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

Predicting RNA folding thermodynamics with a reduced chain representation model

PubMed Central

CAO, SONG; CHEN, SHI-JIE

2005-01-01

Based on the virtual bond representation for the nucleotide backbone, we develop a reduced conformational model for RNA. We use the experimentally measured atomic coordinates to model the helices and use the self-avoiding walks in a diamond lattice to model the loop conformations. The atomic coordinates of the helices and the lattice representation for the loops are matched at the loop–helix junction, where steric viability is accounted for. Unlike the previous simplified lattice-based models, the present virtual bond model can account for the atomic details of realistic three-dimensional RNA structures. Based on the model, we develop a statistical mechanical theory for RNA folding energy landscapes and folding thermodynamics. Tests against experiments show that the theory can give much more improved predictions for the native structures, the thermal denaturation curves, and the equilibrium folding/unfolding pathways than the previous models. The application of the model to the P5abc region of Tetrahymena group I ribozyme reveals the misfolded intermediates as well as the native-like intermediates in the equilibrium folding process. Moreover, based on the free energy landscape analysis for each and every loop mutation, the model predicts five lethal mutations that can completely alter the free energy landscape and the folding stability of the molecule. PMID:16251382

Robustness of atomistic Gō models in predicting native-like folding intermediates

NASA Astrophysics Data System (ADS)

Estácio, S. G.; Fernandes, C. S.; Krobath, H.; Faísca, P. F. N.; Shakhnovich, E. I.

2012-08-01

Gō models are exceedingly popular tools in computer simulations of protein folding. These models are native-centric, i.e., they are directly constructed from the protein's native structure. Therefore, it is important to understand up to which extent the atomistic details of the native structure dictate the folding behavior exhibited by Gō models. Here we address this challenge by performing exhaustive discrete molecular dynamics simulations of a Gō potential combined with a full atomistic protein representation. In particular, we investigate the robustness of this particular type of Gō models in predicting the existence of intermediate states in protein folding. We focus on the N47G mutational form of the Spc-SH3 folding domain (x-ray structure) and compare its folding pathway with that of alternative native structures produced in silico. Our methodological strategy comprises equilibrium folding simulations, structural clustering, and principal component analysis.

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

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.

Predicting RNA pseudoknot folding thermodynamics

PubMed Central

Cao, Song; Chen, Shi-Jie

2006-01-01

Based on the experimentally determined atomic coordinates for RNA helices and the self-avoiding walks of the P (phosphate) and C4 (carbon) atoms in the diamond lattice for the polynucleotide loop conformations, we derive a set of conformational entropy parameters for RNA pseudoknots. Based on the entropy parameters, we develop a folding thermodynamics model that enables us to compute the sequence-specific RNA pseudoknot folding free energy landscape and thermodynamics. The model is validated through extensive experimental tests both for the native structures and for the folding thermodynamics. The model predicts strong sequence-dependent helix-loop competitions in the pseudoknot stability and the resultant conformational switches between different hairpin and pseudoknot structures. For instance, for the pseudoknot domain of human telomerase RNA, a native-like and a misfolded hairpin intermediates are found to coexist on the (equilibrium) folding pathways, and the interplay between the stabilities of these intermediates causes the conformational switch that may underlie a human telomerase disease. PMID:16709732

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

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate.

PubMed

Thomas, Sabrina; Bakkes, Patrick J; Smits, Sander H J; Schmitt, Lutz

2014-09-01

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca(2+) binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca(2+) is essential for stability as well as correct folding of the whole protein. In the absence of Ca(2+), pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca(2+) addition, indicating that these are not dead-end species and that Ca(2+) drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6-7M urea, relates to Ca(2+) dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca(2+) induced folding is not restricted to the RTX domain. Taken together, Ca(2+) binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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

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

A thermodynamic definition of protein domains.

PubMed

Porter, Lauren L; Rose, George D

2012-06-12

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.

Equilibrium Ensembles for Insulin Folding from Bias-Exchange Metadynamics.

PubMed

Singh, Richa; Bansal, Rohit; Rathore, Anurag Singh; Goel, Gaurav

2017-04-25

Earliest events in the aggregation process, such as single molecule reconfiguration, are extremely important and the most difficult to characterize in experiments. To this end, we have used well-tempered bias exchange metadynamics simulations to determine the equilibrium ensembles of an insulin molecule under amyloidogenic conditions of low pH and high temperature. A bin-based clustering method that uses statistics accumulated in bias exchange metadynamics trajectories was employed to construct a detailed thermodynamic and kinetic model of insulin folding. The highest lifetime, lowest free-energy ensemble identified consisted of native conformations adopted by a folded insulin monomer in solution, namely, the R-, the R f -, and the T-states of insulin. The lowest free-energy structure had a root mean square deviation of only 0.15 nm from native x-ray structure. The second longest-lived metastable state was an unfolded, compact monomer with little similarity to the native structure. We have identified three additional long-lived, metastable states from the bin-based model. We then carried out an exhaustive structural characterization of metastable states on the basis of tertiary contact maps and per-residue accessible surface areas. We have also determined the lowest free-energy path between two longest-lived metastable states and confirm earlier findings of non-two-state folding for insulin through a folding intermediate. The ensemble containing the monomeric intermediate retained 58% of native hydrophobic contacts, however, accompanied by a complete loss of native secondary structure. We have discussed the relative importance of nativelike versus nonnative tertiary contacts for the folding transition. We also provide a simple measure to determine the importance of an individual residue for folding transition. Finally, we have compared and contrasted this intermediate with experimental data obtained in spectroscopic, crystallographic, and calorimetric measurements during early stages of insulin aggregation. We have also determined stability of monomeric insulin by incubation at a very low concentration to isolate protein-protein interaction effects. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Determination of an ensemble of structures representing the intermediate state of the bacterial immunity protein Im7.

PubMed

Gsponer, Joerg; Hopearuoho, Harri; Whittaker, Sara B-M; Spence, Graham R; Moore, Geoffrey R; Paci, Emanuele; Radford, Sheena E; Vendruscolo, Michele

2006-01-03

We present a detailed structural characterization of the intermediate state populated during the folding and unfolding of the bacterial immunity protein Im7. We achieve this result by incorporating a variety of experimental data available for this species in molecular dynamics simulations. First, we define the structure of the exchange-competent intermediate state of Im7 by using equilibrium hydrogen-exchange protection factors. Second, we use this ensemble to predict Phi-values and compare the results with the experimentally determined Phi-values of the kinetic refolding intermediate. Third, we predict chemical-shift measurements and compare them with the measured chemical shifts of a mutational variant of Im7 for which the kinetic folding intermediate is the most stable state populated at equilibrium. Remarkably, we found that the properties of the latter two species are predicted with high accuracy from the exchange-competent intermediate that we determined, suggesting that these three states are characterized by a similar architecture in which helices I, II, and IV are aligned in a native-like, but reorganized, manner. Furthermore, the structural ensemble that we obtained enabled us to rationalize the results of tryptophan fluorescence experiments in the WT protein and a series of mutational variants. The results show that the integration of diverse sets of experimental data at relatively low structural resolution is a powerful approach that can provide insights into the structural organization of this conformationally heterogeneous three-helix intermediate with unprecedented detail and highlight the importance of both native and non-native interactions in stabilizing its structure.

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.

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

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

PubMed

Fossat, Martin J; Dao, Thuy P; Jenkins, Kelly; Dellarole, Mariano; Yang, Yinshan; McCallum, Scott A; Garcia, Angel E; Barrick, Doug; Roumestand, Christian; Royer, Catherine A

2016-12-06

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1 H- 15 N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway

PubMed Central

Dyson, H. Jane; Wright, Peter E.

2017-01-01

Conspectus Although each type of protein fold and in some cases individual proteins within a fold classification can have very different mechanisms of folding, the underlying biophysical and biochemical principles that operate to cause a linear polypeptide chain to fold into a globular structure must be the same. In an aqueous solution, the protein takes up the thermodynamically most stable structure, but the pathway along which the polypeptide proceeds in order to reach that structure is a function of the amino acid sequence, which must be the final determining factor, not only in shaping the final folded structure, but in dictating the folding pathway. A number of groups have focused on a single protein or group of proteins, to determine in detail the factors that influence the rate and mechanism of folding in a defined system, with the hope that hypothesis-driven experiments can elucidate the underlying principles governing the folding process. Our research group has focused on the folding of the globin family of proteins, and in particular on the monomeric protein apomyoglobin. Apomyoglobin (apoMb) folds relatively slowly (~2 seconds) via an ensemble of obligatory intermediates that form rapidly after the initiation of folding. The folding pathway can be dissected using rapid-mixing techniques, which can probe processes in the millisecond time range. Stopped-flow measurements detected by circular dichroism (CD) or fluorescence spectroscopy give information on the rates of folding events. Quench-flow experiments utilize the differential rates of hydrogen-deuterium exchange of amide protons protected in parts of the structure that are folded early; protection of amides can be detected by mass spectrometry or proton nuclear magnetic resonance spectroscopy (NMR). In addition, apoMb forms an intermediate at equilibrium at pH ~ 4, which is sufficiently stable for it to be structurally characterized by solution methods such as CD, fluorescence and NMR spectroscopies, and the conformational ensembles formed in the presence of denaturing agents and low pH can be characterized as models for the unfolded states of the protein. Newer NMR techniques such as measurement of residual dipolar couplings in the various partly folded states, and relaxation dispersion measurements to probe invisible states present at low concentrations, have contributed to providing a detailed picture of the apomyoglobin folding pathway. The research summarized in this review was aimed at characterizing and comparing the equilibrium and kinetic intermediates both structurally and dynamically, as well as delineating the complete folding pathway at a residue-specific level, in order to answer the question “What is it about the amino acid sequence that causes each molecule in the unfolded protein ensemble to start folding, and, once started, to proceed towards the formation of the correctly folded three-dimensional structure?” PMID:28032989

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

Mathematical modeling of HIV-like particle assembly in vitro.

PubMed

Liu, Yuewu; Zou, Xiufen

2017-06-01

In vitro, the recombinant HIV-1 Gag protein can generate spherical particles with a diameter of 25-30 nm in a fully defined system. It has approximately 80 building blocks, and its intermediates for assembly are abundant in geometry. Accordingly, there are a large number of nonlinear equations in the classical model. Therefore, it is difficult to compute values of geometry parameters for intermediates and make the mathematical analysis using the model. In this work, we develop a new model of HIV-like particle assembly in vitro by using six-fold symmetry of HIV-like particle assembly to decrease the number of geometry parameters. This method will greatly reduce computational costs and facilitate the application of the model. Then, we prove the existence and uniqueness of the positive equilibrium solution for this model with 79 nonlinear equations. Based on this model, we derive the interesting result that concentrations of all intermediates at equilibrium are independent of three important parameters, including two microscopic on-rate constants and the size of nucleating structure. Before equilibrium, these three parameters influence the concentration variation rates of all intermediates. We also analyze the relationship between the initial concentration of building blocks and concentrations of all intermediates. Furthermore, the bounds of concentrations of free building blocks and HIV-like particles are estimated. These results will be helpful to guide HIV-like particle assembly experiments and improve our understanding of the assembly dynamics of HIV-like particles in vitro. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural basis of urea-induced unfolding: Unraveling the folding pathway of hemochromatosis factor E.

PubMed

Khan, Parvez; Prakash, Amresh; Haque, Md Anzarul; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

2016-10-01

Hereditary hemochromatosis factor E (HFE) is a type 1 transmembrane protein, and acts as a negative regulator of iron-uptake. The equilibrium unfolding and conformational stability of the HFE protein was examined in the presence of urea. The folding and unfolding transitions were monitored with the help of circular dichroism (CD), intrinsic fluorescence and absorption spectroscopy. Analysis of transition curves revealed that the folding of HFE is not a two-state process. However, it involved stable intermediates. Transition curves (plot of fluorescence (F346) and CD signal at 222nm (θ222) versus [Urea], the molar urea concentration) revealed a biphasic transition with midpoint (Cm) values at 2.88M and 4.95M urea. Whereas, absorption analysis shows one two-state transition centered at 2.96M. To estimate the protein stability, denaturation curves were analyzed for Gibbs free energy change in the absence of urea (ΔGD(0)) associated with the equilibrium of denaturation exist between native state↔denatured state. The intermediate state was further characterized by hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS-binding). For seeing the effect of urea on the structure and dynamics of HFE, molecular dynamics simulation for 60ns was also performed. A clear correspondence was established between the in vitro and in silico studies. Copyright © 2016 Elsevier B.V. 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

Chemical Frustration in the Protein Folding Landscape: Grand Canonical Ensemble Simulations of Cytochrome c

PubMed Central

Weinkam, Patrick; Romesberg, Floyd E.; Wolynes, Peter G.

2010-01-01

A grand canonical formalism is developed to combine discrete simulations for chemically distinct species in equilibrium. Each simulation is based on a perturbed funneled landscape. The formalism is illustrated using the alkaline-induced transitions of cytochrome c as observed by FTIR spectroscopy and with various other experimental approaches. The grand canonical simulation method accounts for the acid/base chemistry of deprotonation, the inorganic chemistry of heme ligation and misligation, and the minimally frustrated folding energy landscape, thus elucidating the physics of protein folding involved with an acid/base titration of a protein. The formalism combines simulations for each of the relevant chemical species, varying by protonation and ligation states. In contrast to models based on perfectly funneled energy landscapes that contain only contacts found in the native structure, the current study introduces “chemical frustration” from deprotonation and misligation that gives rise to many intermediates at alkaline pH. While the nature of these intermediates cannot be easily inferred from available experimental data, the current study provides specific structural details of these intermediates thus extending our understanding of how cytochrome c changes with increasing pH. The results demonstrate the importance of chemical frustration for understanding biomolecular energy landscapes. PMID:19199810

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 the RNA Recognition Motif (RRM) Domains of the Amyotrophic Lateral Sclerosis (ALS)-linked Protein TDP-43 Reveals an Intermediate State*

PubMed Central

Mackness, Brian C.; Tran, Meme T.; McClain, Shannan P.; Matthews, C. Robert; Zitzewitz, Jill A.

2014-01-01

Pathological alteration of TDP-43 (TAR DNA-binding protein-43), a protein involved in various RNA-mediated processes, is a hallmark feature of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments of TDP-43, composed of the second RNA recognition motif (RRM2) and the disordered C terminus, have been observed in cytoplasmic inclusions in sporadic amyotrophic lateral sclerosis cases, suggesting that conformational changes involving RRM2 together with the disordered C terminus play a role in aggregation and toxicity. The biophysical data collected by CD and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2, with a partially folded intermediate state that is not observed in RRM1. Strikingly, a portion of RRM2 beginning at position 208, which mimics a cleavage site observed in patient tissues, increases the population of this intermediate state. Mutually stabilizing interactions between the domains in the tethered RRM1 and RRM2 construct reduce the population of the intermediate state and enhance DNA/RNA binding. Despite the high sequence homology of the two domains, a network of large hydrophobic residues in RRM2 provides a possible explanation for the increased stability of RRM2 compared with RRM1. The cluster analysis suggests that the intermediate state may play a functional role by enhancing access to the nuclear export signal contained within its sequence. The intermediate state may also serve as a molecular hazard linking productive folding and function with pathological misfolding and aggregation that may contribute to disease. PMID:24497641

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.

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

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.

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

Two-step crystal growth mechanism during crystallization of an undercooled Ni50Al50 alloy

NASA Astrophysics Data System (ADS)

An, Simin; Li, Jiahao; Li, Yang; Li, Shunning; Wang, Qi; Liu, Baixin

2016-08-01

Crystallization processes are always accompanied by the emergence of multiple intermediate states, of which the structures and transition dynamics are far from clarity, since it is difficult to experimentally observe the microscopic pathway. To insight the structural evolution and the crystallization dynamics, we perform large-scale molecular dynamics simulations to investigate the time-dependent crystallization behavior of the NiAl intermetallic upon rapid solidification. The simulation results reveal that the crystallization process occurs via a two-step growth mechanism, involving the formation of initial non-equilibrium long range order (NLRO) regions and of the subsequent equilibrium long range order (ELRO) regions. The formation of the NLRO regions makes the grains rather inhomogeneous, while the rearrangement of the NLRO regions into the ELRO regions makes the grains more ordered and compact. This two-step growth mechanism is actually controlled by the evolution of the coordination polyhedra, which are characterized predominantly by the transformation from five-fold symmetry to four-fold and six-fold symmetry. From liquids to NLRO and further to ELRO, the five-fold symmetry of these polyhedra gradually fades, and finally vanishes when B2 structure is distributed throughout the grain bulk. The energy decrease along the pathway further implies the reliability of the proposed crystallization processes.

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

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.

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

Discrete structure of an RNA folding intermediate revealed by cryo-electron microscopy.

PubMed

Baird, Nathan J; Ludtke, Steven J; Khant, Htet; Chiu, Wah; Pan, Tao; Sosnick, Tobin R

2010-11-24

RNA folding occurs via a series of transitions between metastable intermediate states. It is unknown whether folding intermediates are discrete structures folding along defined pathways or heterogeneous ensembles folding along broad landscapes. We use cryo-electron microscopy and single-particle image reconstruction to determine the structure of the major folding intermediate of the specificity domain of a ribonuclease P ribozyme. Our results support the existence of a discrete conformation for this folding intermediate.

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

Energetic Coupling between Ligand Binding and Dimerization in E. coli Phosphoglycerate Mutase

PubMed Central

Gardner, Nathan W.; Monroe, Lyman K.; Kihara, Daisuke; Park, Chiwook

2016-01-01

Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding in an exceptional quality. The homodimeric E. coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. In the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active-site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization. PMID:26919584

Membrane-Based Functions in the Origin of Cellular Life

NASA Technical Reports Server (NTRS)

Wilson, Michael

1997-01-01

If peptides consist of nonpolar residues only, they become inserted into the nonpolar phase. As demonstrated by the example of the leucine undecamer, such peptides fold into an a-helix as they partition into the nonpolar medium. The folding proceeds through an intermediate, called the 310-helix, which remains in equilibrium with the a-helix. This process represents a simple, protobiologically relevant example of environmentally-mediated self-organization of biological molecules. Once in the nonpolar environment, the peptides can readily change their orientation with respect to the interface from parallel to perpendicular, especially in response to local electric fields. The ability of nonpolar peptides to modify both the structure and orientation with changing external conditions may have provided a simple mechanism of transmitting signals from the environment to the interior of a protocell.

Protein vivisection reveals elusive intermediates in folding

PubMed Central

Zheng, Zhongzhou; Sosnick, Tobin R.

2010-01-01

Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu→Glu−) to destabilize and unfold a specific region of the protein. We apply this strategy to Ubiquitin, reversibly trapping a folding intermediate in which the β5 strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high energy states. PMID:20144618

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

PubMed Central

Betts, S. D.; King, J.

1998-01-01

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly. PMID:9684883

The H2A.Z/H2B dimer is unstable compared to the dimer containing the major H2A isoform.

