinetic energy distributions are found in good agreement with the data.
An immersed-boundary method for flow–structure interaction in biological systems with application to phonation

PubMed Central

Luo, Haoxiang; Mittal, Rajat; Zheng, Xudong; Bielamowicz, Steven A.; Walsh, Raymond J.; Hahn, James K.

2008-01-01

A new numerical approach for modeling a class of flow–structure interaction problems typically encountered in biological systems is presented. In this approach, a previously developed, sharp-interface, immersed-boundary method for incompressible flows is used to model the fluid flow and a new, sharp-interface Cartesian grid, immersed boundary method is devised to solve the equations of linear viscoelasticity that governs the solid. The two solvers are coupled to model flow–structure interaction. This coupled solver has the advantage of simple grid generation and efficient computation on simple, single-block structured grids. The accuracy of the solid-mechanics solver is examined by applying it to a canonical problem. The solution methodology is then applied to the problem of laryngeal aerodynamics and vocal fold vibration during human phonation. This includes a three-dimensional eigen analysis for a multi-layered vocal fold prototype as well as two-dimensional, flow-induced vocal fold vibration in a modeled larynx. Several salient features of the aerodynamics as well as vocal-fold dynamics are presented. PMID:19936017
Folding and Stabilization of Native-Sequence-Reversed Proteins

PubMed Central

Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

2016-01-01

Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844
Folding and Stabilization of Native-Sequence-Reversed Proteins

NASA Astrophysics Data System (ADS)

Zhang, Yuanzhao; Weber, Jeffrey K.; Zhou, Ruhong

2016-04-01

Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.
Folding mechanism of β-hairpin trpzip2: heterogeneity, transition state and folding pathways.

PubMed

Xiao, Yi; Chen, Changjun; He, Yi

2009-06-22

We review the studies on the folding mechanism of the beta-hairpin tryptophan zipper 2 (trpzip2) and present some additional computational results to refine the picture of folding heterogeneity and pathways. We show that trpzip2 can have a two-state or a multi-state folding pattern, depending on whether it folds within the native basin or through local state basins on the high-dimensional free energy surface; Trpzip2 can fold along different pathways according to the packing order of tryptophan pairs. We also point out some important problems related to the folding mechanism of trpzip2 that still need clarification, e.g., a wide distribution of the computed conformations for the transition state ensemble.
ALCHEMIC: Advanced time-dependent chemical kinetics

NASA Astrophysics Data System (ADS)

Semenov, Dmitry A.

2017-08-01

ALCHEMIC solves chemical kinetics problems, including gas-grain interactions, surface reactions, deuterium fractionization, and transport phenomena and can model the time-dependent chemical evolution of molecular clouds, hot cores, corinos, and protoplanetary disks.
Linearized-moment analysis of the temperature jump and temperature defect in the Knudsen layer of a rarefied gas.

PubMed

Gu, Xiao-Jun; Emerson, David R

2014-06-01

Understanding the thermal behavior of a rarefied gas remains a fundamental problem. In the present study, we investigate the predictive capabilities of the regularized 13 and 26 moment equations. In this paper, we consider low-speed problems with small gradients, and to simplify the analysis, a linearized set of moment equations is derived to explore a classic temperature problem. Analytical solutions obtained for the linearized 26 moment equations are compared with available kinetic models and can reliably capture all qualitative trends for the temperature-jump coefficient and the associated temperature defect in the thermal Knudsen layer. In contrast, the linearized 13 moment equations lack the necessary physics to capture these effects and consistently underpredict kinetic theory. The deviation from kinetic theory for the 13 moment equations increases significantly for specular reflection of gas molecules, whereas the 26 moment equations compare well with results from kinetic theory. To improve engineering analyses, expressions for the effective thermal conductivity and Prandtl number in the Knudsen layer are derived with the linearized 26 moment equations.
Physics of protein folding

NASA Astrophysics Data System (ADS)

Finkelstein, A. V.; Galzitskaya, O. V.

2004-04-01

Protein physics is grounded on three fundamental experimental facts: protein, this long heteropolymer, has a well defined compact three-dimensional structure; this structure can spontaneously arise from the unfolded protein chain in appropriate environment; and this structure is separated from the unfolded state of the chain by the “all-or-none” phase transition, which ensures robustness of protein structure and therefore of its action. The aim of this review is to consider modern understanding of physical principles of self-organization of protein structures and to overview such important features of this process, as finding out the unique protein structure among zillions alternatives, nucleation of the folding process and metastable folding intermediates. Towards this end we will consider the main experimental facts and simple, mostly phenomenological theoretical models. We will concentrate on relatively small (single-domain) water-soluble globular proteins (whose structure and especially folding are much better studied and understood than those of large or membrane and fibrous proteins) and consider kinetic and structural aspects of transition of initially unfolded protein chains into their final solid (“native”) 3D structures.
Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

PubMed

Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

2016-01-12

We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.
Coarse kMC-based replica exchange algorithms for the accelerated simulation of protein folding in explicit solvent.

PubMed

Peter, Emanuel K; Shea, Joan-Emma; Pivkin, Igor V

2016-05-14

In this paper, we present a coarse replica exchange molecular dynamics (REMD) approach, based on kinetic Monte Carlo (kMC). The new development significantly can reduce the amount of replicas and the computational cost needed to enhance sampling in protein simulations. We introduce 2 different methods which primarily differ in the exchange scheme between the parallel ensembles. We apply this approach on folding of 2 different β-stranded peptides: the C-terminal β-hairpin fragment of GB1 and TrpZip4. Additionally, we use the new simulation technique to study the folding of TrpCage, a small fast folding α-helical peptide. Subsequently, we apply the new methodology on conformation changes in signaling of the light-oxygen voltage (LOV) sensitive domain from Avena sativa (AsLOV2). Our results agree well with data reported in the literature. In simulations of dialanine, we compare the statistical sampling of the 2 techniques with conventional REMD and analyze their performance. The new techniques can reduce the computational cost of REMD significantly and can be used in enhanced sampling simulations of biomolecules.
What amyloidoses may tell us about normal protein folding: The Alzheimer's disease story

NASA Astrophysics Data System (ADS)

Teplow, David B.

2002-03-01

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by severe neuronal injury and death. A prominent histopathologic feature of AD is disseminated parenchymal and vascular amyloid deposition. The fibrils in these deposits are composed of the amyloid β-protein (Aβ), a peptide of 4 kDa mass. In vitro and in vivo studies of Aβ fibril formation have shown that both oligomeric and polymeric Aβ assemblies have neurotoxic activity. Understanding how these assemblies form thus could be of direct therapeutic relevance. However, the aggregation and fibril-forming propensities of Aβ have complicated structure determination. Nevertheless, careful morphologic, spectroscopic, protein chemical, and physiologic analyses of the time-dependent changes in Aβ conformation, assembly state, and biological activity which occur during fibrillogenesis have significantly advanced our understanding of this clinically important process. Here, I will discuss recent findings about the pathway(s) of Aβ folding and assembly and about key structural features of Aβ which control the associated kinetics. Interestingly, the amyloidogenic folding pathway of Aβ is in some respects the mirror image of that through which natively folded amyloidogenic proteins proceed.
LSENS: A General Chemical Kinetics and Sensitivity Analysis Code for homogeneous gas-phase reactions. Part 1: Theory and numerical solution procedures

NASA Technical Reports Server (NTRS)

Radhakrishnan, Krishnan

1994-01-01

LSENS, the Lewis General Chemical Kinetics and Sensitivity Analysis Code, has been developed for solving complex, homogeneous, gas-phase chemical kinetics problems and contains sensitivity analysis for a variety of problems, including nonisothermal situations. This report is part 1 of a series of three reference publications that describe LENS, provide a detailed guide to its usage, and present many example problems. Part 1 derives the governing equations and describes the numerical solution procedures for the types of problems that can be solved. The accuracy and efficiency of LSENS are examined by means of various test problems, and comparisons with other methods and codes are presented. LSENS is a flexible, convenient, accurate, and efficient solver for chemical reaction problems such as static system; steady, one-dimensional, inviscid flow; reaction behind incident shock wave, including boundary layer correction; and perfectly stirred (highly backmixed) reactor. In addition, the chemical equilibrium state can be computed for the following assigned states: temperature and pressure, enthalpy and pressure, temperature and volume, and internal energy and volume. For static problems the code computes the sensitivity coefficients of the dependent variables and their temporal derivatives with respect to the initial values of the dependent variables and/or the three rate coefficient parameters of the chemical reactions.
Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.