PubMed

Placek, Brandon J; Harrison, L Nicole; Villers, Brooke M; Gloss, Lisa M

2005-02-01

The nucleosome, the basic fundamental repeating unit of chromatin, contains two H2A/H2B dimers and an H3/H4 tetramer. Modulation of the structure and dynamics of the nucleosome is an important regulation mechanism of DNA-based chemistries in the eukaryotic cell, such as transcription and replication. One means of altering the properties of the nucleosome is by incorporation of histone variants. To provide insights into how histone variants may impact the thermodynamics of the nucleosome, the stability of the heterodimer between the H2A.Z variant and H2B was determined by urea-induced denaturation, monitored by far-UV circular dichroism, intrinsic Tyr fluorescence intensity, and anisotropy. In the absence of stabilizing agents, the H2A.Z/H2B dimer is only partially folded. The stabilizing cosolute, trimethylamine-N-oxide (TMAO) was used to promote folding of the unstable heterodimer. The equilibrium stability of the H2A.Z/H2B dimer is compared to that of the H2A/H2B dimer. The equilibrium folding of both histone dimers is highly reversible and best described by a two-state model, with no detectable equilibrium intermediates populated. The free energies of unfolding, in the absence of denaturant, of H2A.Z/H2B and H2A/H2B are 7.3 kcal mol(-1) and 15.5 kcal mol(-1), respectively, in 1 M TMAO. The H2A.Z/H2B dimer is the least stable histone fold characterized to date, while H2A/H2B appears to be the most stable. It is speculated that this difference in stability may contribute to the different biophysical properties of nucleosomes containing the major H2A and the H2A.Z variant.

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

Revealing the distinct folding phases of an RNA three-helix junction.

PubMed

Plumridge, Alex; Katz, Andrea M; Calvey, George D; Elber, Ron; Kirmizialtin, Serdal; Pollack, Lois

2018-05-14

Remarkable new insight has emerged into the biological role of RNA in cells. RNA folding and dynamics enable many of these newly discovered functions, calling for an understanding of RNA self-assembly and conformational dynamics. Because RNAs pass through multiple structures as they fold, an ensemble perspective is required to visualize the flow through fleetingly populated sets of states. Here, we combine microfluidic mixing technology and small angle X-ray scattering (SAXS) to measure the Mg-induced folding of a small RNA domain, the tP5abc three helix junction. Our measurements are interpreted using ensemble optimization to select atomically detailed structures that recapitulate each experimental curve. Structural ensembles, derived at key stages in both time-resolved studies and equilibrium titrations, reproduce the features of known intermediates, and more importantly, offer a powerful new structural perspective on the time-progression of folding. Distinct collapse phases along the pathway appear to be orchestrated by specific interactions with Mg ions. These key interactions subsequently direct motions of the backbone that position the partners of tertiary contacts for later bonding, and demonstrate a remarkable synergy between Mg and RNA across numerous time-scales.

Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites.

PubMed Central

Gasset, María; Alfonso, Carlos; Neira, José L; Rivas, Germán; Pajares, María A

2002-01-01

The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24+/-0.15. The variations of free energy in the absence of denaturant [DeltaG(H(2)O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15+/-2.3 kJ.mol(-1) and 19.87+/-0.71 kJ.mol(-1).M(-1) for the dissociation of the native dimer and 14.77+/-1.63 kJ.mol(-1) and 5.23+/-0.21 kJ.mol(-1).M(-1) for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69 kJ.mol(-1) and 30.33 kJ.mol(-1).M(-1) respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established. PMID:11772402

Conformational stability and thermodynamic characterization of the lipoic acid bearing domain of human mitochondrial branched chain α-ketoacid dehydrogenase

PubMed Central

Naik, Mandar T.; Huang, Tai-Huang

2004-01-01

The lipoic acid bearing domain (hbLBD) of human mitochondrial branched chain α-ketoacid dehydrogenase (BCKD) plays important role of substrate channeling in oxidative decarboxylation of the branched chain α-ketoacids. Recently hbLBD has been found to follow two-step folding mechanism without detectable presence of stable or kinetic intermediates. The present study describes the conformational stability underlying the folding of this small β-barrel domain. Thermal denaturation in presence of urea and isothermal urea denaturation titrations are used to evaluate various thermodynamic parameters defining the equilibrium unfolding. The linear extrapolation model successfully describes the two-step; native state ↔denatured state unfolding transition of hbLBD. The average temperature of maximum stability of hbLBD is estimated as 295.6 ± 0.9 K. Cold denaturation of hbLBD is also predicted and discussed. PMID:15322287

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

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.

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

PubMed Central

Phillips, J J; Javadi, Y; Millership, C; Main, E R G

2012-01-01

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium. PMID:22170589

Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation

PubMed Central

Nasedkin, Alexandr; Marcellini, Moreno; Religa, Tomasz L.; Freund, Stefan M.; Menzel, Andreas; Fersht, Alan R.; Jemth, Per; van der Spoel, David; Davidsson, Jan

2015-01-01

The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution. PMID:25946337

Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation.

PubMed

Nasedkin, Alexandr; Marcellini, Moreno; Religa, Tomasz L; Freund, Stefan M; Menzel, Andreas; Fersht, Alan R; Jemth, Per; van der Spoel, David; Davidsson, Jan

2015-01-01

The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution.

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

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.

Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach

PubMed Central

Lang, Kathrin; Rieder, Renate; Micura, Ronald

2007-01-01

Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures. PMID:17693433

Transient intermediates are populated in the folding pathways of single-domain two-state folding protein L

NASA Astrophysics Data System (ADS)

Maity, Hiranmay; Reddy, Govardhan

2018-04-01

Small single-domain globular proteins, which are believed to be dominantly two-state folders, played an important role in elucidating various aspects of the protein folding mechanism. However, recent single molecule fluorescence resonance energy transfer experiments [H. Y. Aviram et al. J. Chem. Phys. 148, 123303 (2018)] on a single-domain two-state folding protein L showed evidence for the population of an intermediate state and it was suggested that in this state, a β-hairpin present near the C-terminal of the native protein state is unfolded. We performed molecular dynamics simulations using a coarse-grained self-organized-polymer model with side chains to study the folding pathways of protein L. In agreement with the experiments, an intermediate is populated in the simulation folding pathways where the C-terminal β-hairpin detaches from the rest of the protein structure. The lifetime of this intermediate structure increased with the decrease in temperature. In low temperature conditions, we also observed a second intermediate state, which is globular with a significant fraction of the native-like tertiary contacts satisfying the features of a dry molten globule.

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

Dynamics Analysis of Anti-predator Model on Intermediate Predator With Ratio Dependent Functional Responses

NASA Astrophysics Data System (ADS)

Savitri, D.

2018-01-01

This articel discusses a predator prey model with anti-predator on intermediate predator using ratio dependent functional responses. Dynamical analysis performed on the model includes determination of equilibrium point, stability and simulation. Three kinds of equilibrium points have been discussed, namely the extinction of prey point, the extinction of intermediate predator point and the extinction of predator point are exists under certain conditions. It can be shown that the result of numerical simulations are in accordance with analitical results

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.

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

PubMed Central

Surana, Parag

2016-01-01

Abstract The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly‐Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process. PMID:27111887

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

Folding and stability of the isolated Greek key domains of the long-lived human lens proteins γD-crystallin and γS-crystallin

PubMed Central

Mills, Ishara A.; Flaugh, Shannon L.; Kosinski-Collins, Melissa S.; King, Jonathan A.

2007-01-01

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric γ-crystallins and oligomeric β-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human γD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human γD-crystallin, as well as the isolated domains of human γS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37°C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human γD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein. PMID:17905830

Force generation by titin folding.

PubMed

Mártonfalvi, Zsolt; Bianco, Pasquale; Naftz, Katalin; Ferenczy, György G; Kellermayer, Miklós

2017-07-01

Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (<10 pN), extension fluctuated without resolvable discrete events. In position-clamp experiments, the time-dependent force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles. © 2017 The Protein Society.

Discovery and characterization of a sulfoquinovose mutarotase using kinetic analysis at equilibrium by exchange spectroscopy

PubMed Central

Abayakoon, Palika; Lingford, James P.; Jin, Yi; Bengt, Christopher; Davies, Gideon J.; Yao, Shenggen; Goddard-Borger, Ethan D.

2018-01-01

Bacterial sulfoglycolytic pathways catabolize sulfoquinovose (SQ), or glycosides thereof, to generate a three-carbon metabolite for primary cellular metabolism and a three-carbon sulfonate that is expelled from the cell. Sulfoglycolytic operons encoding an Embden–Meyerhof–Parnas-like or Entner–Doudoroff (ED)-like pathway harbor an uncharacterized gene (yihR in Escherichia coli; PpSQ1_00415 in Pseudomonas putida) that is up-regulated in the presence of SQ, has been annotated as an aldose-1-epimerase and which may encode an SQ mutarotase. Our sequence analyses and structural modeling confirmed that these proteins possess mutarotase-like active sites with conserved catalytic residues. We overexpressed the homolog from the sulfo-ED operon of Herbaspirillum seropedicaea (HsSQM) and used it to demonstrate SQ mutarotase activity for the first time. This was accomplished using nuclear magnetic resonance exchange spectroscopy, a method that allows the chemical exchange of magnetization between the two SQ anomers at equilibrium. HsSQM also catalyzed the mutarotation of various aldohexoses with an equatorial 2-hydroxy group, including d-galactose, d-glucose, d-glucose-6-phosphate (Glc-6-P), and d-glucuronic acid, but not d-mannose. HsSQM displayed only 5-fold selectivity in terms of efficiency (kcat/KM) for SQ versus the glycolysis intermediate Glc-6-P; however, its proficiency [kuncat/(kcat/KM)] for SQ was 17 000-fold better than for Glc-6-P, revealing that HsSQM preferentially stabilizes the SQ transition state. PMID:29535276

Equilibrium Thermodynamics, Formation, and Dissociation Kinetics of Trivalent Iron and Gallium Complexes of Triazacyclononane-Triphosphinate (TRAP) Chelators: Unraveling the Foundations of Highly Selective Ga-68 Labeling.

PubMed

Vágner, Adrienn; Forgács, Attila; Brücher, Ernő; Tóth, Imre; Maiocchi, Alessandro; Wurzer, Alexander; Wester, Hans-Jürgen; Notni, Johannes; Baranyai, Zsolt

2018-01-01

In order to rationalize the influence of Fe III contamination on labeling with the 68 Ga eluted from 68 Ge/ 68 Ga- g enerator, a detailed investigation was carried out on the equilibrium properties, formation and dissociation kinetics of Ga III - and Fe III -complexes of 1,4,7-triazacyclononane-1,4,7-tris(methylene[2-carboxyethylphosphinic acid]) (H 6 TRAP). The stability and protonation constants of the [Fe(TRAP)] 3- complex were determined by pH-potentiometry and spectrophotometry by following the competition reaction between the TRAP ligand and benzhydroxamic acid (0.15 M NaNO 3 , 25°C). The formation rates of [Fe(TRAP)] and [Ga(TRAP)] complexes were determined by spectrophotometry and 31 P-NMR spectroscopy in the pH range 4.5-6.5 in the presence of 5-40 fold H x TRAP (x-6) excess (x = 1 and 2, 0.15 M NaNO 3 , 25°C). The kinetic inertness of [Fe(TRAP)] 3- and [Ga(TRAP)] 3- was examined by the trans-chelation reactions with 10 to 20-fold excess of H x HBED (x-4) ligand by spectrophotometry at 25°C in 0.15 M NaCl (x = 0,1 and 2). The stability constant of [Fe(TRAP)] 3- (log K FeL = 26.7) is very similar to that of [Ga(TRAP)] 3- (log K GaL = 26.2). The rates of ligand exchange reaction of [Fe(TRAP)] 3- and [Ga(TRAP)] 3- with H x HBED (x-4) are similar. The reactions take place quite slowly via spontaneous dissociation of [M(TRAP)] 3- , [M(TRAP)OH] 4- and [M(TRAP)(OH) 2 ] 5- species. Dissociation half-lives ( t 1/2 ) of [Fe(TRAP)] 3- and [Ga(TRAP)] 3- complexes are 1.1 × 10 5 and 1.4 × 10 5 h at pH = 7.4 and 25°C. The formation reactions of [Fe(TRAP)] 3- and [Ga(TRAP)] 3- are also slow due to the formation of the unusually stable monoprotonated [ * M(HTRAP)] 2- intermediates [ * log K Ga(HL) = 10.4 and * log K Fe(HL) = 9.9], which are much more stable than the [ * Ga(HNOTA)] + intermediate [ * log K Ga(HL) = 4.2]. Deprotonation and transformation of the monoprotonated [ * M(HTRAP)] 2- intermediates into the final complex occur via OH - -assisted reactions. Rate constants ( k OH ) characterizing the OH - -driven deprotonation and transformation of [ * Ga(HTRAP)] 2- and [ * Fe(HTRAP)] 2- intermediates are 1.4 × 10 5 M -1 s -1 and 3.4 × 10 4 M -1 s -1 , respectively. In conclusion, the equilibrium and kinetic properties of [Fe(TRAP)] and [Ga(TRAP)] complexes are remarkably similar due to the close physico-chemical properties of Fe III and Ga III -ions. However, a slightly faster formation of [Ga(TRAP)] over [Fe(TRAP)] provides a rationale for a previously observed, selective complexation of 68 Ga III in presence of excess Fe III .

Equilibrium Thermodynamics, Formation, and Dissociation Kinetics of Trivalent Iron and Gallium Complexes of Triazacyclononane-Triphosphinate (TRAP) Chelators: Unraveling the Foundations of Highly Selective Ga-68 Labeling

PubMed Central

Vágner, Adrienn; Forgács, Attila; Brücher, Ernő; Tóth, Imre; Maiocchi, Alessandro; Wurzer, Alexander; Wester, Hans-Jürgen; Notni, Johannes; Baranyai, Zsolt

2018-01-01

In order to rationalize the influence of FeIII contamination on labeling with the 68Ga eluted from 68Ge/68Ga-generator, a detailed investigation was carried out on the equilibrium properties, formation and dissociation kinetics of GaIII- and FeIII-complexes of 1,4,7-triazacyclononane-1,4,7-tris(methylene[2-carboxyethylphosphinic acid]) (H6TRAP). The stability and protonation constants of the [Fe(TRAP)]3− complex were determined by pH-potentiometry and spectrophotometry by following the competition reaction between the TRAP ligand and benzhydroxamic acid (0.15 M NaNO3, 25°C). The formation rates of [Fe(TRAP)] and [Ga(TRAP)] complexes were determined by spectrophotometry and 31P-NMR spectroscopy in the pH range 4.5–6.5 in the presence of 5–40 fold HxTRAP(x−6) excess (x = 1 and 2, 0.15 M NaNO3, 25°C). The kinetic inertness of [Fe(TRAP)]3− and [Ga(TRAP)]3− was examined by the trans-chelation reactions with 10 to 20-fold excess of HxHBED(x−4) ligand by spectrophotometry at 25°C in 0.15 M NaCl (x = 0,1 and 2). The stability constant of [Fe(TRAP)]3− (logKFeL = 26.7) is very similar to that of [Ga(TRAP)]3− (logKGaL = 26.2). The rates of ligand exchange reaction of [Fe(TRAP)]3− and [Ga(TRAP)]3− with HxHBED(x−4) are similar. The reactions take place quite slowly via spontaneous dissociation of [M(TRAP)]3−, [M(TRAP)OH]4− and [M(TRAP)(OH)2]5− species. Dissociation half-lives (t1/2) of [Fe(TRAP)]3− and [Ga(TRAP)]3− complexes are 1.1 × 105 and 1.4 × 105 h at pH = 7.4 and 25°C. The formation reactions of [Fe(TRAP)]3− and [Ga(TRAP)]3− are also slow due to the formation of the unusually stable monoprotonated [*M(HTRAP)]2− intermediates [*logKGa(HL) = 10.4 and *logKFe(HL) = 9.9], which are much more stable than the [*Ga(HNOTA)]+ intermediate [*logKGa(HL) = 4.2]. Deprotonation and transformation of the monoprotonated [*M(HTRAP)]2− intermediates into the final complex occur via OH−-assisted reactions. Rate constants (kOH) characterizing the OH−-driven deprotonation and transformation of [* Ga(HTRAP)]2− and [*Fe(HTRAP)]2− intermediates are 1.4 × 105 M−1s−1 and 3.4 × 104 M−1s−1, respectively. In conclusion, the equilibrium and kinetic properties of [Fe(TRAP)] and [Ga(TRAP)] complexes are remarkably similar due to the close physico-chemical properties of FeIII and GaIII-ions. However, a slightly faster formation of [Ga(TRAP)] over [Fe(TRAP)] provides a rationale for a previously observed, selective complexation of 68GaIII in presence of excess FeIII. PMID:29876344

Building polyhedra by self-assembly: theory and experiment.

PubMed

Kaplan, Ryan; Klobušický, Joseph; Pandey, Shivendra; Gracias, David H; Menon, Govind

2014-01-01

We investigate the utility of a mathematical framework based on discrete geometry to model biological and synthetic self-assembly. Our primary biological example is the self-assembly of icosahedral viruses; our synthetic example is surface-tension-driven self-folding polyhedra. In both instances, the process of self-assembly is modeled by decomposing the polyhedron into a set of partially formed intermediate states. The set of all intermediates is called the configuration space, pathways of assembly are modeled as paths in the configuration space, and the kinetics and yield of assembly are modeled by rate equations, Markov chains, or cost functions on the configuration space. We review an interesting interplay between biological function and mathematical structure in viruses in light of this framework. We discuss in particular: (i) tiling theory as a coarse-grained description of all-atom models; (ii) the building game-a growth model for the formation of polyhedra; and (iii) the application of these models to the self-assembly of the bacteriophage MS2. We then use a similar framework to model self-folding polyhedra. We use a discrete folding algorithm to compute a configuration space that idealizes surface-tension-driven self-folding and analyze pathways of assembly and dominant intermediates. These computations are then compared with experimental observations of a self-folding dodecahedron with side 300 μm. In both models, despite a combinatorial explosion in the size of the configuration space, a few pathways and intermediates dominate self-assembly. For self-folding polyhedra, the dominant intermediates have fewer degrees of freedom than comparable intermediates, and are thus more rigid. The concentration of assembly pathways on a few intermediates with distinguished geometric properties is biologically and physically important, and suggests deeper mathematical structure.

Consequences of localized frustration for the folding mechanism of the IM7 protein

PubMed Central

Sutto, Ludovico; Lätzer, Joachim; Hegler, Joseph A.; Ferreiro, Diego U.; Wolynes, Peter G.

2007-01-01

In the laboratory, IM7 has been found to have an unusual folding mechanism in which an “on-pathway” intermediate with nonnative interactions is formed. We show that this intermediate is a consequence of an unusual cluster of highly frustrated interactions in the native structure. This cluster is involved in the binding of IM7 to its target, Colicin E7. Redesign of residues in this cluster to eliminate frustration is predicted by simulations to lead to faster folding without the population of an intermediate ensemble. PMID:18077415

Dynamics of one-state downhill protein folding.

PubMed

Li, Peng; Oliva, Fabiana Y; Naganathan, Athi N; Muñoz, Victor

2009-01-06

The small helical protein BBL has been shown to fold and unfold in the absence of a free energy barrier according to a battery of quantitative criteria in equilibrium experiments, including probe-dependent equilibrium unfolding, complex coupling between denaturing agents, characteristic DSC thermogram, gradual melting of secondary structure, and heterogeneous atom-by-atom unfolding behaviors spanning the entire unfolding process. Here, we present the results of nanosecond T-jump experiments probing backbone structure by IR and end-to-end distance by FRET. The folding dynamics observed with these two probes are both exponential with common relaxation times but have large differences in amplitude following their probe-dependent equilibrium unfolding. The quantitative analysis of amplitude and relaxation time data for both probes shows that BBL folding dynamics are fully consistent with the one-state folding scenario and incompatible with alternative models involving one or several barrier crossing events. At 333 K, the relaxation time for BBL is 1.3 micros, in agreement with previous folding speed limit estimates. However, late folding events at room temperature are an order of magnitude slower (20 micros), indicating a relatively rough underlying energy landscape. Our results in BBL expose the dynamic features of one-state folding and chart the intrinsic time-scales for conformational motions along the folding process. Interestingly, the simple self-averaging folding dynamics of BBL are the exact dynamic properties required in molecular rheostats, thus supporting a biological role for one-state folding.

Unravelling the mysteries of sub-second biochemical processes using time-resolved mass spectrometry.

PubMed

Lento, Cristina; Wilson, Derek J

2017-05-21

Many important chemical and biochemical phenomena proceed on sub-second time scales before entering equilibrium. In this mini-review, we explore the history and recent advancements of time-resolved mass spectrometry (TRMS) for the characterization of millisecond time-scale chemical reactions and biochemical processes. TRMS allows for the simultaneous tracking of multiple reactants, intermediates and products with no chromophoric species required, high sensitivity and temporal resolution. The method has most recently been used for the characterization of several short-lived reaction intermediates in rapid chemical reactions. Most of the reactions that occur in living organisms are accelerated by enzymes, with pre-steady state kinetics only attainable using time-resolved methods. TRMS has been increasingly used to monitor the conversion of substrates to products and the resulting changes to the enzyme during catalytic turnover. Early events in protein folding systems have also been elucidated, along with the characterization of dynamics and transient secondary structures in intrinsically disordered proteins. In this review, we will highlight representative examples where TRMS has been applied to study these phenomena.

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.

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

Folding and translocation of the undecamer of poly-L-leucine across the water-hexane interface. A molecular dynamics study

NASA Technical Reports Server (NTRS)

Chipot, C.; Pohorille, A.

1998-01-01

The undecamer of poly-L-leucine at the water-hexane interface is studied by molecular dynamics simulations. This represents a simple model relevant to folding and insertion of hydrophobic peptides into membranes. The peptide, initially placed in a random coil conformation on the aqueous side of the system, rapidly translocates toward the hexane phase and undergoes interfacial folding into an alpha-helix in the subsequent 36 ns. Folding is nonsequential and highly dynamic. The initially formed helical segment at the N-terminus of the undecamer becomes transiently broken and, subsequently, reforms before the remainder of the peptide folds from the C-terminus. The formation of intramolecular hydrogen bonds during the folding of the peptide is preceded by a dehydration of the participating polar groups, as they become immersed in hexane. Folding proceeds through a short-lived intermediate, a 3(10)-helix, which rapidly interconverts to an alpha-helix. Both helices contribute to the equilibrium ensemble of folded structures. The helical peptide is largely buried in hexane, yet remains adsorbed at the interface. Its preferred orientation is parallel to the interface, although the perpendicular arrangement with the N-terminus immersed in hexane is only slightly less favorable. In contrast, the reversed orientation is highly unfavorable, because it would require dehydration of C-terminus carbonyl groups that do not participate in intramolecular hydrogen bonding. For the same reason, the transfer of the undecamer from the interface to the bulk hexane is also unfavorable. The results suggest that hydrophobic peptides fold in the interfacial region and, simultaneously, translocate into the nonpolar side of the interface. It is further implied that peptide insertion into the membrane is accomplished by rotating from the parallel to the perpendicular orientation, most likely in such a way that the N-terminus penetrates the bilayer.

An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis.

PubMed Central

Szewczak, A A; Cech, T R

1997-01-01

RNA molecules commonly consist of helical regions separated by internal loops, and in many cases these internal loops have been found to assume stable structures. We have examined the function and dynamics of an internal loop, J5/5a, that joins the two halves of the P4-P6 domain of the Tetrahymena self-splicing group I intron. P4-P6 RNAs with mutations in the J5/5a region showed nondenaturing gel electrophoretic mobilities and levels of Fe(II)-EDTA cleavage protection intermediate between those of wild-type RNA and a mutant incapable of folding into the native P4-P6 tertiary structure. Mutants with the least structured J5/5a loops behaved the most like wild-type P4-P6, and required smaller amounts of Mg2+ to rescue folding. The activity of reconstituted introns containing mutant P4-P6 RNAs correlated similarly with the nature of the J5/5a mutation. Our results suggest that, in solution, the P4-P6 RNA is in a two-state equilibrium between folded and unfolded states. We conclude that this internal loop mainly acts as a flexible hinge, allowing the coaxially stacked helical regions on either side of it to interact via specific tertiary contacts. To a lesser extent, the specific bases within the loop contribute to folding. Furthermore, it is crucial that the junction remain unstructured in the unfolded state. These conclusions cannot be derived from a simple examination of the P4-P6 crystal structure (Cate JH et al., 1996, Science 273:1678-1685), showing once again that structure determination must be supplemented with mutational and thermodynamic analysis to provide a complete picture of a folded macromolecule. PMID:9257643

Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates.

PubMed

Jain, Neha; Knowles, Timothy J; Lund, Peter A; Chaudhuri, Tapan K

2018-06-02

The isolated apical domain of GroEL consisting of residues 191-345 (known as "minichaperone") binds and assists the folding of a wide variety of client proteins without GroES and ATP, but the mechanism of its action is still unknown. In order to probe into the matter, we have examined minichaperone-mediated folding of a large aggregation prone protein Maltodextrin-glucosidase (MalZ). The key objective was to identify whether MalZ exists free in solution, or remains bound to, or cycling on and off the minichaperone during the refolding process. When GroES was introduced during refolding process, production of the native MalZ was inhibited. We also observed the same findings with a trap mutant of GroEL, which stably captures a predominantly non-native MalZ released from minichaperone during refolding process, but does not release it. Tryptophan and ANS fluorescence measurements indicated that refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. Surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ± 0.04 μM, which are significantly higher than for most GroEL clients. This showed that minichaperone interacts loosely with MalZ to allow the protein to change its conformation and fold while bound during the refolding process. These observations suggest that the minichaperone works by carrying out repeated cycles of binding aggregation-prone protein MalZ in a relatively compact conformation and in a partially folded but active state, and releasing them to attempt to fold in solution. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

In silico direct folding of thrombin-binding aptamer G-quadruplex at all-atom level

PubMed Central

Yang, Changwon; Kulkarni, Mandar; Lim, Manho

2017-01-01

Abstract The reversible folding of the thrombin-binding DNA aptamer G-quadruplexes (GQs) (TBA-15) starting from fully unfolded states was demonstrated using a prolonged time scale (10–12 μs) parallel tempering metadynamics (PTMetaD) simulation method in conjunction with a modified version of the AMBER bsc1 force field. For unbiased descriptions of the folding free energy landscape of TBA-15, this force field was minimally modified. From this direct folding simulation using the modified bsc1 force field, reasonably converged free energy landscapes were obtained in K+-rich aqueous solution (150 mM), providing detailed atomistic pictures of GQ folding mechanisms for TBA-15. This study found that the TBA folding occurred via multiple folding pathways with two major free energy barriers of 13 and 15 kcal/mol in the presence of several intermediate states of G-triplex variants. The early formation of these intermediates was associated with a single K+ ion capturing. Interestingly, these intermediate states appear to undergo facile transitions among themselves through relatively small energy barriers. PMID:29112755

Enhanced sampling molecular dynamics simulation captures experimentally suggested intermediate and unfolded states in the folding pathway of Trp-cage miniprotein.

PubMed

Shao, Qiang; Shi, Jiye; Zhu, Weiliang

2012-09-28

The ability of molecular dynamics simulation to capturing the transient states within the folding pathway of protein is important to the understanding of protein folding mechanism. In the present study, the integrated-tempering-sampling molecular dynamics (ITS-MD) simulation was performed to investigate the transient states including intermediate and unfolded ones in the folding pathway of a miniprotein, Trp-cage. Three force fields (FF03, FF99SB, and FF96) were tested, and both intermediate and unfolded states with their characteristics in good agreement with experiments were observed during the simulations, which supports the hypothesis that observable intermediates might present in the folding pathway of small polypeptides. In addition, it was demonstrated that FF03 force field as combined with ITS-MD is in overall a more proper force field than the others in reproducing experimentally recorded properties in UVRS, ECD, and NMR, Photo-CIDNP NMR, and IR T-jump experiments, and the folding∕unfolding thermodynamics parameters, such as ΔG(U), ΔC(p), and ΔH(U) (T(m)). In summary, the present study showed that using suitable force field and energy sampling method, molecular dynamics simulation could capture the transient states within the folding pathway of protein which are consistent with the experimental measurements, and thus provide information of protein folding mechanism and thermodynamics.

Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase.

PubMed

Scholl, Zackary N; Yang, Weitao; Marszalek, Piotr E

2017-05-09

Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarse-grained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Dominant role of local dipolar interactions in phosphate binding to a receptor cleft with an electronegative charge surface: equilibrium, kinetic, and crystallographic studies.

PubMed

Ledvina, P S; Tsai, A L; Wang, Z; Koehl, E; Quiocho, F A

1998-12-01

Stringent specificity and complementarity between the receptor, a periplasmic phosphate-binding protein (PBP) with a two-domain structure, and the completely buried and dehydrated phosphate are achieved by hydrogen bonding or dipolar interactions. We recently found that the surface charge potential of the cleft between the two domains that contains the anion binding site is intensely electronegative. This novel finding prompted the study reported here of the effect of ionic strength on the equilibrium and rapid kinetics of phosphate binding. To facilitate this study, Ala197, located on the edge of the cleft, was replaced by a Trp residue (A197W PBP) to generate a fluorescence reporter group. The A197W PBP-phosphate complex retains wild-type Kd and X-ray structure beyond the replacement residue. The Kd (0.18 microM) at no salt is increased by 20-fold at greater than 0.30 M NaCl. Stopped-flow fluorescence kinetic studies indicate a two-step binding process: (1) The phosphate (L) binds, at near diffusion-controlled rate, to the open cleft form (Po) of PBP to produce an intermediate, PoL. This rate decreases with increasing ionic strength. (2) The intermediate isomerizes to the closed-conformation form, PcL. The results indicate that the high specificity, affinity, and rate of phosphate binding are not influenced by the noncomplementary electronegative surface potential of the cleft. That binding depends almost entirely on local dipolar interactions with the receptor has important ramification in electrostatic interactions in protein structures and in ligand recognition.

The dual-basin landscape in GFP folding

PubMed Central

Andrews, Benjamin T.; Gosavi, Shachi; Finke, John M.; Onuchic, José N.; Jennings, Patricia A.

2008-01-01

Recent experimental studies suggest that the mature GFP has an unconventional landscape composed of an early folding event with a typical funneled landscape, followed by a very slow search and rearrangement step into the locked, active chromophore-containing structure. As we have shown previously, the substantial difference in time scales is what generates the observed hysteresis in thermodynamic folding. The interconversion between locked and the soft folding structures at intermediate denaturant concentrations is so slow that it is not observed under the typical experimental observation time. Simulations of a coarse-grained model were used to describe the fast folding event as well as identify native-like intermediates on energy landscapes enroute to the fluorescent native fold. Interestingly, these simulations reveal structural features of the slow dynamic transition to chromophore activation. Experimental evidence presented here shows that the trapped, native-like intermediate has structural heterogeneity in residues previously linked to chromophore formation. We propose that the final step of GFP folding is a “locking” mechanism leading to chromophore formation and high stability. The combination of previous experimental work and current simulation work is explained in the context of a dual-basin folding mechanism described above. PMID:18713871

Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD–Transfer Reaction†

PubMed Central

Morrison, Eliot; Kantz, Auric; Gassner, George T.; Sazinsky, Matthew H.

2013-01-01

The two–component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH– and FAD–dependent epoxidation of styrene to styrene oxide. In this study we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to epoxidase (NSMOA) component and report our findings in light of the 2.2–Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH→FADox charge–transfer intermediate and catalyzes a hydride–transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s−1, in a step that is coupled to the rapid dissociation of NAD+. Electrochemical and equilibrium–binding studies indicate that NSMOA binds FADhq ~13–times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)–hydroperoxide intermediate. The half–life of apoSMOB generated in the FAD–transfer reaction is increased ~21–fold, supporting the model of a protein–protein interaction between apoSMOB and NSMOA with the peroxide intermediate. The mechanisms of FAD–dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. Based on these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S–state) and more reactive, transfer state (T–state). Dissociation of NAD+ after the hydride transfer–reaction transiently populates the T–state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB–FADhq shifts the FADhq–binding equilibrium from the T–state to the S–state. Additionally, the 2.2–Å crystal structure of SMOB–FADox reported in this work is discussed in light of the pyridine nucleotide–gated flavin–transfer and electron–transfer reactions. PMID:23909369

Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model

NASA Astrophysics Data System (ADS)

Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong

2016-04-01

Thrombin-binding aptamer (TBA) with the sequence 5‧GGTTGGTGTGGTTGG3‧ could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.

Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

PubMed

Bedouelle, Hugues

2016-02-01

The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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.

Insilico direct folding of thrombin-binding aptamer G-quadruplex at all-atom level.

PubMed

Yang, Changwon; Kulkarni, Mandar; Lim, Manho; Pak, Youngshang

2017-12-15

The reversible folding of the thrombin-binding DNA aptamer G-quadruplexes (GQs) (TBA-15) starting from fully unfolded states was demonstrated using a prolonged time scale (10-12 μs) parallel tempering metadynamics (PTMetaD) simulation method in conjunction with a modified version of the AMBER bsc1 force field. For unbiased descriptions of the folding free energy landscape of TBA-15, this force field was minimally modified. From this direct folding simulation using the modified bsc1 force field, reasonably converged free energy landscapes were obtained in K+-rich aqueous solution (150 mM), providing detailed atomistic pictures of GQ folding mechanisms for TBA-15. This study found that the TBA folding occurred via multiple folding pathways with two major free energy barriers of 13 and 15 kcal/mol in the presence of several intermediate states of G-triplex variants. The early formation of these intermediates was associated with a single K+ ion capturing. Interestingly, these intermediate states appear to undergo facile transitions among themselves through relatively small energy barriers. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

GroEL actively stimulates folding of the endogenous substrate protein PepQ.

PubMed

Weaver, Jeremy; Jiang, Mengqiu; Roth, Andrew; Puchalla, Jason; Zhang, Junjie; Rye, Hays S

2017-06-30

Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role.

Geometrical Frustration in Interleukin-33 Decouples the Dynamics of the Functional Element from the Folding Transition State Ensemble

PubMed Central

Fisher, Kaitlin M.; Haglund, Ellinor; Noel, Jeffrey K.; Hailey, Kendra L.; Onuchic, José N.; Jennings, Patricia A.

2015-01-01

Interleukin-33 (IL-33) is currently the focus of multiple investigations into targeting pernicious inflammatory disorders. This mediator of inflammation plays a prevalent role in chronic disorders such as asthma, rheumatoid arthritis, and progressive heart disease. In order to better understand the possible link between the folding free energy landscape and functional regions in IL-33, a combined experimental and theoretical approach was applied. IL-33 is a pseudo- symmetrical protein composed of three distinct structural elements that complicate the folding mechanism due to competition for nucleation on the dominant folding route. Trefoil 1 constitutes the majority of the binding interface with the receptor whereas Trefoils 2 and 3 provide the stable scaffold to anchor Trefoil 1. We identified that IL-33 folds with a three-state mechanism, leading to a rollover in the refolding arm of its chevron plots in strongly native conditions. In addition, there is a second slower refolding phase that exhibits the same rollover suggesting similar limitations in folding along parallel routes. Characterization of the intermediate state and the rate limiting steps required for folding suggests that the rollover is attributable to a moving transition state, shifting from a post- to pre-intermediate transition state as you move from strongly native conditions to the midpoint of the transition. On a structural level, we found that initially, all independent Trefoil units fold equally well until a QCA of 0.35 when Trefoil 1 will backtrack in order to allow Trefoils 2 and 3 to fold in the intermediate state, creating a stable scaffold for Trefoil 1 to fold onto during the final folding transition. The formation of this intermediate state and subsequent moving transition state is a result of balancing the difficulty in folding the functionally important Trefoil 1 onto the remainder of the protein. Taken together our results indicate that the functional element of the protein is geometrically frustrated, requiring the more stable elements to fold first, acting as a scaffold for docking of the functional element to allow productive folding to the native state. PMID:26630011

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.

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.

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

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

Dodging the crisis of folding proteins with knots

NASA Astrophysics Data System (ADS)

Sulkowska, Joanna

2009-03-01

Proteins with nontrivial topology, containing knots and slipknots, have the ability to fold to their native states without any additional external forces invoked. A mechanism is suggested for folding of these proteins, such as YibK and YbeA, which involves an intermediate configuration with a slipknot. It elucidates the role of topological barriers and backtracking during the folding event. It also illustrates that native contacts are sufficient to guarantee folding in around 1-2% of the simulations, and how slipknot intermediates are needed to reduce the topological bottlenecks. As expected, simulations of proteins with similar structure but with knot removed fold much more efficiently, clearly demonstrating the origin of these topological barriers. Although these studies are based on a simple coarse-grained model, they are already able to extract some of the underlying principles governing folding in such complex topologies.

Effect of mammalian kidney osmolytes on the folding pathway of sheep serum albumin.

PubMed

Dar, Mohammad Aasif; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

2017-04-01

Recently, we had published that urea-induced denaturation curves of optical properties of sheep serum albumin (SSA) are biphasic with a stable intermediate that has characteristics of molten globule (MG) state. In this study, we have extended the work by carrying out urea- and guanidinium chloride (GdmCl)-induced denaturations of SSA in the presence of naturally occurring mammalian kidney osmolytes, namely, sorbitol, myo-inositol and glycine betaine. We have observed that all these osmolytes (i) transform this biphasic transition into a co-operative, two-state transition and (ii) increase the stability of the protein in terms of midpoint of denaturation (C m ) and Gibbs free energy change in the absence of both denaturants (ΔG D 0 ). The relative effectiveness of different osmolytes on the stability of SSA follows the order: glycine betaine>myo-inositol>sorbitol. In this paper, we also report that kidney osmolytes destabilize MG state by shifting the equilibrium, native state↔MG state toward the left. This study will be helpful in understanding the existence of osmolytes in kidney and their role in folding of kidney proteins soaked with urea. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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

Isocyanides inhibit human heme oxygenases at the verdoheme stage.

PubMed

Evans, John P; Kandel, Sylvie; Ortiz de Montellano, Paul R

2009-09-22

Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.

Isocyanides Inhibit Human Heme Oxygenases at the Verdoheme Stage†

PubMed Central

Evans, John P.; Kandel, Sylvie; Ortiz de Montellano, Paul R.

2010-01-01

Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides; isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides, and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 μM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design. PMID:19694439

Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.

PubMed

Robinson, Philip J; Pringle, Marie Anne; Woolhead, Cheryl A; Bulleid, Neil J

2017-04-28

The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase*

PubMed Central

Albert, Armando; Yunta, Cristina; Arranz, Rocío; Peña, Álvaro; Salido, Eduardo; Valpuesta, José María; Martín-Benito, Jaime

2010-01-01

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism. PMID:20056599

Structure of GroEL in complex with an early folding intermediate of alanine glyoxylate aminotransferase.

PubMed

Albert, Armando; Yunta, Cristina; Arranz, Rocío; Peña, Alvaro; Salido, Eduardo; Valpuesta, José María; Martín-Benito, Jaime

2010-02-26

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.

Nucleation via an unstable intermediate phase.

PubMed

Sear, Richard P

2009-08-21

The pathway for crystallization from dilute vapors and solutions is often observed to take a detour via a liquid or concentrated-solution phase. For example, in moist subzero air, droplets of liquid water form, which then freeze. In this example and in many others, an intermediate phase (here liquid water) is dramatically accelerating the kinetics of a phase transition between two other phases (water vapor and ice). Here we study this phenomenon via exact computer simulations of a simple lattice model. Surprisingly, we find that the rate of nucleation of the new equilibrium phase is actually fastest when the intermediate phase is slightly unstable in the bulk, i.e., has a slightly higher free energy than the phase we start in. Nucleation occurs at a concave part of the surface and microscopic amounts of the intermediate phase can form there even before the phase is stable in the bulk. As the nucleus of the equilibrium phase is microscopic, this allows nucleation to occur effectively in the intermediate phase before it is stable in the bulk.