PubMed

Kim, Jinsook; Song, Insil; Jo, Ara; Shin, Joo-Ho; Cho, Hana; Eoff, Robert L; Guengerich, F Peter; Choi, Jeong-Yun

2014-10-20

DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The Δ1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1-25 variant, was ∼7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens.
Biochemical Analysis of Six Genetic Variants of Error-Prone Human DNA Polymerase ι Involved in Translesion DNA Synthesis

PubMed Central

2015-01-01

DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N2-ethyl(Et)G, O6-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1–445) proteins and DNA templates containing a G, N2-EtG, O6-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg2+ (but not with Mn2+), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg2+). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg2+ or Mn2+, except for that opposite N2-EtG with Mn2+ (showing a 9-fold increase for dCTP incorporation). The Δ1–25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg2+), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1–25 variant, was ∼7-fold stronger with 0.15 mM Mn2+ than with Mg2+. The results indicate that the R96G variation severely impairs most of the Mg2+- and Mn2+-dependent TLS abilities of pol ι, whereas the Δ1–25 variation selectively and substantially enhances the Mg2+-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens. PMID:25162224
Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein.

PubMed

Chung, Hoi Sung; Gopich, Irina V; McHale, Kevin; Cellmer, Troy; Louis, John M; Eaton, William A

2011-04-28

Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (<10%) population of the minor component where the cross-correlation function was too noisy to obtain any useful information. The rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded molecules are present, this method yields rate coefficients in very good agreement with those obtained with the maximum likelihood method. As a first step toward characterizing transition paths, the Viterbi algorithm was used to locate the most probable transition points in the photon trajectories.
Update of KDBI: Kinetic Data of Bio-molecular Interaction database

PubMed Central

Kumar, Pankaj; Han, B. C.; Shi, Z.; Jia, J.; Wang, Y. P.; Zhang, Y. T.; Liang, L.; Liu, Q. F.; Ji, Z. L.; Chen, Y. Z.

2009-01-01

Knowledge of the kinetics of biomolecular interactions is important for facilitating the study of cellular processes and underlying molecular events, and is essential for quantitative study and simulation of biological systems. Kinetic Data of Bio-molecular Interaction database (KDBI) has been developed to provide information about experimentally determined kinetic data of protein–protein, protein–nucleic acid, protein–ligand, nucleic acid–ligand binding or reaction events described in the literature. To accommodate increasing demand for studying and simulating biological systems, numerous improvements and updates have been made to KDBI, including new ways to access data by pathway and molecule names, data file in System Biology Markup Language format, more efficient search engine, access to published parameter sets of simulation models of 63 pathways, and 2.3-fold increase of data (19 263 entries of 10 532 distinctive biomolecular binding and 11 954 interaction events, involving 2635 proteins/protein complexes, 847 nucleic acids, 1603 small molecules and 45 multi-step processes). KDBI is publically available at http://bidd.nus.edu.sg/group/kdbi/kdbi.asp. PMID:18971255

Kinetics of Photoelectrochemical Oxidation of Methanol on Hematite Photoanodes

PubMed Central

2017-01-01

The kinetics of photoelectrochemical (PEC) oxidation of methanol, as a model organic substrate, on α-Fe2O3 photoanodes are studied using photoinduced absorption spectroscopy and transient photocurrent measurements. Methanol is oxidized on α-Fe2O3 to formaldehyde with near unity Faradaic efficiency. A rate law analysis under quasi-steady-state conditions of PEC methanol oxidation indicates that rate of reaction is second order in the density of surface holes on hematite and independent of the applied potential. Analogous data on anatase TiO2 photoanodes indicate similar second-order kinetics for methanol oxidation with a second-order rate constant 2 orders of magnitude higher than that on α-Fe2O3. Kinetic isotope effect studies determine that the rate constant for methanol oxidation on α-Fe2O3 is retarded ∼20-fold by H/D substitution. Employing these data, we propose a mechanism for methanol oxidation under 1 sun irradiation on these metal oxide surfaces and discuss the implications for the efficient PEC methanol oxidation to formaldehyde and concomitant hydrogen evolution. PMID:28735533
Kinetic and Potential Sputtering of Lunar Regolith: Contribution of Solar-Wind Heavy Ions

NASA Technical Reports Server (NTRS)

Meyer, F. W.; Harris, P. R.; Meyer, H. M., III; Hijiazi, H.; Barghouty, A. F.

2013-01-01

Sputtering of lunar regolith by protons as well as solar-wind heavy ions is considered. From preliminary measurements of H+, Ar+1, Ar+6 and Ar+9 ion sputtering of JSC-1A AGGL lunar regolith simulant at solar wind velocities, and TRIM simulations of kinetic sputtering yields, the relative contributions of kinetic and potential sputtering contributions are estimated. An 80-fold enhancement of oxygen sputtering by Ar+ over same-velocity H+, and an additional x2 increase for Ar+9 over same-velocity Ar+ was measured. This enhancement persisted to the maximum fluences investigated is approximately 1016/cm (exp2). Modeling studies including the enhanced oxygen ejection by potential sputtering due to the minority heavy ion multicharged ion solar wind component, and the kinetic sputtering contribution of all solar wind constituents, as determined from TRIM sputtering simulations, indicate an overall 35% reduction of near-surface oxygen abundance. XPS analyses of simulant samples exposed to singly and multicharged Ar ions show the characteristic signature of reduced (metallic) Fe, consistent with the preferential ejection of oxygen atoms that can occur in potential sputtering of some metal oxides.
Extremely elevated room-temperature kinetic isotope effects quantify the critical role of barrier width in enzymatic C-H activation.

PubMed

Hu, Shenshen; Sharma, Sudhir C; Scouras, Alexander D; Soudackov, Alexander V; Carr, Cody A Marcus; Hammes-Schiffer, Sharon; Alber, Tom; Klinman, Judith P

2014-06-11

The enzyme soybean lipoxygenase (SLO) has served as a prototype for hydrogen-tunneling reactions, as a result of its unusual kinetic isotope effects (KIEs) and their temperature dependencies. Using a synergy of kinetic, structural, and theoretical studies, we show how the interplay between donor-acceptor distance and active-site flexibility leads to catalytic behavior previously predicted by quantum tunneling theory. Modification of the size of two hydrophobic residues by site-specific mutagenesis in SLO reduces the reaction rate 10(4)-fold and is accompanied by an enormous and unprecedented room-temperature KIE. Fitting of the kinetic data to a non-adiabatic model implicates an expansion of the active site that cannot be compensated by donor-acceptor distance sampling. A 1.7 Å resolution X-ray structure of the double mutant further indicates an unaltered backbone conformation, almost identical side-chain conformations, and a significantly enlarged active-site cavity. These findings show the compelling property of room-temperature hydrogen tunneling within a biological context and demonstrate the very high sensitivity of such tunneling to barrier width.
Stabilization of a protein conferred by an increase in folded state entropy.

PubMed

Dagan, Shlomi; Hagai, Tzachi; Gavrilov, Yulian; Kapon, Ruti; Levy, Yaakov; Reich, Ziv

2013-06-25

Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.
Kinetics studies of rapid strain-promoted [3 + 2]-cycloadditions of nitrones with biaryl-aza-cyclooctynone.

PubMed

McKay, Craig S; Chigrinova, Mariya; Blake, Jessie A; Pezacki, John Paul

2012-04-21

Strain-promoted cycloadditions of cyclic nitrones with biaryl-aza-cyclooctynone (BARAC) proceed with rate constants up to 47.3 M(-1) s(-1), this corresponds to a 47-fold rate enhancement relative to reaction of BARAC with benzyl azide and a 14-fold enhancement over previously reported strain promoted alkyne-nitrone cycloadditions (SPANC). Studies of the SPANC reaction using the linear free energy relationship defined by the Hammett equation demonstrated that the cycloaddition reaction has a rho value of 0.25 ± 0.04, indicating that reaction is not sensitive to substituents and thus should have broad applicability. This journal is © The Royal Society of Chemistry 2012
The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization

PubMed Central

Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

2016-01-01

Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell’s capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified. PMID:27812109
Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin.

PubMed

Okochi, Mina; Nomura, Tomoko; Zako, Tamotsu; Arakawa, Takatoshi; Iizuka, Ryo; Ueda, Hiroshi; Funatsu, Takashi; Leroux, Michel; Yohda, Masafumi

2004-07-23

Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N- and C-terminal regions of the prefoldin beta sub-unit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.
Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

PubMed Central

Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

2016-01-01

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768
Blueberry polyphenol oxidase: Characterization and the kinetics of thermal and high pressure activation and inactivation.