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

The Oxygen Equilibrium of Mammalian Hemoglobin

PubMed Central

Roughton, F. J. W.

1965-01-01

The three chief physicochemical theories of the oxygen-hemoglobin equilibrium in vogue 40 years ago still influence current thought on the problem. Although the Hill theory lost its fundamental basis some 40 years ago, the famous empiric equation to which it gave rise is still much used, as a useful phenomenological expression, only involving two disposable constants. The Haldane theory, of which a difference in aggregation of oxygenated and deoxygenated hemoglobin was a fundamental feature, lay for many years dormant but has recently had an astonishing reawakening through the work on lamprey hemoglobin, which clearly reveals such differences in aggregation. Lamprey hemoglobin might thus be called a "Haldane type" hemoglobin. Adair's four-stage intermediate compound theory still seems applicable in the case of hemoglobins such as those of sheep, whose tetramer molecules do not tend to dissociate into dimers, and which might therefore be called "Adair type" hemoglobins. Horse and human hemoglobins appear to reveal both "Haldane" and "Adair" behaviour. The effects of pH, temperature, and protein concentration on the oxygen-equilibrium of sheep hemoglobin are summarised, and it is shown that, although the equilibrium curves are often isomorphous over their middle range, intensive work at the top and bottom of the curves reveals considerable differences in the relative effects of these factors on the several equilibrium constants of Adair's four intermediate equations. In the last section an account is given of preliminary experimental attempts to interpret the oxygen- and carbon monoxide—equilibrium curves of whole human blood, under physiological conditions in terms of the Adair intermediate compound hypothesis. PMID:5859923

Atomistic Picture for the Folding Pathway of a Hybrid-1 Type Human Telomeric DNA G-quadruplex

PubMed Central

Bian, Yunqiang; Tan, Cheng; Wang, Jun; Sheng, Yuebiao; Zhang, Jian; Wang, Wei

2014-01-01

In this work we studied the folding process of the hybrid-1 type human telomeric DNA G-quadruplex with solvent and ions explicitly modeled. Enabled by the powerful bias-exchange metadynamics and large-scale conventional molecular dynamic simulations, the free energy landscape of this G-DNA was obtained for the first time and four folding intermediates were identified, including a triplex and a basically formed quadruplex. The simulations also provided atomistic pictures for the structures and cation binding patterns of the intermediates. The results showed that the structure formation and cation binding are cooperative and mutually supporting each other. The syn/anti reorientation dynamics of the intermediates was also investigated. It was found that the nucleotides usually take correct syn/anti configurations when they form native and stable hydrogen bonds with the others, while fluctuating between two configurations when they do not. Misfolded intermediates with wrong syn/anti configurations were observed in the early intermediates but not in the later ones. Based on the simulations, we also discussed the roles of the non-native interactions. Besides, the formation process of the parallel conformation in the first two G-repeats and the associated reversal loop were studied. Based on the above results, we proposed a folding pathway for the hybrid-1 type G-quadruplex with atomistic details, which is new and more complete compared with previous ones. The knowledge gained for this type of G-DNA may provide a general insight for the folding of the other G-quadruplexes. PMID:24722458

The Unfolding and Refolding Reactions of Triosephosphate Isomerase from Trypanosoma Cruzi Follow Similar Pathways. Guanidinium Hydrochloride Studies

NASA Astrophysics Data System (ADS)

Vázquez-Contreras, Edgar; Pérez Hernández, Gerardo; Sánchez-Rebollar, Brenda Guadalupe; Chánez-Cárdenas, María Elena

2005-04-01

The unfolding and refolding reactions of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was studied under equilibrium conditions at increasing guanidinium hydrochloride concentrations. The changes in activity intrinsic fluorescence and far-ultraviolet circular dichroism as a function of denaturant were used as a quaternary, tertiary and secondary structural probes respectively. The change in extrinsic ANS fluorescence intensity was also investigated. The results show that the transition between the homodimeric native enzyme to the unfolded monomers (unfolding), and its inverse reaction (refolding) are described by similar pathways and two equilibrium intermediates were detected in both reactions. The mild denaturant concentrations intermediate is active and contains significant amount of secondary and tertiary structures. The medium denaturant concentrations intermediate is inactive and able to bind the fluorescent dye. This intermediates are maybe related with those observed in the denaturation pattern of TIMs from other species; the results are discussed in this context.

Evolution and polymorphism in the multilocus Levene model with no or weak epistasis.

PubMed

Bürger, Reinhard

2010-09-01

Evolution and the maintenance of polymorphism under the multilocus Levene model with soft selection are studied. The number of loci and alleles, the number of demes, the linkage map, and the degree of dominance are arbitrary, but epistasis is absent or weak. We prove that, without epistasis and under mild, generic conditions, every trajectory converges to a stationary point in linkage equilibrium. Consequently, the equilibrium and stability structure can be determined by investigating the much simpler gene-frequency dynamics on the linkage-equilibrium manifold. For a haploid species an analogous result is shown. For weak epistasis, global convergence to quasi-linkage equilibrium is established. As an application, the maintenance of multilocus polymorphism is explored if the degree of dominance is intermediate at every locus and epistasis is absent or weak. If there are at least two demes, then arbitrarily many multiallelic loci can be maintained polymorphic at a globally asymptotically stable equilibrium. Because this holds for an open set of parameters, such equilibria are structurally stable. If the degree of dominance is not only intermediate but also deme independent, and loci are diallelic, an open set of parameters yielding an internal equilibrium exists only if the number of loci is strictly less than the number of demes. Otherwise, a fully polymorphic equilibrium exists only nongenerically, and if it exists, it consists of a manifold of equilibria. Its dimension is determined. In the absence of genotype-by-environment interaction, however, a manifold of equilibria occurs for an open set of parameters. In this case, the equilibrium structure is not robust to small deviations from no genotype-by-environment interaction. In a quantitative-genetic setting, the assumptions of no epistasis and intermediate dominance are equivalent to assuming that in every deme directional selection acts on a trait that is determined additively, i.e., by nonepistatic loci with dominance. Some of our results are exemplified in this quantitative-genetic context. Copyright 2010 Elsevier Inc. All rights reserved.

Differential effects of antifungal agents on expression of genes related to formation of Candida albicans biofilms.

PubMed

Chatzimoschou, Athanasios; Simitsopoulou, Maria; Antachopoulos, Charalampos; Walsh, Thomas J; Roilides, Emmanuel

2016-01-01

The purpose of this study was to analyse specific molecular mechanisms involved in the intrinsic resistance of C. albicans biofilms to antifungals. We investigated the transcriptional profile of three genes (BGL2, SUN41, ECE1) involved in Candida cell wall formation in response to voriconazole or anidulafungin after the production of intermediate and mature biofilms. C. albicans M61, a well-documented biofilm producer strain, was used for the development of intermediate (12 h and 18 h) and completely mature biofilms (48 h). After exposure of cells from each biofilm growth mode to voriconazole (128 and 512 mg l(-1)) or anidulafungin (0.25 and 1 mg l(-1)) for 12-24 h, total RNA samples extracted from biofilm cells were analysed by RT-PCR. The voriconazole and anidulafungin biofilm MIC was 512 and 0.5 mg l(-1) respectively. Anidulafungin caused significant up-regulation of SUN41 (3.7-9.3-fold) and BGL2 (2.2-2.8 fold) in intermediately mature biofilms; whereas, voriconazole increased gene expression in completely mature biofilms (SUN41 2.3-fold, BGL2 2.1-fold). Gene expression was primarily down-regulated by voriconazole in intermediately, but not completely mature biofilms. Both antifungals caused down-regulation of ECE1 in intermediately mature biofilms. © 2015 Blackwell Verlag GmbH.

[The accuracy of rapid equilibrium assumption in steady-state enzyme kinetics is the function of equilibrium segment structure and properties].

PubMed

Vrzheshch, P V

2015-01-01

Quantitative evaluation of the accuracy of the rapid equilibrium assumption in the steady-state enzyme kinetics was obtained for an arbitrary mechanism of an enzyme-catalyzed reaction. This evaluation depends only on the structure and properties of the equilibrium segment, but doesn't depend on the structure and properties of the rest (stationary part) of the kinetic scheme. The smaller the values of the edges leaving equilibrium segment in relation to values of the edges within the equilibrium segment, the higher the accuracy of determination of intermediate concentrations and reaction velocity in a case of the rapid equilibrium assumption.

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.

Cooperative alpha-helix formation of beta-lactoglobulin induced by sodium n-alkyl sulfates.

PubMed

Chamani, J; Moosavi-Movahedi, A A; Rajabi, O; Gharanfoli, M; Momen-Heravi, M; Hakimelahi, G H; Neamati-Baghsiah, A; Varasteh, A R

2006-01-01

It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of beta-lactoglobulin was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at special condition. The effect of n-alkyl sulfates on the structure of native beta-lactoglobulin at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of non-native alpha-helical intermediate. The addition of various concentrations of n-alkyl sulfates to the native state of beta-lactoglobulin (pH 2) appears to support the stabilized form of non-native alpha-helical intermediate at pH 2. The m values of the intermediate state of beta-lactoglobulin by SOS, SDeS, SDS and STS showed substantial variation. The enhancement of m values as the stability criterion of non-native alpha-helical intermediate state corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the folding reaction of beta-lactoglobulin follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the non-native alpha-helical intermediate state.

Factors controlling dimethylsulfide emission from salt marshes

NASA Technical Reports Server (NTRS)

Dacey, John W. H.; Wakeham, S. G.; Howes, B. L.

1985-01-01

The factors that control the emission of methylated gases from salt marshes are being studied. Research focusses on dimethylsulfide (DMS) formation and the mechanism of DMS and CH4 emission to the atmosphere. The approach is to consider the plants as valves regulating the emission of methylated gases to the atmosphere with the goal of developing appropriate methods for emission measurement. In the case of CH4, the sediment is the source and transport to the atmosphere occurs primarily through the internal gas spaces in the plants. The source of DMS appears to be dimethyl sulfoniopropionate (DMSP) which may play a role in osmoregulation in plant tissues. Concentrations of DMSP in leaves are typically several-fold higher than in roots and rhizomes. Even so, the large below ground biomass of this plant means that 2/3 of the DMSP in the ecosystem is below ground on the aerial basis. Upon introduction to sediment water, DMSP rapidly decomposes to DMS and acrylic acid. The solubility of a gas (its equilibrium vapor pressure) is a fundamental aspect of gas exchange kinetics. The first comprehensive study was conducted of DMS solubility in freshwater and seawater. Data suggest that the Setchenow relation holds for H at intermediate salinities collected. These data support the concept that the concentration of DMS in the atmosphere is far from equilibrium with seawater.

Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation.

PubMed

Goldberg, M E; Expert-Bezançon, N; Vuillard, L; Rabilloud, T

1996-01-01

Attempts to renature proteins often yield aggregates rather than native protein. To minimize aggregation, low protein concentrations and/or solubilizing agents are used. Here, we test new solubilizing molecules, non-detergent sulphobetaines, to improve the renaturation of two very different enzymes, hen egg white lysozyme and bacterial beta-D-galactosidase. The renaturation was conducted in the presence of five different sulphobetaines and the yield of active enzyme was measured. The five sulphobetaines improved the yield of native lysozyme up to 12-fold. Some sulphobetaines improved the yield of galactosidase up to 80-fold, but one reduced it 100-fold. Non-detergent sulphobetaines strongly affect the balance between aggregation and folding. Their effect depends on their structure and on their interactions with folding intermediates. These results should serve as a basis for designing more efficient sulphobetaines; for designing improved renaturation protocols using existing sulphobetaines; and for characterizing folding intermediates that interact with sulphobetaines.

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.

Analysis of MHC class I folding: novel insights into intermediate forms

PubMed Central

Simone, Laura C.; Tuli, Amit; Simone, Peter D.; Wang, Xiaojian; Solheim, Joyce C.

2012-01-01

Folding around a peptide ligand is integral to the antigen presentation function of major histocompatibility complex (MHC) class I molecules. Several lines of evidence indicate that the broadly cross-reactive 34-1-2 antibody is sensitive to folding of the MHC class I peptide-binding groove. Here, we show that peptide-loading complex proteins associated with the murine MHC class I molecule Kd are found primarily in association with the 34-1-2+ form. This led us to hypothesize that the 34-1-2 antibody may recognize intermediately, as well as fully, folded MHC class I molecules. In order to further characterize the form(s) of MHC class I molecules recognized by 34-1-2, we took advantage of its cross-reactivity with Ld. Recognition of the open and folded forms of Ld by the 64-3-7 and 30-5-7 antibodies, respectively, has been extensively characterized, providing us with parameters against which to compare 34-1-2 reactivity. We found that the 34-1-2+ Ld molecules displayed characteristics indicative of incomplete folding, including increased tapasin association, endoplasmic reticulum retention, and instability at the cell surface. Moreover, we demonstrate that an Ld-specific peptide induced folding of the 34-1-2+ Ld intermediate. Altogether, these results yield novel insights into the nature of MHC class I molecules recognized by the 34-1-2 antibody. PMID:22329842

Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence.

PubMed

Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B; Puckett, Latisha M; Phan, N H; Starch, Joel A; Okimoto, Mark R; Gidden, Jennifer; Stites, Wesley E; Lay, Jackson O

2012-12-15

Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured and denatured protein molecules are equilibrated at easily detectable ratios, namely 1:1. It also requires that closed and open states have lifetimes measurable in the time frame of the HDX experiment. Because both conditions are met by PEPS, these measurements can provide an accurate assessment of closed/open state populations and thus protein folding energies/rates.

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

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.

Native Contact Density and Nonnative Hydrophobic Effects in the Folding of Bacterial Immunity Proteins

PubMed Central

Chen, Tao; Chan, Hue Sun

2015-01-01

The bacterial colicin-immunity proteins Im7 and Im9 fold by different mechanisms. Experimentally, at pH 7.0 and 10°C, Im7 folds in a three-state manner via an intermediate but Im9 folding is two-state-like. Accordingly, Im7 exhibits a chevron rollover, whereas the chevron arm for Im9 folding is linear. Here we address the biophysical basis of their different behaviors by using native-centric models with and without additional transferrable, sequence-dependent energies. The Im7 chevron rollover is not captured by either a pure native-centric model or a model augmented by nonnative hydrophobic interactions with a uniform strength irrespective of residue type. By contrast, a more realistic nonnative interaction scheme that accounts for the difference in hydrophobicity among residues leads simultaneously to a chevron rollover for Im7 and an essentially linear folding chevron arm for Im9. Hydrophobic residues identified by published experiments to be involved in nonnative interactions during Im7 folding are found to participate in the strongest nonnative contacts in this model. Thus our observations support the experimental perspective that the Im7 folding intermediate is largely underpinned by nonnative interactions involving large hydrophobics. Our simulation suggests further that nonnative effects in Im7 are facilitated by a lower local native contact density relative to that of Im9. In a one-dimensional diffusion picture of Im7 folding with a coordinate- and stability-dependent diffusion coefficient, a significant chevron rollover is consistent with a diffusion coefficient that depends strongly on native stability at the conformational position of the folding intermediate. PMID:26016652

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.

Negative Charge Neutralization in the Loops and Turns of Outer Membrane Phospholipase A Impacts Folding Hysteresis at Neutral pH.

PubMed

McDonald, Sarah K; Fleming, Karen G

2016-11-08

Hysteresis in equilibrium protein folding titrations is an experimental barrier that must be overcome to extract meaningful thermodynamic quantities. Traditional approaches to solving this problem involve testing a spectrum of solution conditions to find ones that achieve path independence. Through this procedure, a specific pH of 3.8 was required to achieve path independence for the water-to-bilayer equilibrium folding of outer membrane protein OmpLA. We hypothesized that the neutralization of negatively charged side chains (Asp and Glu) at pH 3.8 could be the physical basis for path-independent folding at this pH. To test this idea, we engineered variants of OmpLA with Asp → Asn and Glu → Gln mutations to neutralize the negative charges within various regions of the protein and tested for reversible folding at neutral pH. Although not fully resolved, our results show that these mutations in the periplasmic turns and extracellular loops are responsible for 60% of the hysteresis in wild-type folding. Overall, our study suggests that negative charges impact the folding hysteresis in outer membrane proteins and their neutralization may aid in protein engineering applications.

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

Equilibrium unfolding of A. niger RNase: pH dependence of chemical and thermal denaturation.

PubMed

Kumar, Gundampati Ravi; Sharma, Anurag; Kumari, Moni; Jagannadham, Medicherla V; Debnath, Mira

2011-08-01

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A(MG1)) into another (I(MG2)).

General Equilibrium Models: Improving the Microeconomics Classroom

ERIC Educational Resources Information Center

Nicholson, Walter; Westhoff, Frank

2009-01-01

General equilibrium models now play important roles in many fields of economics including tax policy, environmental regulation, international trade, and economic development. The intermediate microeconomics classroom has not kept pace with these trends, however. Microeconomics textbooks primarily focus on the insights that can be drawn from the…

Chemical Equilibrium and Polynomial Equations: Beware of Roots.

ERIC Educational Resources Information Center

Smith, William R.; Missen, Ronald W.

1989-01-01

Describes two easily applied mathematical theorems, Budan's rule and Rolle's theorem, that in addition to Descartes's rule of signs and intermediate-value theorem, are useful in chemical equilibrium. Provides examples that illustrate the use of all four theorems. Discusses limitations of the polynomial equation representation of chemical…

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

Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

PubMed

Santra, Manas Kumar; Banerjee, Abhijit; Krishnakumar, Shyam Sundar; Rahaman, Obaidur; Panda, Dulal

2004-05-01

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.

Topological frustration in βα-repeat proteins: sequence diversity modulates the conserved folding mechanisms of α/β/α sandwich proteins

PubMed Central

Hills, Ronald D.; Kathuria, Sagar V.; Wallace, Louise A.; Day, Iain J.; Brooks, Charles L.; Matthews, C. Robert

2010-01-01

The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of non-local interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of βα–repeat proteins, which are dominated by local interactions, a combined experimental and simulation analysis was performed on two members of the flavodoxin-like, α/β/α sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured sub-millisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine and valine (ILV) side-chains that form a large sequence-local cluster involving the central β-sheet and helices α2, α3 and α4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side-chains in its corresponding hydrophobic cluster. The location of the hydrophobic clusters in the structure may also be related to the differing functional properties of these response regulators. Comparison with the results of previous experimental and simulation analyses on the homologous CheY argues that prematurely-folded unproductive intermediates are a common property of the βα-repeat motif. PMID:20226790

Modulating DNA configuration by interfacial traction: an elastic rod model to characterize DNA folding and unfolding.

PubMed

Huang, Zaixing

2011-01-01

As a continuum model of DNA, a thin elastic rod subjected to interfacial interactions is used to investigate the equilibrium configuration of DNA in intracellular solution. The interfacial traction between the rod and the solution environment is derived in detail. Kirchhoff's theory of elastic rods is used to analyze the equilibrium configuration of a DNA segment under the action of the interfacial traction. The influences of the interfacial energy factor and bending stiffness on the toroidal spool formation of the DNA segment are discussed. The results show that the equilibrium configuration of DNA is mainly determined by competition between the interfacial energy and elastic strain energy of the DNA itself, and the interfacial traction is one of the forces that drives DNA folding and unfolding.

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.