PubMed

Terefe, Netsanet Shiferaw; Delon, Antoine; Buckow, Roman; Versteeg, Cornelis

2015-12-01

Partially purified blueberry polyphenol oxidase (PPO) in Mcllvaine buffer (pH=3.6, typical pH of blueberry juice) was subjected to processing at isothermal-isobaric conditions at temperatures from 30 to 80 °C and pressure from 0.1 to 700 MPa. High pressure processing at 30-50 °C at all pressures studied caused irreversible PPO activity increase with a maximum of 6.1 fold increase at 500 MPa and 30 °C. Treatments at mild pressure-mild temperature conditions (0.1-400 MPa, 60 °C) also caused up to 3 fold PPO activity increase. Initial activity increase followed by a decrease occurred at relatively high pressure-mild temperature (400-600 MPa, 60 °C) and mild pressure-high temperature (0.1-400 MPa, 70-80 °C) combinations. At temperatures higher than 76 °C, monotonic decrease in PPO activity occurred at 0.1 MPa and pressures higher than 500 MPa. The activation/inactivation kinetics of the enzyme was successfully modelled assuming consecutive reactions in series with activation followed by inactivation. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.
Exploration of the susceptibility of AChE from the poultry red mite Dermanyssus gallinae (Acari: Mesostigmata) to organophosphates in field isolates from France.

PubMed

Roy, Lise; Chauve, Claude; Delaporte, Jean; Inizan, Gilbert; Buronfosse, Thierry

2009-06-01

The red fowl mite Dermanyssus gallinae (De Geer, 1778) is a hematophagous mite species, which is very commonly found in layer facilities in Europe. The economic and animal health impact of this parasite is quite important. In laying hen houses, organophosphates are almost the only legally usable chemicals. Detecting a target resistance can be useful in order to limit the emergence of resistant populations. The acetylcholinesterase (AChE) activity and the enzyme sensitivity to paraoxon was investigated in 39 field samples and compared to a susceptible reference strain (SSK). Insensitivity factor values (expressed as IC50 ratio) obtained from field isolates compared to SSK revealed some polymorphism but not exceeding a 6-fold difference. The kinetic characteristics of AChE from some field samples showed some difference in KM values for acetylthiocholine and inhibition kinetics performed with diethyl paraoxon exhibited a 5.5-fold difference in the bimolecular rate constant in one field isolate. Taken together, these data suggested that differences in AChE susceptibility to organophosphates may exist in D. gallinae but no resistant population was found.
Symmetry Relations in Chemical Kinetics Arising from Microscopic Reversibility

NASA Astrophysics Data System (ADS)

Adib, Artur B.

2006-01-01

It is shown that the kinetics of time-reversible chemical reactions having the same equilibrium constant but different initial conditions are closely related to one another by a directly measurable symmetry relation analogous to chemical detailed balance. In contrast to detailed balance, however, this relation does not require knowledge of the elementary steps that underlie the reaction, and remains valid in regimes where the concept of rate constants is ill defined, such as at very short times and in the presence of low activation barriers. Numerical simulations of a model of isomerization in solution are provided to illustrate the symmetry under such conditions, and potential applications in protein folding or unfolding are pointed out.
A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories

PubMed Central

2015-01-01

Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351
A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories.

PubMed

Ramanathan, Ravishankar; Muñoz, Victor

2015-06-25

Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics.
A crawling robot driven by multi-stable origami

NASA Astrophysics Data System (ADS)

Pagano, Alexander; Yan, Tongxi; Chien, Brian; Wissa, A.; Tawfick, S.

2017-09-01

Using origami folding to construct and actuate mechanisms and machines offers attractive opportunities from small, scalable, and cheap robots to deployable adaptive structures. This paper presents the design of a bio-inspired origami crawling robot constructed by folding sheets of paper. The origami building block structure is based on the Kresling crease pattern (CP), a chiral tower with a polygonal base, which expands and contracts through coupled longitudinal and rotational motion similar to a screw. We design the origami to have multi-stable structural equilibria which can be tuned by changing the folding CP. Kinematic analysis of these structures based on rigid-plates and hinges at fold lines precludes the shape transformation associated with the bistability of the physical models. To capture the kinematics of the bi-stable origami, the panels’ deformation behavior is modeled utilizing principles of virtual folds. Virtual folds approximate material bending by hinged, rigid panels, which facilitates the development of a kinematic solution via rigid-plate rotation analysis. As such, the kinetics and stability of folded structures are investigated by assigning suitable torsional spring constants to the fold lines. The results presented demonstrate the effect of fold-pattern geometries on the snapping behavior of the bi-stable origami structure based on the Kresling pattern. The crawling robot is presented as a case study for the use of this origami structure to mimic crawling locomotion. The robot is comprised of two origami towers nested inside a paper bellow, and connected by 3D printed end plates. DC motors are used to actuate the expansion and contraction of the internal origami structures to achieve forward locomotion and steering. Beyond locomotion, this simple design can find applications in manipulators, booms, and active structures.
Evidence for the principle of minimal frustration in the evolution of protein folding landscapes.

PubMed

Tzul, Franco O; Vasilchuk, Daniel; Makhatadze, George I

2017-02-28

Theoretical and experimental studies have firmly established that protein folding can be described by a funneled energy landscape. This funneled energy landscape is the result of foldable protein sequences evolving following the principle of minimal frustration, which allows proteins to rapidly fold to their native biologically functional conformations. For a protein family with a given functional fold, the principle of minimal frustration suggests that, independent of sequence, all proteins within this family should fold with similar rates. However, depending on the optimal living temperature of the organism, proteins also need to modulate their thermodynamic stability. Consequently, the difference in thermodynamic stability should be primarily caused by differences in the unfolding rates. To test this hypothesis experimentally, we performed comprehensive thermodynamic and kinetic analyses of 15 different proteins from the thioredoxin family. Eight of these thioredoxins were extant proteins from psychrophilic, mesophilic, or thermophilic organisms. The other seven protein sequences were obtained using ancestral sequence reconstruction and can be dated back over 4 billion years. We found that all studied proteins fold with very similar rates but unfold with rates that differ up to three orders of magnitude. The unfolding rates correlate well with the thermodynamic stability of the proteins. Moreover, proteins that unfold slower are more resistant to proteolysis. These results provide direct experimental support to the principle of minimal frustration hypothesis.
A stoichiometry driven universal spatial organization of backbones of folded proteins: are there Chargaff's rules for protein folding?

PubMed

Mittal, A; Jayaram, B; Shenoy, Sandhya; Bawa, Tejdeep Singh

2010-10-01

Protein folding is at least a six decade old problem, since the times of Pauling and Anfinsen. However, rules of protein folding remain elusive till date. In this work, rigorous analyses of several thousand crystal structures of folded proteins reveal a surprisingly simple unifying principle of backbone organization in protein folding. We find that protein folding is a direct consequence of a narrow band of stoichiometric occurrences of amino-acids in primary sequences, regardless of the size and the fold of a protein. We observe that "preferential interactions" between amino-acids do not drive protein folding, contrary to all prevalent views. We dedicate our discovery to the seminal contribution of Chargaff which was one of the major keys to elucidation of the stoichiometry-driven spatially organized double helical structure of DNA.
Kinetic Studies of Calcium-Induced Calcium Release in Cardiac Sarcoplasmic Reticulum Vesicles

PubMed Central

Sánchez, Gina; Hidalgo, Cecilia; Donoso, Paulina

2003-01-01

Fast Ca2+ release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca2+. Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca2+] and [Mg2+]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa ≤5. These results suggest that channels respond differently when exposed to sudden [Ca2+] changes than when exposed to Ca2+ for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg2+ produced a marked inhibition of release kinetics at pCa 6 (K0.5 = 63 μM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg2+] tested and increased at pCa 6 the K0.5 for Mg2+ inhibition, from 63 μM to 136 μM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca2+ and Mg2+ followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing. PMID:12668440
Effects of Step Length, Age, and Fall History on Hip and Knee Kinetics and Knee Co-contraction during the Maximum Step Length Test

PubMed Central

Schulz, Brian W.; Jongprasithporn, Manutchanok; Hart-Hughes, Stephanie J.; Bulat, Tatjana

2017-01-01

Background Maximum step length is a brief clinical test involving stepping out and back as far as possible with the arms folded across the chest. This test has been shown to predict fall risk, but the biomechanics of this test are not fully understood. Knee and hip kinetics (moments and powers) are greater for longer steps and for younger subjects, but younger subjects also step farther. Methods To separate effects of step length, age, and fall history on joint kinetics; 14 healthy younger, 14 older non-fallers, and 11 older fallers (27(5), 72(5), 75(6) years respectively) all stepped to the same relative target distances of 20-80% of their height. Knee and hip kinetics and knee co-contraction were calculated. Findings Hip and knee kinetics and knee co-contraction all increased with step length, but older non-fallers and fallers utilized greater stepping hip and less stepping knee extensor kinetics. Fallers had greater stepping knee co-contraction than non-fallers. Stance knee co-contraction of non-fallers was similar to young for shorter steps and similar to fallers for longer steps. Interpretation Age had minimal effects and fall history had no effects on joint kinetics of steps to similar distances. Effects of age and fall history on knee co-contraction may contribute to age-related kinetic differences and shorter maximal step lengths of older non-fallers and fallers, but step length correlated with every variable tested. Thus, declines in maximum step length could indicate declines in hip and knee extensor kinetics and impaired performance on similar tasks like recovering from a trip. PMID:23978310
UFO (UnFold Operator) computer program abstract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kissel, L.; Biggs, F.

UFO (UnFold Operator) is an interactive user-oriented computer program designed to solve a wide range of problems commonly encountered in physical measurements. This document provides a summary of the capabilities of version 3A of UFO.
On Riemann solvers and kinetic relations for isothermal two-phase flows with surface tension

NASA Astrophysics Data System (ADS)

Rohde, Christian; Zeiler, Christoph

2018-06-01

We consider a sharp interface approach for the inviscid isothermal dynamics of compressible two-phase flow that accounts for phase transition and surface tension effects. Kinetic relations are frequently used to fix the mass exchange and entropy dissipation rate across the interface. The complete unidirectional dynamics can then be understood by solving generalized two-phase Riemann problems. We present new well-posedness theorems for the Riemann problem and corresponding computable Riemann solvers that cover quite general equations of state, metastable input data and curvature effects. The new Riemann solver is used to validate different kinetic relations on physically relevant problems including a comparison with experimental data. Riemann solvers are building blocks for many numerical schemes that are used to track interfaces in two-phase flow. It is shown that the new Riemann solver enables reliable and efficient computations for physical situations that could not be treated before.

Folding domain B of protein A on a dynamically partitioned free energy landscape.

PubMed

Nelson, Erik D; Grishin, Nick V

2008-02-05

The B domain of staphylococcal protein A (BdpA) is a small helical protein that has been studied intensively in kinetics experiments and detailed computer simulations that include explicit water. The simulations indicate that BdpA needs to reorganize in crossing the transition barrier to facilitate folding its C-terminal helix (H3) onto the nucleus formed from helices H1 and H2. This process suggests frustration between two partially ordered forms of the protein, but recent varphi value measurements indicate that the transition structure is relatively constant over a broad range of temperatures. Here we develop a simplistic model to investigate the folding transition in which properties of the free energy landscape can be quantitatively compared with experimental data. The model is a continuation of the Muñoz-Eaton model to include the intermittency of contacts between structured parts of the protein, and the results compare variations in the landscape with denaturant and temperature to varphi value measurements and chevron plots of the kinetic rates. The topography of the model landscape (in particular, the feature of frustration) is consistent with detailed simulations even though variations in the varphi values are close to measured values. The transition barrier is smaller than indicated by the chevron data, but it agrees in order of magnitude with a similar alpha-carbon type of model. Discrepancies with the chevron plots are investigated from the point of view of solvent effects, and an approach is suggested to account for solvent participation in the model.
Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester.

PubMed

Chen, Ke-Cai; Zheng, Ming-Min; Pan, Jiang; Li, Chun-Xiu; Xu, Jian-He

2017-10-01

The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipA L315S and LipA S271F , were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipA L315S , LipA S271F , and their combination LipA L315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipA S271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipA S271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m . Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.
DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds

PubMed Central

Potapova, Olga; Chan, Chikio; DeLucia, Angela M.; Helquist, Sandra A.; Kool, Eric T.; Grindley, Nigel D. F.; Joyce, Catherine M.

2008-01-01

We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair in order to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA Polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen bonding ability in the nascent pair, the efficiency (kpol/Kd) of the polymerase reaction is decreased by 30-fold, affecting ground state (Kd) and transition state (kpol) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. By contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions, and more dependent on hydrogen bonding between base-paired partners. PMID:16411765
Exploring the origins of topological frustration: Design of a minimally frustrated model of fragment B of protein A

PubMed Central

Shea, Joan-Emma; Onuchic, José N.; Brooks, Charles L.

1999-01-01

Topological frustration in an energetically unfrustrated off-lattice model of the helical protein fragment B of protein A from Staphylococcus aureus was investigated. This Gō-type model exhibited thermodynamic and kinetic signatures of a well-designed two-state folder with concurrent collapse and folding transitions and single exponential kinetics at the transition temperature. Topological frustration is determined in the absence of energetic frustration by the distribution of Fersht φ values. Topologically unfrustrated systems present a unimodal distribution sharply peaked at intermediate φ, whereas highly frustrated systems display a bimodal distribution peaked at low and high φ values. The distribution of φ values in protein A was determined both thermodynamically and kinetically. Both methods yielded a unimodal distribution centered at φ = 0.3 with tails extending to low and high φ values, indicating the presence of a small amount of topological frustration. The contacts with high φ values were located in the turn regions between helices I and II and II and III, intimating that these hairpins are in large part required in the transition state. Our results are in good agreement with all-atom simulations of protein A, as well as lattice simulations of a three- letter code 27-mer (which can be compared with a 60-residue helical protein). The relatively broad unimodal distribution of φ values obtained from the all-atom simulations and that from the minimalist model for the same native fold suggest that the structure of the transition state ensemble is determined mostly by the protein topology and not energetic frustration. PMID:10535953
A second-order accurate kinetic-theory-based method for inviscid compressible flows

NASA Technical Reports Server (NTRS)

Deshpande, Suresh M.

1986-01-01

An upwind method for the numerical solution of the Euler equations is presented. This method, called the kinetic numerical method (KNM), is based on the fact that the Euler equations are moments of the Boltzmann equation of the kinetic theory of gases when the distribution function is Maxwellian. The KNM consists of two phases, the convection phase and the collision phase. The method is unconditionally stable and explicit. It is highly vectorizable and can be easily made total variation diminishing for the distribution function by a suitable choice of the interpolation strategy. The method is applied to a one-dimensional shock-propagation problem and to a two-dimensional shock-reflection problem.
Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι.

PubMed

Choi, Jeong-Yun; Patra, Amritaj; Yeom, Mina; Lee, Young-Sam; Zhang, Qianqian; Egli, Martin; Guengerich, F Peter

2016-09-30

DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn 2+ than Mg 2+ The human germline R96G variant impairs both Mn 2+ -dependent and Mg 2+ -dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg 2+ -dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1-445) proteins. The presence of Mn 2+ (0.15 mm) instead of Mg 2+ (2 mm) caused a 770-fold increase in efficiency (k pol /K d ,dCTP ) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in K d ,dCTP The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in k pol /K d ,dCTP for dCTP insertion opposite G with Mg 2+ when compared with wild type, substantially attenuated by substitution with Mn 2+ Crystal structures of pol ι ternary complexes, including the primer terminus 3'-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg 2+ or Mn 2+ , revealed that Mn 2+ achieves more optimal octahedral coordination geometry than Mg 2+ , with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι*

PubMed Central

Choi, Jeong-Yun; Patra, Amritaj; Yeom, Mina; Lee, Young-Sam; Zhang, Qianqian; Egli, Martin; Guengerich, F. Peter

2016-01-01

DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn2+ than Mg2+. The human germline R96G variant impairs both Mn2+-dependent and Mg2+-dependent activities of pol ι, whereas the Δ1–25 variant selectively enhances its Mg2+-dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1–445) proteins. The presence of Mn2+ (0.15 mm) instead of Mg2+ (2 mm) caused a 770-fold increase in efficiency (kpol/Kd,dCTP) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in Kd,dCTP. The R96G and Δ1–25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in kpol/Kd,dCTP for dCTP insertion opposite G with Mg2+ when compared with wild type, substantially attenuated by substitution with Mn2+. Crystal structures of pol ι ternary complexes, including the primer terminus 3′-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg2+ or Mn2+, revealed that Mn2+ achieves more optimal octahedral coordination geometry than Mg2+, with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation. PMID:27555320
Comparison of the Inhibitory Profiles of Itraconazole and Cimetidine in Cytochrome P450 3A4 Genetic Variants

PubMed Central

Akiyoshi, Takeshi; Saito, Takashi; Murase, Saori; Miyazaki, Mitsue; Murayama, Norie; Yamazaki, Hiroshi; Guengerich, F. Peter; Nakamura, Katsunori; Yamamoto, Koujirou

2011-01-01

CYP3A4, an important drug-metabolizing enzyme, is known to have genetic variants. We have previously reported that CYP3A4 variants such as CYP3A4.2, 7, 16, and 18 show different enzymatic kinetics from CYP3A4.1 (wild type). In this study, we quantitatively investigated the inhibition kinetics of two typical inhibitors, itraconazole (ITCZ) and cimetidine (CMD), on CYP3A4 variants and evaluated whether the genetic variation leads to interindividual differences in the extent of CYP3A4-mediated drug interactions. The inhibitory profiles of ITCZ and CMD on the metabolism of testosterone (TST) were analyzed by using recombinant CYP3A4 variants. The genetic variation of CYP3A4 significantly affected the inhibition profiles of the two inhibitors. In CYP3A4.7, the Ki value for ITCZ was 2.4-fold higher than that for the wild-type enzyme, whereas the Ki value for CMD was 0.64-fold lower. In CYP3A4.16, the Ki value for ITCZ was 0.54-fold lower than that for wild-type CYP3A4, whereas the Ki value for CMD was 3.2-fold higher. The influence of other genetic variations also differed between the two inhibitors. Docking simulations could explain the changes in the Ki values, based on the accessibility of TST and inhibitors to the heme moiety of the CYP3A4 molecule. In conclusion, the inhibitory effects of an inhibitor differ among CYP3A4 variants, suggesting that the genetic variation of CYP3A4 may contribute, at least in part, to interindividual differences in drug interactions mediated by CYP3A4 inhibition, and the pattern of the influences of genetic variation differs among inhibitors as well as substrates. PMID:21212239
The V122I cardiomyopathy variant of transthyretin increases the velocity of rate-limiting tetramer dissociation, resulting in accelerated amyloidosis

PubMed Central

Jiang, Xin; Buxbaum, Joel N.; Kelly, Jeffery W.