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

Facile chemical synthesis and equilibrium unfolding properties of CopG

PubMed Central

Wales, Thomas E.; Richardson, Jane S.; Fitzgerald, Michael C.

2004-01-01

The 45-amino acid polypeptide chain of the homodimeric transcriptional repressor, CopG, was chemically synthesized by stepwise solid phase peptide synthesis (SPPS) using a protocol based on Boc-chemistry. The product obtained from the synthesis was readily purified by reversed-phase HPLC to give a good overall yield (21% by weight). Moreover, the synthetic CopG constructs prepared in this work folded into three-dimensional structures similar to the wild-type protein prepared using conventional recombinant methods as judged by far UV-CD spectroscopy. A fluorescent CopG analog, (Y39W)CopG, was also designed and chemically synthesized to facilitate biophysical studies of CopG’s protein folding and assembly reaction. The guanidinium chloride-induced equilibrium unfolding properties of the wild-type CopG and (Y39W)CopG constructs in this work were characterized and used to develop a model for CopG’s equilibrium unfolding reaction. Our results indicate that CopG’s folding and assembly reaction is well modeled by a two-state process involving folded dimer and unfolded monomer. Using this model, ΔGf and m-values of −13.42 ± 0.04 kcal/mole dimer and 1.92 ± 0.01 kcal/(mole M) were calculated for CopG. PMID:15169951

Calculating Shocks In Flows At Chemical Equilibrium

NASA Technical Reports Server (NTRS)

Eberhardt, Scott; Palmer, Grant

1988-01-01

Boundary conditions prove critical. Conference paper describes algorithm for calculation of shocks in hypersonic flows of gases at chemical equilibrium. Although algorithm represents intermediate stage in development of reliable, accurate computer code for two-dimensional flow, research leading up to it contributes to understanding of what is needed to complete task.

Reversible Folding of Human Peripheral Myelin Protein 22, a Tetraspan Membrane Protein†

PubMed Central

Schlebach, Jonathan P.; Peng, Dungeng; Kroncke, Brett M.; Mittendorf, Kathleen F.; Narayan, Malathi; Carter, Bruce D.; Sanders, Charles R.

2013-01-01

Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in mixed micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22. PMID:23639031

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.

Molecular Dynamics of Peptide Folding at Aqueous Interfaces

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Chipot, Christophe; Chang, Sherwood (Technical Monitor)

1997-01-01

Even though most monomeric peptides are disordered in water they can adopt sequence-dependent, ordered structures, such as a-helices, at aqueous interfaces. This property is relevant to cellular signaling, membrane fusion, and the action of toxins and antibiotics. The mechanism of folding nonpolar peptides at the water-hexane interface was studied in the example of an 11-mer, of poly-L-leucine. Initially placed as a random coil on the water side of the interface, the peptide folded into an a-helix in 36 ns. Simultaneously, the peptide translocated into the hexane side of the interface. Folding was not sequential and involved a 3/10-helix as an intermediate. The folded peptide was either parallel to the interface or had its C-terminus exposed to water. An 11-mer, LQQLLQQLLQL, composed of leucine (L) and glutamine (G), was taken as a model amphiphilic peptide. It rapidly adopted an amphiphilic, disordered structure at the interface. Further folding proceeded through a series of amphiphilic intermediates.

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.

Effects of tethering a multistate folding protein to a surface

NASA Astrophysics Data System (ADS)

Wei, Shuai; Knotts, Thomas A.

2011-05-01

Protein/surface interactions are important in a variety of fields and devices, yet fundamental understanding of the relevant phenomena remains fragmented due to resolution limitations of experimental techniques. Molecular simulation has provided useful answers, but such studies have focused on proteins that fold through a two-state process. This study uses simulation to show how surfaces can affect proteins which fold through a multistate process by investigating the folding mechanism of lysozyme (PDB ID: 7LZM). The results demonstrate that in the bulk 7LZM folds through a process with four stable states: the folded state, the unfolded state, and two stable intermediates. The folding mechanism remains the same when the protein is tethered to a surface at most residues; however, in one case the folding mechanism changes in such a way as to eliminate one of the intermediates. An analysis of the molecular configurations shows that tethering at this site is advantageous for protein arrays because the active site is both presented to the bulk phase and stabilized. Taken as a whole, the results offer hope that rational design of protein arrays is possible once the behavior of the protein on the surface is ascertained.

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.

An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins

PubMed Central

Cao, Zheng; Bowie, James U

2014-01-01

Equilibrium H/D fractionation factors have been extensively employed to qualitatively assess hydrogen bond strengths in protein structure, enzyme active sites, and DNA. It remains unclear how fractionation factors correlate with hydrogen bond free energies, however. Here we develop an empirical relationship between fractionation factors and free energy, allowing for the simple and quantitative measurement of hydrogen bond free energies. Applying our empirical relationship to prior fractionation factor studies in proteins, we find: [1] Within the folded state, backbone hydrogen bonds are only marginally stronger on average in α-helices compared to β-sheets by ∼0.2 kcal/mol. [2] Charge-stabilized hydrogen bonds are stronger than neutral hydrogen bonds by ∼2 kcal/mol on average, and can be as strong as –7 kcal/mol. [3] Changes in a few hydrogen bonds during an enzyme catalytic cycle can stabilize an intermediate state by –4.2 kcal/mol. [4] Backbone hydrogen bonds can make a large overall contribution to the energetics of conformational changes, possibly playing an important role in directing conformational changes. [5] Backbone hydrogen bonding becomes more uniform overall upon ligand binding, which may facilitate participation of the entire protein structure in events at the active site. Our energetic scale provides a simple method for further exploration of hydrogen bond free energies. PMID:24501090

An Oligomeric Equilibrium Intermediate as the Precursory Nucleus of Globular and Fibrillar Supramacromolecular Assemblies in a PDZ Domain

PubMed Central

Murciano-Calles, Javier; Cobos, Eva S.; Mateo, Pedro L.; Camara-Artigas, Ana; Martinez, Jose C.

2010-01-01

Abstract The equilibrium unfolding at neutral pH of the third PDZ domain of PSD95, as followed by DSC, is characterized by the presence of an equilibrium intermediate with clear signs of oligomerization. DLS and SEC measurements indicate that at 60–70°C small oligomers populate, showing a typical β-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of ∼12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of ∼50 nm and protofibrils, which are subsequently fully transformed into fibrils. The fibrils can also disaggregate after the addition of 1:1 buffer dilution followed by cooling to room temperature, thus returning to the initial monomeric state. Growth kinetics, as shown by ThT and ANS fluorescence, show that the organization of the different supramacromolecular structures comes from a common nucleation unit, the small oligomers, which organize themselves before reaching the incubation temperature of 60°C. Our experiments point toward the existence of a well-defined reversible, stepwise, and downhill organization of the processes involved in the association-dissociation of the intermediate. We estimate the enthalpy change accompanying the association-dissociation equilibria to be 130 kJ × mol−1. Furthermore, the coalescence under essentially reversible conditions of different kinds of supramacromolecular assemblies renders this protein system highly interesting for biophysical studies aimed at our further understanding of amyloid pathological conditions. PMID:20655855

Conformation and Dynamics of Human Urotensin II and Urotensin Related Peptide in Aqueous Solution.

PubMed

Haensele, Elke; Mele, Nawel; Miljak, Marija; Read, Christopher M; Whitley, David C; Banting, Lee; Delépée, Carla; Sopkova-de Oliveira Santos, Jana; Lepailleur, Alban; Bureau, Ronan; Essex, Jonathan W; Clark, Timothy

2017-02-27

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, open and folded, with rapidly interchanging subtypes. The open states are characterized by turns of various types centered at K 8 Y 9 or F 6 W 7 predominantly with no or only sparsely populated transannular hydrogen bonds. The folded conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F 6 W 7 K 8 or W 7 K 8 Y 9 . Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% open:28% folded conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an open:folded equilibrium shifted further toward open conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR.

Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli

PubMed Central

2005-01-01

MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5′-phosphate) enzyme acting as a β-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2–4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation. PMID:15823094

Crystal structure of group II intron domain 1 reveals a template for RNA assembly

DOE PAGES

Zhao, Chen; Rajashankar, Kanagalaghatta R.; Marcia, Marco; ...

2015-10-26

Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. In this paper, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Composed of domain 1 from the Oceanobacillus iheyensis group II intron (266 nucleotides), this intermediate retains native-like features but adopts a compact conformation in which the active site cleft is closed. Transition between this closed andmore » the open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. Finally, the open state is then stabilized by sequential docking of downstream intron domains, suggesting a 'first come, first folded' strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures.« less

[Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States].

PubMed

Melnik, T N; Nagibina, G S; Surin, A K; Glukhova, K A; Melnik, B S

2018-01-01

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

[Effect of hypoxia on glycolysis in the adductor muscle and hepatopancreas of the marine mussel Mytilus galloprovincialis Lmk].

PubMed

Ibarguren, I; Villamarín, J A; Barcia, R; Ramos-Martínez, J I

1989-12-01

Concentrations of glycolytic intermediates and adenine nucleotides have been estimated in adductor muscle and hepatopancreas from the sea mussel Mytilus galloprovincialis Lmk. after various periods of valve closure. Mass action ratios of enzyme steps involved in the metabolism of these components are compared with their equilibrium constants. This reveals hexokinase, phosphofructokinase, pyruvate kinase and fructose-1,6-bisphosphatase catalyze non-equilibrium reactions. The changes in the concentrations of the glycolytic intermediates and in the rate M.A.R./Keq during hypoxia suggest that the carbon flow after valve closure is first controlled by phophofructokinase, but later on the rate of transformation of phosphoenolyruvate regulates this flow.

Non-equilibrium electrokinetic micromixer with 3D nanochannel networks.

PubMed

Choi, Eunpyo; Kwon, Kilsung; Lee, Seung Jun; Kim, Daejoong; Park, Jungyul

2015-04-21

We report an active micromixer which utilizes vortex generation due to non-equilibrium electrokinetics near the interface between a microchannnel and a nanochannel networks membrane (NCNM), constructed from geometrically controlled in situ self-assembled nanoparticles. A large interfacing area where it is possible to generate vortices can be realized, because nano-interstices between the assembled nanoparticles are intrinsically collective three-dimensional nanochannel networks, which may be compared to typical silicon-based 2D nanochannels. The proposed mixer shows a 2-fold shorter mixing time (~0.78 ms) and a 34-fold shorter mixing length (~7.86 μm) compared to conventional 2D nanochannels.

Hidden Structural Codes in Protein Intrinsic Disorder.

PubMed

Borkosky, Silvia S; Camporeale, Gabriela; Chemes, Lucía B; Risso, Marikena; Noval, María Gabriela; Sánchez, Ignacio E; Alonso, Leonardo G; de Prat Gay, Gonzalo

2017-10-17

Intrinsic disorder is a major structural category in biology, accounting for more than 30% of coding regions across the domains of life, yet consists of conformational ensembles in equilibrium, a major challenge in protein chemistry. Anciently evolved papillomavirus genomes constitute an unparalleled case for sequence to structure-function correlation in cases in which there are no folded structures. E7, the major transforming oncoprotein of human papillomaviruses, is a paradigmatic example among the intrinsically disordered proteins. Analysis of a large number of sequences of the same viral protein allowed for the identification of a handful of residues with absolute conservation, scattered along the sequence of its N-terminal intrinsically disordered domain, which intriguingly are mostly leucine residues. Mutation of these led to a pronounced increase in both α-helix and β-sheet structural content, reflected by drastic effects on equilibrium propensities and oligomerization kinetics, and uncovers the existence of local structural elements that oppose canonical folding. These folding relays suggest the existence of yet undefined hidden structural codes behind intrinsic disorder in this model protein. Thus, evolution pinpoints conformational hot spots that could have not been identified by direct experimental methods for analyzing or perturbing the equilibrium of an intrinsically disordered protein ensemble.

Effects of the impurity-host interactions on the nonradiative processes in ZnS:Cr

NASA Astrophysics Data System (ADS)

Tablero, C.

2010-11-01

There is a great deal of controversy about whether the behavior of an intermediate band in the gap of semiconductors is similar or not to the deep-gap levels. It can have significant consequences, for example, on the nonradiative recombination. In order to analyze the behavior of an intermediate band, we have considered the effect of the inward and outward displacements corresponding to breathing and longitudinal modes of Cr-doped ZnS and on the charge density for different processes involved in the nonradiative recombination using first-principles. This metal-doped zinc chalcogenide has a partially filled band within the host semiconductor gap. In contrast to the properties exhibited by deep-gap levels in other systems, we find small variations in the equilibrium configurations, forces, and electronic density around the Cr when the nonradiative recombination mechanisms modify the intermediate band charge. The charge density around the impurity is equilibrated in response to the perturbations in the equilibrium nuclear configuration and the charge of the intermediate band. The equilibration follows a Le Chatelier principle through the modification of the contribution from the impurity to the intermediate band and to the valence band. The intermediate band introduced by Cr in ZnS for the concentrations analyzed makes the electronic capture difficult and later multiphonon emission in the charge-transfer processes, in accordance with experimental results.

The stability of monomeric intermediates controls amyloid formation: Abeta25-35 and its N27Q mutant.

PubMed

Ma, Buyong; Nussinov, Ruth

2006-05-15

The structure and stabilities of the intermediates affect protein folding as well as misfolding and amyloid formation. By applying Kramer's theory of barrier crossing and a Morse-function-like energy landscape, we show that intermediates with medium stability dramatically increase the rate of amyloid formation; on the other hand, very stable and very unstable intermediates sharply decrease amyloid formation. Remarkably, extensive molecular dynamics simulations and conformational energy landscape analysis of Abeta25-35 and its N27Q mutant corroborate the mathematical description. Both experimental and current simulation results indicate that the core of the amyloid structure of Abeta25-35 formed from residues 28-35. A single mutation of N27Q of Abeta25-35 makes the Abeta25-35 N27Q amyloid-free. Energy landscape calculations show that Abeta25-35 has extended intermediates with medium stability that are prone to form amyloids, whereas the extended intermediates for Abeta25-35 N27Q split into stable and very unstable species that are not disposed to form amyloids. The results explain the contribution of both alpha-helical and beta-strand intermediates to amyloid formation. The results also indicate that the structure and stability of the intermediates, as well as of the native folded and the amyloid states can be targeted in drug design. One conceivable approach is to stabilize the intermediates to deter amyloid formation.

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.

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.

Equilibrium denaturation and preferential interactions of an RNA tetraloop with urea

DOE PAGES

Miner, Jacob Carlson; García, Angel Enrique

2017-02-09

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarilymore » show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Lastly, our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.« less

Equilibrium Denaturation and Preferential Interactions of an RNA Tetraloop with Urea.

PubMed

Miner, Jacob C; García, Angel E

2017-04-20

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarily show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.

Equilibrium denaturation and preferential interactions of an RNA tetraloop with urea

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

Miner, Jacob Carlson; García, Angel Enrique

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarilymore » show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Lastly, our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.« less

Dynamical properties of α-amylase in the folded and unfolded state: the role of thermal equilibrium fluctuations for conformational entropy and protein stabilisation

NASA Astrophysics Data System (ADS)

Fitter, J.; Herrmann, R.; Hauß, T.; Lechner, R. E.; Dencher, N. A.

2001-07-01

A comparative analysis of thermal equilibrium fluctuations occurring in a mesophilic and in a thermophilic α-amylase was performed to study the effect of structural fluctuations on thermostability. The thermal fluctuations determining the conformational entropy of both enzymes have been characterised for the folded (at 30°C and 60°C) and for the unfolded state by applying neutron spectroscopy (at 30°C). The folded state shows a higher structural flexibility for the thermophilic protein as compared to the mesophilic homologue. In contrast, the unfolded state of both enzymes is rather similar with respect to the structural fluctuations. On the basis of this result, a mechanism characterised by entropic stabilisation (i.e., smaller Δ S for the unfolding transition of thermophilic α-amylase) can be assumed to be responsible for the higher thermostability of the thermophilic enzyme.

Computational Insights into the Stability and Folding Pathways of Human Telomeric DNA G-Quadruplexes.

PubMed

Luo, Di; Mu, Yuguang

2016-06-09

G-quadruplex is a noncanonical yet crucial secondary structure of nucleic acids, which has proven its importance in cell aging, anticancer therapies, gene expression, and genome stability. In this study, the stability and folding dynamics of human telomeric DNA G-quadruplexes were investigated via enhanced sampling techniques. First, temperature-replica exchange MD (REMD) simulations were employed to compare the thermal stabilities among the five established folding topologies. The hybrid-2 type adopted by extended human telomeric sequence is revealed to be the most stable conformation in our simulations. Next, the free energy landscapes and folding intermediates of the hybrid-1 and -2 types were investigated with parallel tempering metadynamics simulations in the well-tempered ensemble. It was observed that the N-glycosidic conformations of guanines can flip over to accommodate into the cyclic Hoogsteen H-bonding on G-tetrads in which they were not originally involved. Furthermore, a hairpin and a triplex intermediate were identified for the folding of the hybrid-1 type conformation, whereas for the hybrid-2 type, there were no folding intermediates observed from its free energy surface. However, the energy barrier from its native topology to the transition structure is found to be extremely high compared to that of the hybrid-1 type, which is consistent with our stability predictions from the REMD simulations. We hope the insights presented in this work can help to complement current understanding on the stability and dynamics of G-quadruplexes, which is necessary not only to stabilize the structures but also to intervene their formation in genome.

Experimental Phase Equilibria Studies of the Pb-Fe-O System in Air, in Equilibrium with Metallic Lead and at Intermediate Oxygen Potentials

NASA Astrophysics Data System (ADS)

Shevchenko, M.; Jak, E.

2017-12-01

The phase equilibria information on the Pb-Fe-O system is of practical importance for the improvement of the existing thermodynamic database of lead-containing slag systems (Pb-Zn-Fe-Cu-Si-Ca-Al-Mg-O). Phase equilibria of the Pb-Fe-O system have been investigated: (a) in air at temperatures between 1053 K and 1373 K (780 °C and 1100 °C); (b) in equilibrium with metallic lead at temperatures between 1053 K and 1373 K (780 °C and 1100 °C); and (c) at intermediate oxidation conditions for the liquid slag in equilibrium with two solids (spinel + magnetoplumbite), at temperatures between 1093 K and 1373 K (820 °C and 1100 °C). The high-temperature equilibration/quenching/electron probe X-ray microanalysis technique has been used to accurately determine the compositions of the phases in equilibrium in the system. The Pb and Fe concentrations in the phases were determined directly; preliminary thermodynamic modeling with FactSage was used to estimate the ferrous-to-ferric ratios and to present the results in the ternary diagram.

Intermediates detected by visible spectroscopy during the reaction of nitrite with deoxyhemoglobin: the effect of nitrite concentration and diphosphoglycerate.

PubMed

Nagababu, Enika; Ramasamy, Somasundaram; Rifkind, Joseph M

2007-10-16

The reaction of nitrite with deoxyhemoglobin (deoxyHb) results in the reduction of nitrite to NO, which binds unreacted deoxyHb forming Fe(II)-nitrosylhemoglobin (Hb(II)NO). The tight binding of NO to deoxyHb is, however, inconsistent with reports implicating this reaction with hypoxic vasodilation. This dilemma is resolved by the demonstration that metastable intermediates are formed in the course of the reaction of nitrite with deoxyHb. The level of intermediates is quantitated by the excess deoxyHb consumed over the concentrations of the final products formed. The dominant intermediate has a spectrum that does not correspond to that of Hb(III)NO formed when NO reacts with methemoglobin (MetHb), but is similar to metHb resulting in the spectroscopic determinations of elevated levels of metHb. It is a delocalized species involving the heme iron, the NO, and perhaps the beta-93 thiol. The putative role for red cell reacted nitrite on vasodilation is associated with reactions involving the intermediate. (1) The intermediate is less stable with a 10-fold excess of nitrite and is not detected with a 100-fold excess of nitrite. This observation is attributed to the reaction of nitrite with the intermediate producing N2O3. (2) The release of NO quantitated by the formation of Hb(II)NO is regulated by changes in the distal heme pocket as shown by the 4.5-fold decrease in the rate constant in the presence of 2,3-diphosphoglycerate. The regulated release of NO or N2O3 as well as the formation of the S-nitroso derivative of hemoglobin, which has also been reported to be formed from the intermediates generated during nitrite reduction, should be associated with any hypoxic vasodilation attributed to the RBC.

Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase.

PubMed

Borén, Kristina; Grankvist, Hannah; Hammarström, Per; Carlsson, Uno

2004-05-21

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.

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

Characterization of folding intermediates during urea-induced denaturation of human carbonic anhydrase II.

PubMed

Wahiduzzaman; Dar, Mohammad Aasif; Haque, Md Anzarul; Idrees, Danish; Hassan, Md Imtaiyaz; Islam, Asimul; Ahmad, Faizan

2017-02-01

Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29kDa, β-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e., N (native) ↔ X I ↔ X II ↔ D (denatured) with stable intermediates X I and X II populated around 2 and 4M urea, respectively. The far-UV CD was used to characterize the intermediate states (X I and X II ) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced X I state has characteristics of molten globule state while X II state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Hexafluoroisopropanol-induced helix-sheet transition of stem bromelain: correlation to function.

PubMed

Dave, Sandeep; Dkhar, H Kitdorlang; Singh, Manvendra Pratap; Gupta, Garima; Chandra, Vemika; Mahajan, Sahil; Gupta, Pawan

2010-06-01

Stem bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10-30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an alpha-helix to a beta-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in stem bromelain to a form with potentially useful therapeutic properties different from those of the native protein. Copyright 2010 Elsevier Ltd. All rights reserved.

OneG: A Computational Tool for Predicting Cryptic Intermediates in the Unfolding Kinetics of Proteins under Native Conditions

PubMed Central

Richa, Tambi; Sivaraman, Thirunavukkarasu

2012-01-01

Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (ΔG) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (ΔGU) and the free energy of exchange (ΔGHX) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ΔGU and ΔGHX of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ΔGU, ΔGU * and residue-specific ΔGHX determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b562 and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.html PMID:22412877

Protein Folding Free Energy Landscape along the Committor - the Optimal Folding Coordinate.

PubMed

Krivov, Sergei V

2018-06-06

Recent advances in simulation and experiment have led to dramatic increases in the quantity and complexity of produced data, which makes the development of automated analysis tools very important. A powerful approach to analyze dynamics contained in such data sets is to describe/approximate it by diffusion on a free energy landscape - free energy as a function of reaction coordinates (RC). For the description to be quantitatively accurate, RCs should be chosen in an optimal way. Recent theoretical results show that such an optimal RC exists; however, determining it for practical systems is a very difficult unsolved problem. Here we describe a solution to this problem. We describe an adaptive nonparametric approach to accurately determine the optimal RC (the committor) for an equilibrium trajectory of a realistic system. In contrast to alternative approaches, which require a functional form with many parameters to approximate an RC and thus extensive expertise with the system, the suggested approach is nonparametric and can approximate any RC with high accuracy without system specific information. To avoid overfitting for a realistically sampled system, the approach performs RC optimization in an adaptive manner by focusing optimization on less optimized spatiotemporal regions of the RC. The power of the approach is illustrated on a long equilibrium atomistic folding simulation of HP35 protein. We have determined the optimal folding RC - the committor, which was confirmed by passing a stringent committor validation test. It allowed us to determine a first quantitatively accurate protein folding free energy landscape. We have confirmed the recent theoretical results that diffusion on such a free energy profile can be used to compute exactly the equilibrium flux, the mean first passage times, and the mean transition path times between any two points on the profile. We have shown that the mean squared displacement along the optimal RC grows linear with time as for simple diffusion. The free energy profile allowed us to obtain a direct rigorous estimate of the pre-exponential factor for the folding dynamics.

NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.

PubMed Central

De Lorimier, R.; Hellinga, H. W.; Spicer, L. D.

1996-01-01

Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two- and three-dimensional NMR. Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain. The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout. PMID:8976564

Understanding curcumin-induced modulation of protein aggregation.

PubMed

Ahmad, Basir; Borana, Mohanish S; Chaudhary, Ankur P

2017-07-01

Curcumin, a diarylheptanoid compound, found in spice turmeric is known to alter the aggregation of proteins and reduce the toxicity of the aggregates. This review looks at the molecular basis of modulating protein aggregation and toxicity of the aggregates. Foremost, we identify the interaction of curcumin and its derivatives with proteins/peptides and the effect of their interaction on the conformational stability and unfolding/folding pathway(s). The unfolding/folding processes generate partially folded/unfolded intermediate, which serve as aggregation precursor state. Secondly, we discuss the effect of curcumin binding on the kinetics parameters of the aggregation process, which give information about the mechanism of the aggregation inhibition. We describe, in addition, that curcumin can accelerate/promote fibril formation by binding to oligomeric intermediate(s) accumulated in the aggregation pathway. Finally, we discuss the correlation of curcumin-induced monomeric and/or oligomeric precursor states with aggregate structure and toxicity. On the basis of these discussions, we propose a model describing curcumin-induced inhibition/promotion of formation of amyloid-like fibrils. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Quantification of change in vocal fold tissue stiffness relative to depth of artificial damage.

PubMed

Rohlfs, Anna-Katharina; Schmolke, Sebastian; Clauditz, Till; Hess, Markus; Müller, Frank; Püschel, Klaus; Roemer, Frank W; Schumacher, Udo; Goodyer, Eric

2017-10-01

To quantify changes in the biomechanical properties of human excised vocal folds with defined artificial damage. The linear skin rheometer (LSR) was used to obtain a series of rheological measurements of shear modulus from the surface of 30 human cadaver vocal folds. The tissue samples were initially measured in a native condition and then following varying intensities of thermal damage. Histological examination of each vocal fold was used to determine the depth of artificial alteration. The measured changes in stiffness were correlated with the depth of cell damage. For vocal folds in a pre-damage state the shear modulus values ranged from 537 Pa to 1,651 Pa (female) and from 583 Pa to 1,193 Pa (male). With increasing depth of damage from the intermediate layer of the lamina propria (LP), tissue stiffness increased consistently (compared with native values) following application of thermal damage to the vocal folds. The measurement showed an increase of tissue stiffness when the depth of tissue damage was extending from the intermediate LP layer downwards. Changes in the elastic characteristics of human vocal fold tissue following damage at defined depths were demonstrated in an in vitro experiment. In future, reproducible in vivo measurements of elastic vocal fold tissue alterations may enable phonosurgeons to infer the extent of subepithelial damage from changes in surface elasticity.

Role of Tryptophan Side Chain Dynamics on the Trp-Cage Mini-Protein Folding Studied by Molecular Dynamics Simulations

PubMed Central

Kannan, Srinivasaraghavan; Zacharias, Martin

2014-01-01

The 20 residue Trp-cage mini-protein is one of smallest proteins that adopt a stable folded structure containing also well-defined secondary structure elements. The hydrophobic core is arranged around a single central Trp residue. Despite several experimental and simulation studies the detailed folding mechanism of the Trp-cage protein is still not completely understood. Starting from fully extended as well as from partially folded Trp-cage structures a series of molecular dynamics simulations in explicit solvent and using four different force fields was performed. All simulations resulted in rapid collapse of the protein to on average relatively compact states. The simulations indicate a significant dependence of the speed of folding to near-native states on the side chain rotamer state of the central Trp residue. Whereas the majority of intermediate start structures with the central Trp side chain in a near-native rotameric state folded successfully within less than 100 ns only a fraction of start structures reached near-native folded states with an initially non-native Trp side chain rotamer state. Weak restraining of the Trp side chain dihedral angles to the state in the folded protein resulted in significant acceleration of the folding both starting from fully extended or intermediate conformations. The results indicate that the side chain conformation of the central Trp residue can create a significant barrier for controlling transitions to a near native folded structure. Similar mechanisms might be of importance for the folding of other protein structures. PMID:24563686

Magnetospheric equilibrium configurations and slow adiabatic convection

NASA Technical Reports Server (NTRS)

Voigt, Gerd-Hannes

1986-01-01

This review paper demonstrates how the magnetohydrostatic equilibrium (MHE) theory can be used to describe the large-scale magnetic field configuration of the magnetosphere and its time evolution under the influence of magnetospheric convection. The equilibrium problem is reviewed, and levels of B-field modelling are examined for vacuum models, quasi-static equilibrium models, and MHD models. Results from two-dimensional MHE theory as they apply to the Grad-Shafranov equation, linear equilibria, the asymptotic theory, magnetospheric convection and the substorm mechanism, and plasma anisotropies are addressed. Results from three-dimensional MHE theory are considered as they apply to an intermediate analytical magnetospheric model, magnetotail configurations, and magnetopause boundary conditions and the influence of the IMF.

Frailty as a Novel Predictor of Mortality and Hospitalization in Hemodialysis Patients of All Ages

PubMed Central

McAdams-DeMarco, Mara A.; Law, Andrew; Salter, Megan L.; Boyarsky, Brian; Gimenez, Luis; Jaar, Bernard G.; Walston, Jeremy D.; Segev, Dorry L.

2013-01-01

Objectives To quantify the prevalence of frailty in adult patients of all ages undergoing chronic hemodialysis, its relationship to comorbidity and disability, and its association with adverse outcomes of mortality and hospitalization. Design Prospective cohort study. Setting Single hemodialysis center in Baltimore, Maryland. Participants 146 prevalent hemodialysis patients enrolled between January 2009 and March 2010 and followed through August 2012. Measurements Frailty, comorbidity, and disability on enrollment into the study and subsequent mortality and hospitalizations. Results At enrollment, 50.0% of older (age≥65) and 35.4% of younger (age<65) hemodialysis patients were frail; 35.9% and 29.3% were intermediately frail, respectively. The 3-year mortality was 16.2% for non frail, 34.4% for intermediately frail, and 40.2% for frail participants. Intermediate frailty and frailty were associated with a 2.68-fold (95% CI: 1.02-7.07, P=0.046) and 2.60-fold (95%CI: 1.04-6.49, P=0.041) higher risk of death independent of age, sex, comorbidity, and disability. In the year after enrollment, median number of hospitalizations was 1 (IQR 0-3). The proportion with 2 or more hospitalizations was 28.2% for non frail, 25.5% for intermediately frail, and 42.6% for frail participants. While intermediate frailty was not associated with the number of hospitalizations (RR=0.76, 95%CI:0.49-1.16, P=0.21), frailty was associated with a 1.43-fold (95%CI:1.00-2.03, P=0.049) higher number of hospitalizations independent of age, sex, comorbidity, and disability. The association of frailty with mortality and hospitalizations did not differ between older and younger participants (Interaction P=0.64 and P=0.14, respectively). Conclusions Adults of all ages undergoing hemodialysis have a very high prevalence of frailty, more than 5-fold higher than community dwelling older adults. In this population, regardless of age, frailtyis a strong, independent predictor of mortality and number of hospitalizations. PMID:23711111

Antibiotic Effects on Methicillin-Resistant Staphylococcus aureus Cytoplasmic Peptidoglycan Intermediate Levels and Evidence for Potential Metabolite Level Regulatory Loops.

PubMed

Vemula, Harika; Ayon, Navid J; Burton, Alloch; Gutheil, William G

2017-06-01

Cytoplasmic peptidoglycan (PG) precursor levels were determined in methicillin-resistant Staphylococcus aureus (MRSA) after exposure to several cell wall-targeting antibiotics. Three experiments were performed: (i) exposure to 4× MIC levels (acute); (ii) exposure to sub-MIC levels (subacute); (iii) a time course experiment of the effect of vancomycin. In acute exposure experiments, fosfomycin increased UDP-GlcNAc, as expected, and resulted in substantially lower levels of total UDP-linked metabolite accumulation relative to other pathway inhibitors, indicating reduced entry into this pathway. Upstream inhibitors (fosfomycin, d-cycloserine, or d-boroalanine) reduced UDP-MurNAc-pentapeptide levels by more than fourfold. Alanine branch inhibitors (d-cycloserine and d-boroalanine) reduced d-Ala-d-Ala levels only modestly (up to 4-fold) but increased UDP-MurNAc-tripeptide levels up to 3,000-fold. Downstream pathway inhibitors (vancomycin, bacitracin, moenomycin, and oxacillin) increased UDP-MurNAc-pentapeptide levels up to 350-fold and UDP-MurNAc-l-Ala levels up to 80-fold, suggesting reduced MurD activity by downstream inhibitor action. Sub-MIC exposures demonstrated effects even at 1/8× MIC which strongly paralleled acute exposure changes. Time course data demonstrated that UDP-linked intermediate levels respond rapidly to vancomycin exposure, with several intermediates increasing three- to sixfold within minutes. UDP-linked intermediate level changes were also multiphasic, with some increasing, some decreasing, and some increasing and then decreasing. The total (summed) UDP-linked intermediate pool increased by 1,475 μM/min during the first 10 min after vancomycin exposure, providing a revised estimate of flux in this pathway during logarithmic growth. These observations outline the complexity of PG precursor response to antibiotic exposure in MRSA and indicate likely sites of regulation (entry and MurD). Copyright © 2017 American Society for Microbiology.

A two-layer composite model of the vocal fold lamina propria for fundamental frequency regulation.

PubMed

Zhang, Kai; Siegmund, Thomas; Chan, Roger W

2007-08-01

The mechanical properties of the vocal fold lamina propria, including the vocal fold cover and the vocal ligament, play an important role in regulating the fundamental frequency of human phonation. This study examines the equilibrium hyperelastic tensile deformation behavior of cover and ligament specimens isolated from excised human larynges. Ogden's hyperelastic model is used to characterize the tensile stress-stretch behaviors at equilibrium. Several statistically significant differences in the mechanical response differentiating cover and ligament, as well as gender are found. Fundamental frequencies are predicted from a string model and a beam model, both accounting for the cover and the ligament. The beam model predicts nonzero F(0) for the unstretched state of the vocal fold. It is demonstrated that bending stiffness significantly contributes to the predicted F(0), with the ligament contributing to a higher F(0), especially in females. Despite the availability of only a small data set, the model predicts an age dependence of F(0) in males in agreement with experimental findings. Accounting for two mechanisms of fundamental frequency regulation--vocal fold posturing (stretching) and extended clamping--brings predicted F(0) close to the lower bound of the human phonatory range. Advantages and limitations of the current model are discussed.

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.

Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.

PubMed

Chadda, R; Robertson, J L

2016-01-01

Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F - ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl - /H + antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known. © 2016 Elsevier Inc. All rights reserved.

Isometric torque-angle relationship and movement-related activity of human elbow flexors: implications for the equilibrium-point hypothesis.

PubMed

Hasan, Z; Enoka, R M

1985-01-01

Since the moment arms for the elbow-flexor muscles are longest at intermediate positions of the elbow and shorter at the extremes of the range of motion, it was expected that the elbow torque would also show a peak at an intermediate angle provided the activity of the flexor muscles remained constant. We measured the isometric elbow torque at different elbow angles while the subject attempted to keep constant the electromyographic activity (EMG) of the brachioradialis muscle. The torque-angle relationship thus obtained exhibited a peak, as expected, but the shape of the relationship varied widely among subjects. This was due in part to differences in the variation of the biceps brachii EMG with elbow angle among the different subjects. The implications of these observations for the equilibrium-point hypothesis of movement were investigated as follows. The subject performed elbow movements in the presence of an external torque (which tended to extend the elbow joint) provided by a weight-and-pulley arrangement. We found in the case of flexion movements that invariably there was a transient increase in flexor EMG, as would seem necessary for initiating the movement. However, the steady-state EMG after the movement could be greater or less than the pre-movement EMG. Specifically, the least flexor EMG was required for equilibrium in the intermediate range of elbow angles, compared to the extremes of the range of motion. The EMG-angle relationship, however, varied with the muscle and the subject. The observation that the directions of change in the transient and the steady-state EMG are independent of each other militates against the generality of the equilibrium-point hypothesis. However, a form of the hypothesis which includes the effects of the stretch reflex is not contradicted by this observation.

Multiple intermediates on the energy landscape of a 15-HEAT-repeat protein

PubMed Central

Tsytlonok, Maksym; Craig, Patricio O.; Sivertsson, Elin; Serquera, David; Perrett, Sarah; Best, Robert B.; Wolynes, Peter G.; Itzhaki, Laura S.

2014-01-01

Repeat proteins are a special class of modular, non-globular proteins composed of small structural motifs arrayed to form elongated architectures and stabilised solely by short-range contacts. We find a remarkable complexity in the unfolding of the large HEAT repeat protein PR65/A. In contrast to what has been seen for small repeat proteins in which unfolding propagates from one end, the HEAT array of PR65/A ruptures at multiple distant sites, leading to intermediate states with non-contiguous folded subdomains. Kinetic analysis allows us to define a network of intermediates and to delineate the pathways that connect them. There is a dominant sequence of unfolding, reflecting a non-uniform distribution of stability across the repeat array; however the unfolding of certain intermediates is competitive, leading to parallel pathways. Theoretical models accounting for the heterogeneous contact density in the folded structure are able to rationalize the variation in stability across the array. This variation in stability also suggests how folding may direct function in a large repeat protein: The stability distribution enables certain regions to present rigid motifs for molecular recognition while affording others flexibility to broaden the search area as in a fly-casting mechanism. Thus PR65/A uses the two ends of the repeat array to bind diverse partners and thereby coordinate the dephosphorylation of many different substrates and of multiple sites within hyperphosphorylated substrates. PMID:24120762

Formation of Covalently Modified Folding Intermediates of Simian Virus 40 Vp1 in Large T Antigen-Expressing Cells

PubMed Central

Watanabe, Marika; Phamduong, Ellen; Huang, Chu-Han; Itoh, Noriko; Bernal, Janie; Nakanishi, Akira; Rundell, Kathleen; Gjoerup, Ole

2013-01-01

The folding and pentamer assembly of the simian virus 40 (SV40) major capsid protein Vp1, which take place in the infected cytoplasm, have been shown to progress through disulfide-bonded Vp1 folding intermediates. In this report, we further demonstrate the existence of another category of Vp1 folding or assembly intermediates: the nonreducible, covalently modified mdVp1s. These species were present in COS-7 cells that expressed a recombinant SV40 Vp1, Vp1ΔC, through plasmid transfection. The mdVp1s persisted under cell and lysate treatment and SDS-PAGE conditions that are expected to have suppressed the formation of artifactual disulfide cross-links. As shown through a pulse-chase analysis, the mdVp1s were derived from the newly synthesized Vp1ΔC in the same time frame as Vp1's folding and oligomerization. The apparent covalent modifications occurred in the cytoplasm within the core region of Vp1 and depended on the coexpression of the SV40 large T antigen (LT) in the cells. Analogous covalently modified species were found with the expression of recombinant polyomavirus Vp1s and human papillomavirus L1s in COS-7 cells. Furthermore, the mdVp1s formed multiprotein complexes with LT, Hsp70, and Hsp40, and a fraction of the largest mdVp1, md4, was disulfide linked to the unmodified Vp1ΔC. Both mdVp1 formation and most of the multiprotein complex formation were blocked by a Vp1 folding mutation, C87A-C254A. Our observations are consistent with a role for LT in facilitating the folding process of SV40 Vp1 by stimulating certain covalent modifications of Vp1 or by recruiting certain cellular proteins. PMID:23427157

Simulating Metabolism with Statistical Thermodynamics

PubMed Central

Cannon, William R.