2001-01-01

The transthyretin (TTR) amyloid diseases are of keen interest, because there are >80 mutations that cause, and a few mutations that suppress, disease. The V122I variant is the most common amyloidogenic mutation worldwide, producing familial amyloidotic cardiomyopathy primarily in individuals of African descent. The substitution shifts the tetramer-folded monomer equilibrium toward monomer (lowers tetramer stability) and lowers the kinetic barrier associated with rate-limiting tetramer dissociation (pH 7; relative to wild-type TTR) required for amyloid fibril formation. Fibril formation is also accelerated because the folded monomer resulting from the tetramer-folded monomer equilibrium rapidly undergoes partial denaturation and self-assembles into amyloid (in vitro) when subjected to a mild denaturation stress (e.g., pH 4.8). Incorporation of the V122I mutation into a folded monomeric variant of transthyretin reveals that this mutation does not destabilize the tertiary structure or alter the rate of amyloidogenesis relative to the wild-type monomer. The increase in the velocity of rate-limiting tetramer dissociation coupled with the lowered tetramer stability (increasing the mol fraction of folded monomer present at equilibrium) may explain why V122I confers an apparent absolute anatomic risk for cardiac amyloid deposition. PMID:11752443
Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

PubMed Central

Paslawski, Wojciech; Lillelund, Ove K.; Kristensen, Julie Veje; Schafer, Nicholas P.; Baker, Rosanna P.; Urban, Sinisa; Otzen, Daniel E.

2015-01-01

Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2–3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3–6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state–level structure in the transition state. We propose that loops 1–3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated. PMID:26056273
Cisplatin and Oxaliplatin Toxicity: Importance of Cochlear Kinetics as a Determinant for Ototoxicity

PubMed Central

Wallin, Inger; Eriksson, Sofi; Hernlund, Emma; Jerremalm, Elin; Berndtsson, Maria; Eksborg, Staffan; Arnér, Elias S. J.; Shoshan, Maria; Ehrsson, Hans; Laurell, Göran

2009-01-01

Background Cisplatin is a cornerstone anticancer drug with pronounced ototoxicity, whereas oxaliplatin, a platinum derivative with a different clinical profile, is rarely ototoxic. This difference has not been explained. Methods In HCT116 cells, cisplatin (20 μM)-induced apoptosis was reduced by a calcium chelator from 9.9-fold induction (95% confidence interval [CI] = 8.1- to 11.7-fold), to 3.1-fold induction (95% CI = 2.0- to 4.2-fold) and by superoxide scavenging from 9.3-fold (95% CI = 8.8- to 9.8-fold), to 5.1-fold (95% CI = 4.4- to 5.8-fold). A guinea pig model (n = 23) was used to examine pharmacokinetics. Drug concentrations were determined by liquid chromatography with post-column derivatization. The total platinum concentration in cochlear tissue was determined by inductively coupled plasma mass spectrometry. Drug pharmacokinetics was assessed by determining the area under the concentration–time curve (AUC). Statistical tests were two-sided. Results In HCT116 cells, cisplatin (20 μM)-induced apoptosis was reduced by a calcium chelator from 9.9-fold induction (95% confidence interval [CI] = 8.1- to 11.7-fold to 3.1-fold induction) (95% CI = 2.0- to 4.2-fold) and by superoxide scavenging (from 9.3-fold, 95% CI = 8.8- to 9.8-fold, to 5.1-fold, 95% CI = 4.4- to 5.8-fold). Oxaliplatin (20 μM)-induced apoptosis was unaffected by calcium chelation (from 7.1- to 6.2-fold induction) and by superoxide scavenging (from 5.9- to 5.6-fold induction). In guinea pig cochlea, total platinum concentration (0.12 vs 0.63 μg/kg, respectively, P = .008) and perilymphatic drug concentrations (238 vs 515 μM × minute, respectively, P < .001) were lower after intravenous oxaliplatin treatment (16.6 mg/kg) than after equimolar cisplatin treatment (12.5 mg/kg). However, after a non-ototoxic cisplatin dose (5 mg/kg) or the same oxaliplatin dose (16.6 mg/kg), the AUC for perilymphatic concentrations was similar, indicating that the two drugs have different cochlear pharmacokinetics. Conclusion Cisplatin- but not oxaliplatin-induced apoptosis involved superoxide-related pathways. Lower cochlear uptake of oxaliplatin than cisplatin appears to be a major explanation for its lower ototoxicity. PMID:19116379
DOE Office of Scientific and Technical Information (OSTI.GOV)

Alvarez-Ramirez, J.; Aguilar, R.; Lopez-Isunza, F.

FCC processes involve complex interactive dynamics which are difficult to operate and control as well as poorly known reaction kinetics. This work concerns the synthesis of temperature controllers for FCC units. The problem is addressed first for the case where perfect knowledge of the reaction kinetics is assumed, leading to an input-output linearizing state feedback. However, in most industrial FCC units, perfect knowledge of reaction kinetics and composition measurements is not available. To address the problem of robustness against uncertainties in the reaction kinetics, an adaptive model-based nonlinear controller with simplified reaction models is presented. The adaptive strategy makes usemore » of estimates of uncertainties derived from calorimetric (energy) balances. The resulting controller is similar in form to standard input-output linearizing controllers and can be tuned analogously. Alternatively, the controller can be tuned using a single gain parameter and is computationally efficient. The performance of the closed-loop system and the controller design procedure are shown with simulations.« less
Numerical Simulation of Interaction of Human Vocal Folds and Fluid Flow

NASA Astrophysics Data System (ADS)

Kosík, A.; Feistauer, M.; Horáček, J.; Sváček, P.

Our goal is to simulate airflow in human vocal folds and their flow-induced vibrations. We consider two-dimensional viscous incompressible flow in a time-dependent domain. The fluid flow is described by the Navier-Stokes equations in the arbitrary Lagrangian-Eulerian formulation. The flow problem is coupled with the elastic behaviour of the solid bodies. The developed solution of the coupled problem based on the finite element method is demonstrated by numerical experiments.
Integral Design Methodology of Photocatalytic Reactors for Air Pollution Remediation.

PubMed

Passalía, Claudio; Alfano, Orlando M; Brandi, Rodolfo J

2017-06-07

An integral reactor design methodology was developed to address the optimal design of photocatalytic wall reactors to be used in air pollution control. For a target pollutant to be eliminated from an air stream, the proposed methodology is initiated with a mechanistic derived reaction rate. The determination of intrinsic kinetic parameters is associated with the use of a simple geometry laboratory scale reactor, operation under kinetic control and a uniform incident radiation flux, which allows computing the local superficial rate of photon absorption. Thus, a simple model can describe the mass balance and a solution may be obtained. The kinetic parameters may be estimated by the combination of the mathematical model and the experimental results. The validated intrinsic kinetics obtained may be directly used in the scaling-up of any reactor configuration and size. The bench scale reactor may require the use of complex computational software to obtain the fields of velocity, radiation absorption and species concentration. The complete methodology was successfully applied to the elimination of airborne formaldehyde. The kinetic parameters were determined in a flat plate reactor, whilst a bench scale corrugated wall reactor was used to illustrate the scaling-up methodology. In addition, an optimal folding angle of the corrugated reactor was found using computational fluid dynamics tools.
A Rough Energy Landscape to Describe Surface-Linked Antibody and Antigen Bond Formation

NASA Astrophysics Data System (ADS)

Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

2016-10-01

Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2kBT, in the range of previously proposed rough parts of landscapes models during dissociation.
A Rough Energy Landscape to Describe Surface-Linked Antibody and Antigen Bond Formation

PubMed Central

Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

2016-01-01

Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2kBT, in the range of previously proposed rough parts of landscapes models during dissociation. PMID:27731375
A Rough Energy Landscape to Describe Surface-Linked Antibody and Antigen Bond Formation.

PubMed

Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

2016-10-12

Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2k B T, in the range of previously proposed rough parts of landscapes models during dissociation.
N-fold Darboux transformation and double-Wronskian-typed solitonic structures for a variable-coefficient modified Kortweg-de Vries equation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Lei, E-mail: wanglei2239@126.com; Gao, Yi-Tian; State Key Laboratory of Software Development Environment, Beijing University of Aeronautics and Astronautics, Beijing 100191

2012-08-15

Under investigation in this paper is a variable-coefficient modified Kortweg-de Vries (vc-mKdV) model describing certain situations from the fluid mechanics, ocean dynamics and plasma physics. N-fold Darboux transformation (DT) of a variable-coefficient Ablowitz-Kaup-Newell-Segur spectral problem is constructed via a gauge transformation. Multi-solitonic solutions in terms of the double Wronskian for the vc-mKdV model are derived by the reduction of the N-fold DT. Three types of the solitonic interactions are discussed through figures: (1) Overtaking collision; (2) Head-on collision; (3) Parallel solitons. Nonlinear, dispersive and dissipative terms have the effects on the velocities of the solitonic waves while the amplitudes ofmore » the waves depend on the perturbation term. - Highlights: Black-Right-Pointing-Pointer N-fold DT is firstly applied to a vc-AKNS spectral problem. Black-Right-Pointing-Pointer Seeking a double Wronskian solution is changed into solving two systems. Black-Right-Pointing-Pointer Effects of the variable coefficients on the multi-solitonic waves are discussed in detail. Black-Right-Pointing-Pointer This work solves the problem from Yi Zhang [Ann. Phys. 323 (2008) 3059].« less
Statistical mechanics of simple models of protein folding and design.

PubMed Central

Pande, V S; Grosberg, A Y; Tanaka, T

1997-01-01

It is now believed that the primary equilibrium aspects of simple models of protein folding are understood theoretically. However, current theories often resort to rather heavy mathematics to overcome some technical difficulties inherent in the problem or start from a phenomenological model. To this end, we take a new approach in this pedagogical review of the statistical mechanics of protein folding. The benefit of our approach is a drastic mathematical simplification of the theory, without resort to any new approximations or phenomenological prescriptions. Indeed, the results we obtain agree precisely with previous calculations. Because of this simplification, we are able to present here a thorough and self contained treatment of the problem. Topics discussed include the statistical mechanics of the random energy model (REM), tests of the validity of REM as a model for heteropolymer freezing, freezing transition of random sequences, phase diagram of designed ("minimally frustrated") sequences, and the degree to which errors in the interactions employed in simulations of either folding and design can still lead to correct folding behavior. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 6 PMID:9414231
Deterministic folding: The role of entropic forces and steric specificities

NASA Astrophysics Data System (ADS)

da Silva, Roosevelt A.; da Silva, M. A. A.; Caliri, A.

2001-03-01

The inverse folding problem of proteinlike macromolecules is studied by using a lattice Monte Carlo (MC) model in which steric specificities (nearest-neighbors constraints) are included and the hydrophobic effect is treated explicitly by considering interactions between the chain and solvent molecules. Chemical attributes and steric peculiarities of the residues are encoded in a 10-letter alphabet and a correspondent "syntax" is provided in order to write suitable sequences for the specified target structures; twenty-four target configurations, chosen in order to cover all possible values of the average contact order χ (0.2381⩽χ⩽0.4947 for this system), were encoded and analyzed. The results, obtained by MC simulations, are strongly influenced by geometrical properties of the native configuration, namely χ and the relative number φ of crankshafts-type structures: For χ<0.35 the folding is deterministic, that is, the syntax is able to encode successful sequences: The system presents larger encodability, minimum sequence-target degeneracies and smaller characteristic folding time τf. For χ⩾0.35 the above results are not reproduced any more: The folding success is severely reduced, showing strong correlation with φ. Additionally, the existence of distinct characteristic folding times suggests that different mechanisms are acting at the same time in the folding process. The results (all obtained from the same single model, under the same "physiological conditions") resemble some general features of the folding problem, supporting the premise that the steric specificities, in association with the entropic forces (hydrophobic effect), are basic ingredients in the protein folding process.

Dynamic dual-tracer PET reconstruction.

PubMed

Gao, Fei; Liu, Huafeng; Jian, Yiqiang; Shi, Pengcheng

2009-01-01

Although of important medical implications, simultaneous dual-tracer positron emission tomography reconstruction remains a challenging problem, primarily because the photon measurements from dual tracers are overlapped. In this paper, we propose a simultaneous dynamic dual-tracer reconstruction of tissue activity maps based on guidance from tracer kinetics. The dual-tracer reconstruction problem is formulated in a state-space representation, where parallel compartment models serve as continuous-time system equation describing the tracer kinetic processes of dual tracers, and the imaging data is expressed as discrete sampling of the system states in measurement equation. The image reconstruction problem has therefore become a state estimation problem in a continuous-discrete hybrid paradigm, and H infinity filtering is adopted as the estimation strategy. As H infinity filtering makes no assumptions on the system and measurement statistics, robust reconstruction results can be obtained for the dual-tracer PET imaging system where the statistical properties of measurement data and system uncertainty are not available a priori, even when there are disturbances in the kinetic parameters. Experimental results on digital phantoms, Monte Carlo simulations and physical phantoms have demonstrated the superior performance.
Assessment of biological, psychological and adherence factors in the prediction of step-down treatment for patients with well-controlled asthma.

PubMed

Saito, N; Kamata, A; Itoga, M; Tamaki, M; Kayaba, H; Ritz, T

2017-04-01

Inhaled corticosteroids (ICS) and inhaled corticosteroids combined with long-acting beta2-agonist (ICS/LABA) are standard treatments for asthma. However, factors that might help reduce medication in well-controlled asthma are unknown. We classified problems of asthma patients into biological, psychological and adherence factors, and investigated factors associated with the indication and failure of a medication step-down treatment. Two hundred twenty two well-controlled asthma patients receiving ICS or ICS/LABA were assessed for physical and psychiatric problems and followed up for one year from adjustment of their treatment step. Factor B was defined as a presence of chronic upper airway complications. Factor P was defined as presence of psychiatric complications such as sleep disorder, depression, anxiety and somatoform disorders. Factor A was defined as poor adherence to ICS or ICS/LABA inhaler of 75% or less. Success in step-down treatment was defined as maintenance of well-controlled status for over one year after step-down. Factor B was the most important single negative predictive factor for indication for step-down treatment (Odds ratio; 0.19). Factor A increased the risk of failure to maintain step-down treatment most significantly by 23-fold, and factor B increased it by 11-fold. The combination of factors B and A increased failure by 24-fold, factors P and A by 21-fold, all three factors by 36-fold. Factor P only interacted with the other factors to reduce chances of stepping down, but did not constitute a problem factor when present alone. The evaluation of biological, psychological and adherence problems may lead to a more proactive and targeted approach to step-down treatment for patients with well-controlled asthma. © 2017 John Wiley & Sons Ltd.
A third-order gas-kinetic CPR method for the Euler and Navier-Stokes equations on triangular meshes

NASA Astrophysics Data System (ADS)

Zhang, Chao; Li, Qibing; Fu, Song; Wang, Z. J.

2018-06-01

A third-order accurate gas-kinetic scheme based on the correction procedure via reconstruction (CPR) framework is developed for the Euler and Navier-Stokes equations on triangular meshes. The scheme combines the accuracy and efficiency of the CPR formulation with the multidimensional characteristics and robustness of the gas-kinetic flux solver. Comparing with high-order finite volume gas-kinetic methods, the current scheme is more compact and efficient by avoiding wide stencils on unstructured meshes. Unlike the traditional CPR method where the inviscid and viscous terms are treated differently, the inviscid and viscous fluxes in the current scheme are coupled and computed uniformly through the kinetic evolution model. In addition, the present scheme adopts a fully coupled spatial and temporal gas distribution function for the flux evaluation, achieving high-order accuracy in both space and time within a single step. Numerical tests with a wide range of flow problems, from nearly incompressible to supersonic flows with strong shocks, for both inviscid and viscous problems, demonstrate the high accuracy and efficiency of the present scheme.
Kinetic effects in thermal explosion with oscillating ambient conditions.