2014-01-01

New methods are needed for large scale modeling of metabolism that predict metabolite levels and characterize the thermodynamics of individual reactions and pathways. Current approaches use either kinetic simulations, which are difficult to extend to large networks of reactions because of the need for rate constants, or flux-based methods, which have a large number of feasible solutions because they are unconstrained by the law of mass action. This report presents an alternative modeling approach based on statistical thermodynamics. The principles of this approach are demonstrated using a simple set of coupled reactions, and then the system is characterized with respect to the changes in energy, entropy, free energy, and entropy production. Finally, the physical and biochemical insights that this approach can provide for metabolism are demonstrated by application to the tricarboxylic acid (TCA) cycle of Escherichia coli. The reaction and pathway thermodynamics are evaluated and predictions are made regarding changes in concentration of TCA cycle intermediates due to 10- and 100-fold changes in the ratio of NAD+:NADH concentrations. Finally, the assumptions and caveats regarding the use of statistical thermodynamics to model non-equilibrium reactions are discussed. PMID:25089525

Simulating metabolism with statistical thermodynamics.

PubMed

Cannon, William R

2014-01-01

New methods are needed for large scale modeling of metabolism that predict metabolite levels and characterize the thermodynamics of individual reactions and pathways. Current approaches use either kinetic simulations, which are difficult to extend to large networks of reactions because of the need for rate constants, or flux-based methods, which have a large number of feasible solutions because they are unconstrained by the law of mass action. This report presents an alternative modeling approach based on statistical thermodynamics. The principles of this approach are demonstrated using a simple set of coupled reactions, and then the system is characterized with respect to the changes in energy, entropy, free energy, and entropy production. Finally, the physical and biochemical insights that this approach can provide for metabolism are demonstrated by application to the tricarboxylic acid (TCA) cycle of Escherichia coli. The reaction and pathway thermodynamics are evaluated and predictions are made regarding changes in concentration of TCA cycle intermediates due to 10- and 100-fold changes in the ratio of NAD+:NADH concentrations. Finally, the assumptions and caveats regarding the use of statistical thermodynamics to model non-equilibrium reactions are discussed.

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

Structures and energetics of hydrated deprotonated cis-pinonic acid anion clusters and their atmospheric relevance

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

Hou, Gao-Lei; Zhang, Jun; Valiev, Marat

2017-01-01

Pinonic acid, a C10-monocarboxylic acid with a hydrophilic –CO 2H group and a hydrophobic hydrocarbon backbone, is a key intermediate oxidation product of α-pinene – an important monoterpene compound in biogenic emission processes that influences the atmosphere. Molecular interaction between cis-pinonic acid and water is essential for understanding its role in the formation and growth of pinene-derived secondary organic aerosols. In this work, we studied the structures, energetics, and optical properties of hydrated clusters of cis-pinonate anion (cPA–), the deprotonated form of cis-pinonic acid, by negative ion photoelectron spectroscopy and ab initio theoretical calculations. Our results show that cPA– canmore » adopt two different structural configurations – open and folded. In the absence of waters, the open configuration has the lowest energy and provides the best agreement with the experiment. The addition waters, which mainly interact with the negatively charged -CO 2– group, gradually stabilize the folded configuration and lower its energy difference relative to the most stable open-configured structure. Thermochemical and equilibrium hydrate distribution analysis suggests that the mono- and di- hydrates are likely to exist in humid atmospheric environment with high populations. The detailed molecular description of cPA– hydrated clusters unraveled in this study provides a valuable reference for understanding the initial nucleation process and aerosol formation involving organics containing both hydrophilic and hydrophobic groups, as well as for analyzing the optical properties of those organic aerosols.« less

Δ98Δ, a minimalist model of antiparallel β-sheet proteins based on intestinal fatty acid binding protein

PubMed Central

Curto, Lucrecia María; Caramelo, Julio Javier; Franchini, Gisela Raquel; Delfino, José María

2009-01-01

The design of β-barrels has always been a formidable challenge for de novo protein design. For instance, a persistent problem is posed by the intrinsic tendency to associate given by free edges. From the opposite standpoint provided by the redesign of natural motifs, we believe that the intestinal fatty acid binding protein (IFABP) framework allows room for intervention, giving rise to abridged forms from which lessons on β-barrel architecture and stability could be learned. In this context, Δ98Δ (encompassing residues 29–126 of IFABP) emerges as a monomeric variant that folds properly, retaining functional activity, despite lacking extensive stretches involved in the closure of the β-barrel. Spectroscopic probes (fluorescence and circular dichroism) support the existence of a form preserving the essential determinants of the parent structure, albeit endowed with enhanced flexibility. Chemical and physical perturbants reveal cooperative unfolding transitions, with evidence of significant population of intermediate species in equilibrium, structurally akin to those transiently observed in IFABP. The recognition by the natural ligand oleic acid exerts a mild stabilizing effect, being of a greater magnitude than that found for IFABP. In summary, Δ98Δ adopts a monomeric state with a compact core and a loose periphery, thus pointing to the nonintuitive notion that the integrity of the β-barrel can indeed be compromised with no consequence on the ability to attain a native-like and functional fold. PMID:19309727

Improved Simulation of the Pre-equilibrium Triton Emission in Nuclear Reactions Induced by Nucleons

NASA Astrophysics Data System (ADS)

Konobeyev, A. Yu.; Fischer, U.; Pereslavtsev, P. E.; Blann, M.

2014-04-01

A new approach is proposed for the calculation of non-equilibrium triton energy distributions in nuclear reactions induced by nucleons of intermediate energies. It combines models describing the nucleon pick-up, the coalescence and the triton knock-out processes. Emission and absorption rates for excited particles are represented by the pre-equilibrium hybrid model. The model of Sato, Iwamoto, Harada is used to describe the nucleon pick-up and the coalescence of nucleons from exciton configurations starting from (2p,1h) states. The contribution of the direct nucleon pick-up is described phenomenologically. Multiple pre-equilibrium emission of tritons is accounted for. The calculated triton energy distributions are compared with available experimental data.

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.

Solar flare model atmospheres

NASA Technical Reports Server (NTRS)

Hawley, Suzanne L.; Fisher, George H.

1993-01-01

Solar flare model atmospheres computed under the assumption of energetic equilibrium in the chromosphere are presented. The models use a static, one-dimensional plane parallel geometry and are designed within a physically self-consistent coronal loop. Assumed flare heating mechanisms include collisions from a flux of non-thermal electrons and x-ray heating of the chromosphere by the corona. The heating by energetic electrons accounts explicitly for variations of the ionized fraction with depth in the atmosphere. X-ray heating of the chromosphere by the corona incorporates a flare loop geometry by approximating distant portions of the loop with a series of point sources, while treating the loop leg closest to the chromospheric footpoint in the plane-parallel approximation. Coronal flare heating leads to increased heat conduction, chromospheric evaporation and subsequent changes in coronal pressure; these effects are included self-consistently in the models. Cooling in the chromosphere is computed in detail for the important optically thick HI, CaII and MgII transitions using the non-LTE prescription in the program MULTI. Hydrogen ionization rates from x-ray photo-ionization and collisional ionization by non-thermal electrons are included explicitly in the rate equations. The models are computed in the 'impulsive' and 'equilibrium' limits, and in a set of intermediate 'evolving' states. The impulsive atmospheres have the density distribution frozen in pre-flare configuration, while the equilibrium models assume the entire atmosphere is in hydrostatic and energetic equilibrium. The evolving atmospheres represent intermediate stages where hydrostatic equilibrium has been established in the chromosphere and corona, but the corona is not yet in energetic equilibrium with the flare heating source. Thus, for example, chromospheric evaporation is still in the process of occurring.

Optical methods for measuring DNA folding

NASA Astrophysics Data System (ADS)

Smith, Adam D.; Ukogu, Obinna A.; Devenica, Luka M.; White, Elizabeth D.; Carter, Ashley R.

2017-03-01

One of the most important biological processes is the dynamic folding and unfolding of deoxyribonucleic acid (DNA). The folding process is crucial for DNA to fit within the boundaries of the cell, while the unfolding process is essential for DNA replication and transcription. To accommodate both processes, the cell employs a highly active folding mechanism that has been the subject of intense study over the last few decades. Still, many open questions remain. What are the pathways for folding or unfolding? How does the folding equilibrium shift? And, what is the energy landscape for a particular process? Here, we review these emerging questions and the in vitro, optical methods that have provided answers, introducing the topic for those physicists seeking to step into biology. Specifically, we discuss two iconic experiments for DNA folding, the tethered particle motion (TPM) experiment and the optical tweezers experiment.

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.

Information-Theoretic Uncertainty of SCFG-Modeled Folding Space of The Non-coding RNA

PubMed Central

Manzourolajdad, Amirhossein; Wang, Yingfeng; Shaw, Timothy I.; Malmberg, Russell L.

2012-01-01

RNA secondary structure ensembles define probability distributions for alternative equilibrium secondary structures of an RNA sequence. Shannon’s Entropy is a measure for the amount of diversity present in any ensemble. In this work, Shannon’s entropy of the SCFG ensemble on an RNA sequence is derived and implemented in polynomial time for both structurally ambiguous and unambiguous grammars. Micro RNA sequences generally have low folding entropy, as previously discovered. Surprisingly, signs of significantly high folding entropy were observed in certain ncRNA families. More effective models coupled with targeted randomization tests can lead to a better insight into folding features of these families. PMID:23160142

Equilibrium intermediate-state patterns in a type-I superconducting slab in an arbitrarily oriented applied magnetic field

DOE PAGES

Clem, John; Prozorov, Ruslan; Wijngaarden, Rinke J.

2013-09-04

The equilibrium topology of superconducting and normal domains in flat type-I superconductors is investigated. Important improvements with respect to previous work are that (1) the energy of the external magnetic field, as deformed by the presence of superconducting domains, is calculated in the same way for three different topologies and (2) calculations are made for arbitrary orientation of the applied field. A phase diagram is presented for the minimum-energy topology as a function of applied field magnitude and angle. For small (large) applied fields, normal (superconducting) tubes are found, while for intermediate fields, parallel domains have a lower energy. Themore » range of field magnitudes for which the superconducting-tubes structure is favored shrinks when the field is more in-plane oriented.« less

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.

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

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

Protein Folding—How and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry

PubMed Central

Englander, S. Walter; Mayne, Leland; Kan, Zhong-Yuan; Hu, Wenbing

2017-01-01

Advanced hydrogen exchange (HX) methodology can now determine the structure of protein folding intermediates and their progression in folding pathways. Key developments over time include the HX pulse labeling method with nuclear magnetic resonance analysis, development of the fragment separation method, the addition to it of mass spectrometric (MS) analysis, and recent improvements in the HX MS technique and data analysis. Also, the discovery of protein foldons and their role supplies an essential interpretive link. Recent work using HX pulse labeling with HX MS analysis finds that a number of proteins fold by stepping through a reproducible sequence of native-like intermediates in an ordered pathway. The stepwise nature of the pathway is dictated by the cooperative foldon unit construction of the protein. The pathway order is determined by a sequential stabilization principle; prior native-like structure guides the formation of adjacent native-like structure. This view does not match the funneled energy landscape paradigm of a very large number of folding tracks, which was framed before foldons were known. PMID:27145881

Reshaping the folding energy landscape of human carbonic anhydrase II by a single point genetic mutation Pro237His.

PubMed

Jiang, Yan; Su, Jing-Tan; Zhang, Jun; Wei, Xiang; Yan, Yong-Bin; Zhou, Hai-Meng

2008-01-01

Human carbonic anhydrase (HCA) II participates in a variety of important biological processes, and it has long been known that genetic mutations of HCA II are closely correlated to human disease. In this research, we investigated the effects of a genetic single point mutation P237, which is located on the surface of the molecule and does not participate in the HCA II catalysis, on HCA II activity, stability and folding. Spectroscopic studies revealed that the mutation caused more buried Trp residues to become accessible by solvent and caused the NMR signals to become less dispersed, but did not affect the secondary structure or the hydrophobic exposure of the protein. The mutant was less stable than the wild type enzyme against heat- and GdnHCl-induced inactivation, but its pH adaptation was similar to the wild type. The mutation slightly decreased the stability of the molten globular intermediate, but gradually affected the stability of the native state by a 10-fold reduction of the Gibbs free energy for the transition from the native state to the intermediate. This might have led to an accumulation of the aggregation-prone molten globular intermediate, which further trapped the proteins into the off-pathway aggregates during refolding and reduced the levels of active enzyme in vivo. The results herein suggested that the correct positioning of the long loop around P237 might be crucial to the folding of HCA II, particularly the formation of the active site.

Communication: Microphase equilibrium and assembly dynamics.

PubMed

Zhuang, Yuan; Charbonneau, Patrick

2017-09-07

Despite many attempts, ordered equilibrium microphases have yet to be obtained in experimental colloidal suspensions. The recent computation of the equilibrium phase diagram of a microscopic, particle-based microphase former [Zhuang et al., Phys. Rev. Lett. 116, 098301 (2016)] has nonetheless found such mesoscale assemblies to be thermodynamically stable. Here, we consider their equilibrium and assembly dynamics. At intermediate densities above the order-disorder transition, we identify four different dynamical regimes and the structural changes that underlie the dynamical crossovers from one disordered regime to the next. Below the order-disorder transition, we also find that periodic lamellae are the most dynamically accessible of the periodic microphases. Our analysis thus offers a comprehensive view of the dynamics of disordered microphases and a route to the assembly of periodic microphases in a putative well-controlled, experimental system.

Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis

NASA Astrophysics Data System (ADS)

Booth, David R.; Sunde, Margaret; Bellotti, Vittorio; Robinson, Carol V.; Hutchinson, Winston L.; Fraser, Paul E.; Hawkins, Philip N.; Dobson, Christopher M.; Radford, Sheena E.; Blake, Colin C. F.; Pepys, Mark B.

1997-02-01

Tissue deposition of soluble proteins as amyloid fibrils underlies a range of fatal diseases. The two naturally occurring human lysozyme variants are both amyloidogenic, and are shown here to be unstable. They aggregate to form amyloid fibrils with transformation of the mainly helical native fold, observed in crystal structures, to the amyloid fibril cross-β fold. Biophysical studies suggest that partly folded intermediates are involved in fibrillogenesis, and this may be relevant to amyloidosis generally.

Directing folding pathways for multi-component DNA origami nanostructures with complex topology

NASA Astrophysics Data System (ADS)

Marras, A. E.; Zhou, L.; Kolliopoulos, V.; Su, H.-J.; Castro, C. E.

2016-05-01

Molecular self-assembly has become a well-established technique to design complex nanostructures and hierarchical mesoscale assemblies. The typical approach is to design binding complementarity into nucleotide or amino acid sequences to achieve the desired final geometry. However, with an increasing interest in dynamic nanodevices, the need to design structures with motion has necessitated the development of multi-component structures. While this has been achieved through hierarchical assembly of similar structural units, here we focus on the assembly of topologically complex structures, specifically with concentric components, where post-folding assembly is not feasible. We exploit the ability to direct folding pathways to program the sequence of assembly and present a novel approach of designing the strand topology of intermediate folding states to program the topology of the final structure, in this case a DNA origami slider structure that functions much like a piston-cylinder assembly in an engine. The ability to program the sequence and control orientation and topology of multi-component DNA origami nanostructures provides a foundation for a new class of structures with internal and external moving parts and complex scaffold topology. Furthermore, this work provides critical insight to guide the design of intermediate states along a DNA origami folding pathway and to further understand the details of DNA origami self-assembly to more broadly control folding states and landscapes.

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.

Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

PubMed

Anumalla, Bramhini; Prabhu, N Prakash

2018-01-25

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two-state transitions during chemical denaturation. The extent of stability exerted by Glu is higher for RNase A at higher temperature, whereas it provides more stability for α-LA at lower temperature. Thus, the experiments indicate that Glu induces a thermal equilibrium intermediate and increases the thermodynamic stability of proteins irrespective of their surface charges. The extent of stability varies between the proteins in a temperature-dependent manner.

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.

Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding.

PubMed

Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

2015-01-01

Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.

Bi-stable vocal fold adduction: a mechanism of modal-falsetto register shifts and mixed registration.

PubMed

Titze, Ingo R

2014-04-01

The origin of vocal registers has generally been attributed to differential activation of cricothyroid and thyroarytenoid muscles in the larynx. Register shifts, however, have also been shown to be affected by glottal pressures exerted on vocal fold surfaces, which can change with loudness, pitch, and vowel. Here it is shown computationally and with empirical data that intraglottal pressures can change abruptly when glottal adductory geometry is changed relatively smoothly from convergent to divergent. An intermediate shape between large convergence and large divergence, namely, a nearly rectangular glottal shape with almost parallel vocal fold surfaces, is associated with mixed registration. It can be less stable than either of the highly angular shapes unless transglottal pressure is reduced and upper stiffness of vocal fold tissues is balanced with lower stiffness. This intermediate state of adduction is desirable because it leads to a low phonation threshold pressure with moderate vocal fold collision. Achieving mixed registration consistently across wide ranges of F0, lung pressure, and vocal tract shapes appears to be a balancing act of coordinating laryngeal muscle activation with vocal tract pressures. Surprisingly, a large transglottal pressure is not facilitative in this process, exacerbating the bi-stable condition and the associated register contrast.

Bi-stable vocal fold adduction: A mechanism of modal-falsetto register shifts and mixed registration

PubMed Central

Titze, Ingo R.

2014-01-01

The origin of vocal registers has generally been attributed to differential activation of cricothyroid and thyroarytenoid muscles in the larynx. Register shifts, however, have also been shown to be affected by glottal pressures exerted on vocal fold surfaces, which can change with loudness, pitch, and vowel. Here it is shown computationally and with empirical data that intraglottal pressures can change abruptly when glottal adductory geometry is changed relatively smoothly from convergent to divergent. An intermediate shape between large convergence and large divergence, namely, a nearly rectangular glottal shape with almost parallel vocal fold surfaces, is associated with mixed registration. It can be less stable than either of the highly angular shapes unless transglottal pressure is reduced and upper stiffness of vocal fold tissues is balanced with lower stiffness. This intermediate state of adduction is desirable because it leads to a low phonation threshold pressure with moderate vocal fold collision. Achieving mixed registration consistently across wide ranges of F0, lung pressure, and vocal tract shapes appears to be a balancing act of coordinating laryngeal muscle activation with vocal tract pressures. Surprisingly, a large transglottal pressure is not facilitative in this process, exacerbating the bi-stable condition and the associated register contrast. PMID:25235006

Mechanism of insulin fibrillation: the structure of insulin under amyloidogenic conditions resembles a protein-folding intermediate.

PubMed

Hua, Qing-xin; Weiss, Michael A

2004-05-14

Insulin undergoes aggregation-coupled misfolding to form a cross-beta assembly. Such fibrillation has long complicated its manufacture and use in the therapy of diabetes mellitus. Of interest as a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. Here, we describe the solution structure of human insulin under amyloidogenic conditions (pH 2.4 and 60 degrees C). Use of an enhanced sensitivity cryogenic probe at high magnetic field avoids onset of fibrillation during spectral acquisition. A novel partial fold is observed in which the N-terminal segments of the A- and B-chains detach from the core. Unfolding of the N-terminal alpha-helix of the A-chain exposes a hydrophobic surface formed by native-like packing of the remaining alpha-helices. The C-terminal segment of the B-chain, although not well ordered, remains tethered to this partial helical core. We propose that detachment of N-terminal segments makes possible aberrant protein-protein interactions in an amyloidogenic nucleus. Non-cooperative unfolding of the N-terminal A-chain alpha-helix resembles that observed in models of proinsulin folding intermediates and foreshadows the extensive alpha --> beta transition characteristic of mature fibrils.