PubMed

Novozhilov, Vasily

2018-03-05

Thermal explosion problem for a medium with oscillating ambient temperature at its boundaries is a new problem which was introduced in the preceding publication by the present author. It is directly applicable to a range of practical fire autoignition scenarios (e.g. in the storages of organic matter, explosives, propellants, etc.). Effects of kinetic mechanisms, however, need be further investigated as they are expected to alter critical conditions of thermal explosion. We consider several global kinetic mechanisms: first order reaction, second order reaction, and first order autocatalysis. It is demonstrated that kinetic effects related to reactants consumption do indeed shift respective critical boundaries. Effect of kinetics on oscillatory development of thermal explosion is of particular interest. In line with conclusions of the preceding publication, it is confirmed that temperature oscillations may develop during induction phase of thermal explosion when the effect of reactants consumption is properly taken into account. Moreover, development of thermal explosion instability through the prior oscillations is an inevitable and natural scenario. This fact is confirmed by a number of examples. Besides, effects of the other relevant parameter, Zeldovich number on critical conditions are also investigated.
Physical and numerical sources of computational inefficiency in integration of chemical kinetic rate equations: Etiology, treatment and prognosis

NASA Technical Reports Server (NTRS)

Pratt, D. T.; Radhakrishnan, K.

1986-01-01

The design of a very fast, automatic black-box code for homogeneous, gas-phase chemical kinetics problems requires an understanding of the physical and numerical sources of computational inefficiency. Some major sources reviewed in this report are stiffness of the governing ordinary differential equations (ODE's) and its detection, choice of appropriate method (i.e., integration algorithm plus step-size control strategy), nonphysical initial conditions, and too frequent evaluation of thermochemical and kinetic properties. Specific techniques are recommended (and some advised against) for improving or overcoming the identified problem areas. It is argued that, because reactive species increase exponentially with time during induction, and all species exhibit asymptotic, exponential decay with time during equilibration, exponential-fitted integration algorithms are inherently more accurate for kinetics modeling than classical, polynomial-interpolant methods for the same computational work. But current codes using the exponential-fitted method lack the sophisticated stepsize-control logic of existing black-box ODE solver codes, such as EPISODE and LSODE. The ultimate chemical kinetics code does not exist yet, but the general characteristics of such a code are becoming apparent.
Broad-Bandwidth Chiral Sum Frequency Generation Spectroscopy for Probing the Kinetics of Proteins at Interfaces

PubMed Central

2016-01-01

The kinetics of proteins at interfaces plays an important role in biological functions and inspires solutions to fundamental problems in biomedical sciences and engineering. Nonetheless, due to the lack of surface-specific and structural-sensitive biophysical techniques, it still remains challenging to probe protein kinetics in situ and in real time without the use of spectroscopic labels at interfaces. Broad-bandwidth chiral sum frequency generation (SFG) spectroscopy has been recently developed for protein kinetic studies at interfaces by tracking the chiral vibrational signals of proteins. In this article, we review our recent progress in kinetic studies of proteins at interfaces using broad-bandwidth chiral SFG spectroscopy. We illustrate the use of chiral SFG signals of protein side chains in the C–H stretch region to monitor self-assembly processes of proteins at interfaces. We also present the use of chiral SFG signals from the protein backbone in the N–H stretch region to probe the real-time kinetics of proton exchange between protein and water at interfaces. In addition, we demonstrate the applications of spectral features of chiral SFG that are typical of protein secondary structures in both the amide I and the N–H stretch regions for monitoring the kinetics of aggregation of amyloid proteins at membrane surfaces. These studies exhibit the power of broad-bandwidth chiral SFG to study protein kinetics at interfaces and the promise of this technique in research areas of surface science to address fundamental problems in biomedical and material sciences. PMID:26196215
Non-exponential kinetics of unfolding under a constant force.

PubMed

Bell, Samuel; Terentjev, Eugene M

2016-11-14

We examine the population dynamics of naturally folded globular polymers, with a super-hydrophobic "core" inserted at a prescribed point in the polymer chain, unfolding under an application of external force, as in AFM force-clamp spectroscopy. This acts as a crude model for a large class of folded biomolecules with hydrophobic or hydrogen-bonded cores. We find that the introduction of super-hydrophobic units leads to a stochastic variation in the unfolding rate, even when the positions of the added monomers are fixed. This leads to the average non-exponential population dynamics, which is consistent with a variety of experimental data and does not require any intrinsic quenched disorder that was traditionally thought to be at the origin of non-exponential relaxation laws.
Non-exponential kinetics of unfolding under a constant force

NASA Astrophysics Data System (ADS)

Bell, Samuel; Terentjev, Eugene M.

2016-11-01

We examine the population dynamics of naturally folded globular polymers, with a super-hydrophobic "core" inserted at a prescribed point in the polymer chain, unfolding under an application of external force, as in AFM force-clamp spectroscopy. This acts as a crude model for a large class of folded biomolecules with hydrophobic or hydrogen-bonded cores. We find that the introduction of super-hydrophobic units leads to a stochastic variation in the unfolding rate, even when the positions of the added monomers are fixed. This leads to the average non-exponential population dynamics, which is consistent with a variety of experimental data and does not require any intrinsic quenched disorder that was traditionally thought to be at the origin of non-exponential relaxation laws.
Alpha-glucosidase folding during urea denaturation: enzyme kinetics and computational prediction.

PubMed

Wu, Xue-Qiang; Wang, Jun; Lü, Zhi-Rong; Tang, Hong-Min; Park, Daeui; Oh, Sang-Ho; Bhak, Jong; Shi, Long; Park, Yong-Doo; Zou, Fei

2010-03-01

In this study, we investigated structural changes in alpha-glucosidase during urea denaturation. Alpha-glucosidase was inactivated by urea in a dose-dependent manner. The inactivation was a first-order reaction with a monophase process. Urea inhibited alpha-glucosidase in a mixed-type reaction. We found that an increase in the hydrophobic surface of this enzyme induced by urea resulted in aggregation caused by unstable folding intermediates. We also simulated the docking between alpha-glucosidase and urea. The docking simulation suggested that several residues, namely THR9, TRP14, LYS15, THR287, ALA289, ASP338, SER339, and TRP340, interact with urea. Our study provides insights into the alpha-glucosidase unfolding pathway and 3D structure of alpha-glucosidase.
The Role of High-Dimensional Diffusive Search, Stabilization, and Frustration in Protein Folding

PubMed Central

Rimratchada, Supreecha; McLeish, Tom C.B.; Radford, Sheena E.; Paci, Emanuele

2014-01-01

Proteins are polymeric molecules with many degrees of conformational freedom whose internal energetic interactions are typically screened to small distances. Therefore, in the high-dimensional conformation space of a protein, the energy landscape is locally relatively flat, in contrast to low-dimensional representations, where, because of the induced entropic contribution to the full free energy, it appears funnel-like. Proteins explore the conformation space by searching these flat subspaces to find a narrow energetic alley that we call a hypergutter and then explore the next, lower-dimensional, subspace. Such a framework provides an effective representation of the energy landscape and folding kinetics that does justice to the essential characteristic of high-dimensionality of the search-space. It also illuminates the important role of nonnative interactions in defining folding pathways. This principle is here illustrated using a coarse-grained model of a family of three-helix bundle proteins whose conformations, once secondary structure has formed, can be defined by six rotational degrees of freedom. Two folding mechanisms are possible, one of which involves an intermediate. The stabilization of intermediate subspaces (or states in low-dimensional projection) in protein folding can either speed up or slow down the folding rate depending on the amount of native and nonnative contacts made in those subspaces. The folding rate increases due to reduced-dimension pathways arising from the mere presence of intermediate states, but decreases if the contacts in the intermediate are very stable and introduce sizeable topological or energetic frustration that needs to be overcome. Remarkably, the hypergutter framework, although depending on just a few physically meaningful parameters, can reproduce all the types of experimentally observed curvature in chevron plots for realizations of this fold. PMID:24739172
Insights into Stability and Folding of GNRA and UNCG Tetraloops Revealed by Microsecond Molecular Dynamics and Well-Tempered Metadynamics.

PubMed

Haldar, Susanta; Kührová, Petra; Banáš, Pavel; Spiwok, Vojtěch; Šponer, Jiří; Hobza, Pavel; Otyepka, Michal

2015-08-11

RNA hairpins capped by 5'-GNRA-3' or 5'-UNCG-3' tetraloops (TLs) are prominent RNA structural motifs. Despite their small size, a wealth of experimental data, and recent progress in theoretical simulations of their structural dynamics and folding, our understanding of the folding and unfolding processes of these small RNA elements is still limited. Theoretical description of the folding and unfolding processes requires robust sampling, which can be achieved by either an exhaustive time scale in standard molecular dynamics simulations or sophisticated enhanced sampling methods, using temperature acceleration or biasing potentials. Here, we study structural dynamics of 5'-GNRA-3' and 5'-UNCG-3' TLs by 15-μs-long standard simulations and a series of well-tempered metadynamics, attempting to accelerate sampling by bias in a few chosen collective variables (CVs). Both methods provide useful insights. The unfolding and refolding mechanisms of the GNRA TL observed by well-tempered metadynamics agree with the (reverse) folding mechanism suggested by recent replica exchange molecular dynamics simulations. The orientation of the glycosidic bond of the GL4 nucleobase is critical for the UUCG TL folding pathway, and our data strongly support the hypothesis that GL4-anti forms a kinetic trap along the folding pathway. Along with giving useful insight, our study also demonstrates that using only a few CVs apparently does not capture the full folding landscape of the RNA TLs. Despite using several sophisticated selections of the CVs, formation of the loop appears to remain a hidden variable, preventing a full convergence of the metadynamics. Finally, our data suggest that the unfolded state might be overstabilized by the force fields used.
Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA*

PubMed Central

Greenfeld, Max; Solomatin, Sergey V.; Herschlag, Daniel

2011-01-01

RNA folding landscapes have been described alternately as simple and as complex. The limited diversity of RNA residues and the ability of RNA to form stable secondary structures prior to adoption of a tertiary structure would appear to simplify folding relative to proteins. Nevertheless, there is considerable evidence for long-lived misfolded RNA states, and these observations have suggested rugged energy landscapes. Recently, single molecule fluorescence resonance energy transfer (smFRET) studies have exposed heterogeneity in many RNAs, consistent with deeply furrowed rugged landscapes. We turned to an RNA of intermediate complexity, the P4-P6 domain from the Tetrahymena group I intron, to address basic questions in RNA folding. P4-P6 exhibited long-lived heterogeneity in smFRET experiments, but the inability to observe exchange in the behavior of individual molecules led us to probe whether there was a non-conformational origin to this heterogeneity. We determined that routine protocols in RNA preparation and purification, including UV shadowing and heat annealing, cause covalent modifications that alter folding behavior. By taking measures to avoid these treatments and by purifying away damaged P4-P6 molecules, we obtained a population of P4-P6 that gave near-uniform behavior in single molecule studies. Thus, the folding landscape of P4-P6 lacks multiple deep furrows that would trap different P4-P6 molecules in different conformations and contrasts with the molecular heterogeneity that has been seen in many smFRET studies of structured RNAs. The simplicity of P4-P6 allowed us to reliably determine the thermodynamic and kinetic effects of metal ions on folding and to now begin to build more detailed models for RNA folding behavior. PMID:21478155
Topology-based modeling of intrinsically disordered proteins: balancing intrinsic folding and intermolecular interactions.

PubMed

Ganguly, Debabani; Chen, Jianhan

2011-04-01

Coupled binding and folding is frequently involved in specific recognition of so-called intrinsically disordered proteins (IDPs), a newly recognized class of proteins that rely on a lack of stable tertiary fold for function. Here, we exploit topology-based Gō-like modeling as an effective tool for the mechanism of IDP recognition within the theoretical framework of minimally frustrated energy landscape. Importantly, substantial differences exist between IDPs and globular proteins in both amino acid sequence and binding interface characteristics. We demonstrate that established Gō-like models designed for folded proteins tend to over-estimate the level of residual structures in unbound IDPs, whereas under-estimating the strength of intermolecular interactions. Such systematic biases have important consequences in the predicted mechanism of interaction. A strategy is proposed to recalibrate topology-derived models to balance intrinsic folding propensities and intermolecular interactions, based on experimental knowledge of the overall residual structure level and binding affinity. Applied to pKID/KIX, the calibrated Gō-like model predicts a dominant multistep sequential pathway for binding-induced folding of pKID that is initiated by KIX binding via the C-terminus in disordered conformations, followed by binding and folding of the rest of C-terminal helix and finally the N-terminal helix. This novel mechanism is consistent with key observations derived from a recent NMR titration and relaxation dispersion study and provides a molecular-level interpretation of kinetic rates derived from dispersion curve analysis. These case studies provide important insight into the applicability and potential pitfalls of topology-based modeling for studying IDP folding and interaction in general. Copyright © 2011 Wiley-Liss, Inc.
Polymer Uncrossing and Knotting in Protein Folding, and Their Role in Minimal Folding Pathways

PubMed Central

Mohazab, Ali R.; Plotkin, Steven S.

2013-01-01

We introduce a method for calculating the extent to which chain non-crossing is important in the most efficient, optimal trajectories or pathways for a protein to fold. This involves recording all unphysical crossing events of a ghost chain, and calculating the minimal uncrossing cost that would have been required to avoid such events. A depth-first tree search algorithm is applied to find minimal transformations to fold , , , and knotted proteins. In all cases, the extra uncrossing/non-crossing distance is a small fraction of the total distance travelled by a ghost chain. Different structural classes may be distinguished by the amount of extra uncrossing distance, and the effectiveness of such discrimination is compared with other order parameters. It was seen that non-crossing distance over chain length provided the best discrimination between structural and kinetic classes. The scaling of non-crossing distance with chain length implies an inevitable crossover to entanglement-dominated folding mechanisms for sufficiently long chains. We further quantify the minimal folding pathways by collecting the sequence of uncrossing moves, which generally involve leg, loop, and elbow-like uncrossing moves, and rendering the collection of these moves over the unfolded ensemble as a multiple-transformation “alignment”. The consensus minimal pathway is constructed and shown schematically for representative cases of an , , and knotted protein. An overlap parameter is defined between pathways; we find that proteins have minimal overlap indicating diverse folding pathways, knotted proteins are highly constrained to follow a dominant pathway, and proteins are somewhere in between. Thus we have shown how topological chain constraints can induce dominant pathway mechanisms in protein folding. PMID:23365638
From Flapping Birds to Space Telescopes: The Modern Science of Origami (BNL Women in Science Lecture)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lang, Robert J

2010-06-24

During the 1990s, the development and application of mathematical techniques to origami revolutionized this centuries-old Japanese art of paper folding. In his talk, Lang will describe how geometric concepts led to the solution of a broad class of origami-folding problems. Conversely, algorithms and theorems of origami design have shed light on long-standing mathematical questions and have solved practical engineering problems. Lang will discuss how origami has led to huge space telescopes, safer airbags, and more.
Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

PubMed Central

Amyot, Whitney M.; deJesus, Dennise

2013-01-01

Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system. PMID:23798536
Reversible thermal denaturation of a 60-kDa genetically engineered beta-sheet polypeptide.

PubMed

Lednev, Igor K; Ermolenkov, Vladimir V; Higashiya, Seiichiro; Popova, Ludmila A; Topilina, Natalya I; Welch, John T

2006-11-15

A de novo 687-amino-acid residue polypeptide with a regular 32-amino-acid repeat sequence, (GA)(3)GY(GA)(3)GE(GA)(3)GH(GA)(3)GK, forms large beta-sheet assemblages that exhibit remarkable folding properties and, as well, form fibrillar structures. This construct is an excellent tool to explore the details of beta-sheet formation yielding intimate folding information that is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel beta-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125 degrees C, adopting a predominant polyproline II conformation. Deep ultraviolet Raman spectroscopy indicated that melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state, as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of approximately 1 and approximately 60 min, respectively, indicating no detectable formation of hairpin-type nuclei in the millisecond timescale that could be attributed to nonlocal "nonnative" interactions. The polypeptide folding dynamics agree with a general property of beta-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less frustrated free-energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains.
A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction.

PubMed

Kopra, Kari; Ligabue, Alessio; Wang, Qi; Syrjänpää, Markku; Blaževitš, Olga; Veltel, Stefan; van Adrichem, Arjan J; Hänninen, Pekka; Abankwa, Daniel; Härmä, Harri

2014-07-01

A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.
The energy landscape of adenylate kinase during catalysis

DOE PAGES

Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; ...

2015-01-12

Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. In this paper, we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg 2+ cofactor activates two distinct molecular events: phosphoryl transfer (>10 5-fold) and lid opening (10 3-fold). In contrast, mutation of an essential active site arginine decelerates phosphorylmore » transfer 10 3-fold without substantially affecting lid opening. Finally, our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.« less
Conformational rigidity in a lattice model of proteins.

PubMed

Collet, Olivier

2003-06-01

It is shown in this paper that some simulations of protein folding in lattice models, which use an incorrect implementation of the Monte Carlo algorithm, do not converge towards thermal equilibrium. I developed a rigorous treatment for protein folding simulation on a lattice model relying on the introduction of a parameter standing for the rigidity of the conformations. Its properties are discussed and its role during the folding process is elucidated. The calculation of thermal properties of small chains living on a two-dimensional lattice is performed and a Bortz-Kalos-Lebowitz scheme is implemented in the presented method in order to study kinetics of chains at very low temperature. The coefficients of the Arrhenius law obtained with this algorithm are found to be in excellent agreement with the value of the main potential barrier of the system. Finally, a scenario of the mechanisms, including the rigidity parameters, that guide a protein towards its native structure, at medium temperature, is given.

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