Modelling non-equilibrium thermodynamic systems from the speed-gradient principle.

PubMed

Khantuleva, Tatiana A; Shalymov, Dmitry S

2017-03-06

The application of the speed-gradient (SG) principle to the non-equilibrium distribution systems far away from thermodynamic equilibrium is investigated. The options for applying the SG principle to describe the non-equilibrium transport processes in real-world environments are discussed. Investigation of a non-equilibrium system's evolution at different scale levels via the SG principle allows for a fresh look at the thermodynamics problems associated with the behaviour of the system entropy. Generalized dynamic equations for finite and infinite number of constraints are proposed. It is shown that the stationary solution to the equations, resulting from the SG principle, entirely coincides with the locally equilibrium distribution function obtained by Zubarev. A new approach to describe time evolution of systems far from equilibrium is proposed based on application of the SG principle at the intermediate scale level of the system's internal structure. The problem of the high-rate shear flow of viscous fluid near the rigid plane plate is discussed. It is shown that the SG principle allows closed mathematical models of non-equilibrium processes to be constructed.This article is part of the themed issue 'Horizons of cybernetical physics'. © 2017 The Author(s).

Modelling non-equilibrium thermodynamic systems from the speed-gradient principle

NASA Astrophysics Data System (ADS)

Khantuleva, Tatiana A.; Shalymov, Dmitry S.

2017-03-01

The application of the speed-gradient (SG) principle to the non-equilibrium distribution systems far away from thermodynamic equilibrium is investigated. The options for applying the SG principle to describe the non-equilibrium transport processes in real-world environments are discussed. Investigation of a non-equilibrium system's evolution at different scale levels via the SG principle allows for a fresh look at the thermodynamics problems associated with the behaviour of the system entropy. Generalized dynamic equations for finite and infinite number of constraints are proposed. It is shown that the stationary solution to the equations, resulting from the SG principle, entirely coincides with the locally equilibrium distribution function obtained by Zubarev. A new approach to describe time evolution of systems far from equilibrium is proposed based on application of the SG principle at the intermediate scale level of the system's internal structure. The problem of the high-rate shear flow of viscous fluid near the rigid plane plate is discussed. It is shown that the SG principle allows closed mathematical models of non-equilibrium processes to be constructed. This article is part of the themed issue 'Horizons of cybernetical physics'.

Modelling non-equilibrium thermodynamic systems from the speed-gradient principle

PubMed Central

Khantuleva, Tatiana A.

2017-01-01

The application of the speed-gradient (SG) principle to the non-equilibrium distribution systems far away from thermodynamic equilibrium is investigated. The options for applying the SG principle to describe the non-equilibrium transport processes in real-world environments are discussed. Investigation of a non-equilibrium system's evolution at different scale levels via the SG principle allows for a fresh look at the thermodynamics problems associated with the behaviour of the system entropy. Generalized dynamic equations for finite and infinite number of constraints are proposed. It is shown that the stationary solution to the equations, resulting from the SG principle, entirely coincides with the locally equilibrium distribution function obtained by Zubarev. A new approach to describe time evolution of systems far from equilibrium is proposed based on application of the SG principle at the intermediate scale level of the system's internal structure. The problem of the high-rate shear flow of viscous fluid near the rigid plane plate is discussed. It is shown that the SG principle allows closed mathematical models of non-equilibrium processes to be constructed. This article is part of the themed issue ‘Horizons of cybernetical physics’. PMID:28115617

Numerical Simulation of Particle Motion in a Curved Channel

NASA Astrophysics Data System (ADS)

Liu, Yi; Nie, Deming

2018-01-01

In this work the lattice Boltzmann method (LBM) is used to numerically study the motion of a circular particle in a curved channel at intermediate Reynolds numbers (Re). The effects of the Reynolds number and the initial particle position are taken into account. Numerical results include the streamlines, particle trajectories and final equilibrium positions. It has been found that the particle is likely to migrate to a similar equilibrium position irrespective of its initial position when Re is large.

The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides.

PubMed

Haker, Andrea; Hendriks, Johnny; van Stokkum, Ivo H M; Heberle, Joachim; Hellingwerf, Klaas J; Crielaard, Wim; Gensch, Thomas

2003-03-07

The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.

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.

Peptide Folding and Translocation Across the Water-Membrane Interface

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Chang, Sherwood (Technical Monitor)

1997-01-01

The ability of small peptides to organize at aqueous interfaces was examined by performing a series of large-scale, molecular dynamics computer simulations of several peptides composed of two amino acids, nonpolar leucine (L) and polar glutamine (Q). The peptides differed in size and sequence of the amino acids. Studies on dipeptides LL, LQ, QL and QQ were extended to two heptamers, LQQLLQL and LQLQLQL, designed to maximize interfacial stability of an alpha-helix and a beta-strand, respectively, by exposing polar side chains to water and nonpolar side chains to a nonpolar phase. Finally, a transition of an undecamer, composed entirely of leucine residues, from a disordered structure in water to an alpha-helix in a nonpolar phase representing the interior of the membrane was investigated. Complete folding of a peptide in solution was accomplished for the first time in computer simulations. The simulations revealed several basic principles governing the sequence-dependent organization of peptides at interfaces. Short peptides tend to accumulate at interfaces and acquire ordered structures, providing that they have a proper sequence of polar and nonpolar amino acids. The dominant factor determining the interfacial structure of peptides is the hydrophobic effect, which is manifested at aqueous interfaces as a tendency for polar and nonpolar groups of the solute to segregate into the aqueous and nonpolar phases, respectively. If peptides consist of nonpolar residue's only, they become inserted into the nonpolar phase. As demonstrated by the example of the leucine undecamer, such peptides fold into an alpha-helix as they partition into the nonpolar medium. The folding proceeds through an intermediate, called 3-10-helix, which remains in equilibrium with the alpha-helix. Once in the nonpolar environment, the peptides can readily change their orientation with respect to the interface from parallel to perpendicular, especially in response to local electric fields. The ability of nonpolar peptides to modify both the structure and orientation with respect to the interface from parallel to perpendicular, especially in response to local electric fields. The ability of nonpolar peptides to modify both the structure and orientation with changing external conditions may have provided a simple mechanism of transmitting signals from the environment to the interior of a cell.

High-lying intermediate excitations in the nuclear effective interaction with a super-soft-core potential

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

Goode, P.R.; Barrett, B.R.; Portilho, O.

1979-02-01

The earlier calculations of Goode and Barrett are repeated using the super-soft-core potential of Gogny, Pires, and de Tourreil. The particular third-order folded diagram which they calculated now converges in its intermediate-state energy summation, because of the suppression of the strong short-range repulsive effects present in earlier calculations.

A Personal Narrative: The Synergistic Leadership Theory as It Applies to the Leadership of a Principal of a Rural Intermediate School

ERIC Educational Resources Information Center

Manuel, Karlis R.

2010-01-01

The purpose of this narrative study was two-fold. First, the researcher, an African American male principal in a rural, high minority, intermediate school, used to reflect on strategies implemented to enhance the learning environment that subsequently increased student achievement. Second, determined through the study was how personal leadership…

Chaperonin GroEL Reassembly: An Effect of Protein Ligands and Solvent Composition

PubMed Central

Ryabova, Nataliya; Marchenkov, Victor; Kotova, Nina; Semisotnov, Gennady

2014-01-01

Chaperonin GroEL is a complex oligomeric heat shock protein (Hsp60) assisting the correct folding and assembly of other proteins in the cell. An intriguing question is how GroEL folds itself. According to the literature, GroEL reassembly is dependent on chaperonin ligands and solvent composition. Here we demonstrate dependence of GroEL reassembly efficiency on concentrations of the essential factors (Mg2+, ADP, ATP, GroES, ammonium sulfate, NaCl and glycerol). Besides, kinetics of GroEL oligomerization in various conditions was monitored by the light scattering technique and proved to be two-exponential, which suggested accumulation of a certain oligomeric intermediate. This intermediate was resolved as a heptamer by nondenaturing blue electrophoresis of GroEL monomers during their assembly in the presence of both Mg-ATP and co-chaperonin GroES. Presumably, this intermediate heptamer plays a key role in formation of the GroEL tetradecameric particle. The role of co-chaperonin GroES (Hsp10) in GroEL assembly is also discussed. PMID:24970225

NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations.

PubMed

Helmling, Christina; Wacker, Anna; Wolfinger, Michael T; Hofacker, Ivo L; Hengesbach, Martin; Fürtig, Boris; Schwalbe, Harald

2017-02-22

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

Lithium hydride doped intermediate connector for high-efficiency and long-term stable tandem organic light-emitting diodes.

PubMed

Ding, Lei; Tang, Xun; Xu, Mei-Feng; Shi, Xiao-Bo; Wang, Zhao-Kui; Liao, Liang-Sheng

2014-10-22

Lithium hydride (LiH) is employed as a novel n-dopant in the intermediate connector for tandem organic light-emitting diodes (OLEDs) because of its easy coevaporation with other electron transporting materials. The tandem OLEDs with two and three electroluminescent (EL) units connected by a combination of LiH doped 8-hydroxyquinoline aluminum (Alq3) and 1,4,5,8,9,11-hexaazatriphenylene-hexacarbonitrile (HAT-CN) demonstrate approximately 2-fold and 3-fold enhancement in current efficiency, respectively. In addition, no extra voltage drop across the intermediate connector is observed. Particularly, the lifetime (T75%) in the tandem OLED with two and three EL units is substantially improved by 3.8 times and 7.4 times, respectively. The doping effect of LiH into Alq3, the charge injection, and transport characteristics of LiH-doped Alq3 are further investigated by ultraviolet photoelectron spectroscopy (UPS) and X-ray photoemission spectroscopy (XPS).

Synchrotron radiation circular dichroism spectroscopy study of recombinant T β4 folding

NASA Astrophysics Data System (ADS)

Huang, Yung-Chin; Chu, Hsueh-Liang; Chen, Peng-Jen; Chang, Chia-Ching

Thymosin beta 4 (T β4) is a 43-amino acid small peptide, has been demonstrated that it can promote cardiac repair, wound repair, tissue protection, and involve in the proliferation of blood cell precursor stem cells of bone marrow. Moreover, T β4 has been identified as a multifunction intrinsically disordered protein, which is lacking the stable tertiary structure. Owing to the small size and disordered character, the T β4 protein degrades rapidly and the storage condition is critical. Therefore, it is not easy to reveal its folding mechanism of native T β4. However, recombinant T β4 protein (rT β4), which fused with a 5-kDa peptide in its amino-terminal, is stable and possesses identical function of T β4. Therefore, rT β4 can be used to study its folding mechanism. By using over-critical folding process, stable folding intermediates of rT β4 can be obtained. Structure analysis of folding intermediates by synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies indicate that rT β4 is a random coli major protein and its hydrophobic region becomes compact gradually. Moreover, the rT β4 folding is a two state transition. Thermal denaturation analysis indicates that rT β4 lacks stable tertiary structure. These results indicated that rT β4, similar to T β4, is an intrinsically disordered protein. Research is supported by MOST, Taiwan. MOST 103-2112-M-009-011-MY3. Corresponding author: Chia-Ching Chang; ccchang01@faculty.nctu.edu.tw.

The small angle x-ray scattering of globular proteins in solution during heat denaturation

NASA Astrophysics Data System (ADS)

Banuelos, Jose; Urquidi, Jacob

2008-10-01

The ability of proteins to change their conformation in response to changes in their environment has consequences in biological processes like metabolism, chemical regulation in cells, and is believed to play a role in the onset of several neurodegenerative diseases. Factors such as a change in temperature, pressure, and the introduction of ions into the aqueous environment of a protein can give rise to the folding/unfolding of a protein. As a protein unfolds, the ratio of nonpolar to polar groups exposed to water changes, affecting a protein's thermodynamic properties. Using small angle x-ray scattering (SAXS), we are currently studying the intermediate protein conformations that arise during the folding/unfolding process as a function of temperature for five globular proteins. Trends in the observed intermediate structures of these globular proteins, along with correlations with data on protein thermodynamics may help elucidate shared characteristics between all proteins in the folding/unfolding process. Experimental design considerations will be discussed and preliminary results for some of these systems will be presented.

Study of the Spin Dependent 3HE-NUCLEUS Interaction at 450 Mev

NASA Astrophysics Data System (ADS)

Kamiya, J.; Hatanaka, K.; Sakemi, Y.; Wakasa, T.; Yoshida, H. P.; Obayashi, E.; Hara, K.; Kitamura, K.; Shimizu, Y.; Fujita, K.; Sakamoto, N.; Shimbara, Y.; Adachi, T.; Sakaguchi, H.; Yosoi, M.; Uchida, M.; Yasuda, Y.; Kawabata, T.; Noro, T.

2003-04-01

Differential cross sections and induced polarizations of 3He+12C, 58Ni, and 90Zr elastic scattering were measured at E3He = 450 MeV. This is the first measurement of the polarization for 3He scattering at intermediate energies. The optical potential parameters including the spin-orbit potential were determined with small uncertainties. The volume integrals per nucleon of the potentials were investigated for 3He and their energy dependence showed the similar behavior to that for protons at intermediate energies. The single folding calculations were compared with the data. The real central and spin-orbit parts of the folded potentials had to be reduced by a few tens of percent in order to reproduce the experimental results.

Structure and antimicrobial activity of platypus 'intermediate' defensin-like peptide.

PubMed

Torres, Allan M; Bansal, Paramjit; Koh, Jennifer M S; Pagès, Guilhem; Wu, Ming J; Kuchel, Philip W

2014-05-02

The three-dimensional structure of a chemically synthesized peptide that we have called 'intermediate' defensin-like peptide (Int-DLP), from the platypus genome, was determined by nuclear magnetic resonance (NMR) spectroscopy; and its antimicrobial activity was investigated. The overall structural fold of Int-DLP was similar to that of the DLPs and β-defensins, however the presence of a third antiparallel β-strand makes its structure more similar to the β-defensins than the DLPs. Int-DLP displayed potent antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. The four arginine residues at the N-terminus of Int-DLP did not affect the overall fold, but were important for its antimicrobial potency. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo.

PubMed

MacAlpine, D M; Perlman, P S; Butow, R A

1998-06-09

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (rho+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in rho+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (rho-). mtDNA recombination junctions are not observed in rho+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Deltamgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in rho+ mtDNA of Deltamgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by >/= 10-fold in wild-type rho+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of rho+ mtDNA.

Fluctuation Pressure Assisted Ejection of DNA From Bacteriophage

NASA Astrophysics Data System (ADS)

Harrison, Michael J.

2011-03-01

The role of thermal pressure fluctuations excited within tightly packaged DNA while it is ejected from protein capsid shells is discussed in a model calculation. At equilibrium before ejection we assume the DNA is folded many times into a bundle of parallel segments that forms an equilibrium conformation at minimum free energy, which presses tightly against capsid walls. Using a canonical ensemble at temperature T we calculate internal pressure fluctuations against a slowly moving or static capsid mantle for an elastic continuum model of the folded DNA bundle. It is found that fluctuating pressures on the capsid from thermal excitation of longitudinal acoustic vibrations in the bundle whose wavelengths are exceeded by the bend persistence length may have root-mean-square values that are several tens of atmospheres for typically small phage dimensions. Comparisons are given with measured data on three mutants of lambda phage with different base pair lengths and total genome ejection pressures.

All-atom calculation of protein free-energy profiles

NASA Astrophysics Data System (ADS)

Orioli, S.; Ianeselli, A.; Spagnolli, G.; Faccioli, P.

2017-10-01

The Bias Functional (BF) approach is a variational method which enables one to efficiently generate ensembles of reactive trajectories for complex biomolecular transitions, using ordinary computer clusters. For example, this scheme was applied to simulate in atomistic detail the folding of proteins consisting of several hundreds of amino acids and with experimental folding time of several minutes. A drawback of the BF approach is that it produces trajectories which do not satisfy microscopic reversibility. Consequently, this method cannot be used to directly compute equilibrium observables, such as free energy landscapes or equilibrium constants. In this work, we develop a statistical analysis which permits us to compute the potential of mean-force (PMF) along an arbitrary collective coordinate, by exploiting the information contained in the reactive trajectories calculated with the BF approach. We assess the accuracy and computational efficiency of this scheme by comparing its results with the PMF obtained for a small protein by means of plain molecular dynamics.

Modeling chain folding in protein-constrained circular DNA.

PubMed Central

Martino, J A; Olson, W K

1998-01-01

An efficient method for sampling equilibrium configurations of DNA chains binding one or more DNA-bending proteins is presented. The technique is applied to obtain the tertiary structures of minimal bending energy for a selection of dinucleosomal minichromosomes that differ in degree of protein-DNA interaction, protein spacing along the DNA chain contour, and ring size. The protein-bound portions of the DNA chains are represented by tight, left-handed supercoils of fixed geometry. The protein-free regions are modeled individually as elastic rods. For each random spatial arrangement of the two nucleosomes assumed during a stochastic search for the global minimum, the paths of the flexible connecting DNA segments are determined through a numerical solution of the equations of equilibrium for torsionally relaxed elastic rods. The minimal energy forms reveal how protein binding and spacing and plasmid size differentially affect folding and offer new insights into experimental minichromosome systems. PMID:9591675

Atomic-level characterization of the structural dynamics of proteins.

PubMed

Shaw, David E; Maragakis, Paul; Lindorff-Larsen, Kresten; Piana, Stefano; Dror, Ron O; Eastwood, Michael P; Bank, Joseph A; Jumper, John M; Salmon, John K; Shan, Yibing; Wriggers, Willy

2010-10-15

Molecular dynamics (MD) simulations are widely used to study protein motions at an atomic level of detail, but they have been limited to time scales shorter than those of many biologically critical conformational changes. We examined two fundamental processes in protein dynamics--protein folding and conformational change within the folded state--by means of extremely long all-atom MD simulations conducted on a special-purpose machine. Equilibrium simulations of a WW protein domain captured multiple folding and unfolding events that consistently follow a well-defined folding pathway; separate simulations of the protein's constituent substructures shed light on possible determinants of this pathway. A 1-millisecond simulation of the folded protein BPTI reveals a small number of structurally distinct conformational states whose reversible interconversion is slower than local relaxations within those states by a factor of more than 1000.

Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

PubMed Central

Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

2015-01-01

Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

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.

Crystallization of calcium oxalate dihydrate in a buffered calcium-containing glucose solution by irradiation with non-equilibrium atmospheric pressure plasma

NASA Astrophysics Data System (ADS)

Kurake, Naoyuki; Tanaka, Hiromasa; Ishikawa, Kenji; Nakamura, Kae; Kajiyama, Hiroaki; Kikkawa, Fumitaka; Mizuno, Masaaki; Ikehara, Yuzuru; Hori, Masaru

2017-10-01

Oxalate was synthesized in the glucose solution by irradiation with non-equilibrium atmospheric pressure plasma (NEAPP), in which the NEAPP plume contacted the solution surface, via the generation of several intermediate organic products such as gluconic acid. A thermodynamically unstable phase of calcium oxalate dihydrate crystallized rapidly during incubation of a NEAPP-irradiated glucose solution that contained calcium ions and was buffered at neutral pH. Longer irradiation times increased the growth rate and the number of seed crystals.

Rate and Equilibrium Constants for the Addition of N-Heterocyclic Carbenes into Benzaldehydes: A Remarkable 2-Substituent Effect**

PubMed Central

Collett, Christopher J; Massey, Richard S; Taylor, James E; Maguire, Oliver R; O'Donoghue, AnnMarie C; Smith, Andrew D

2015-01-01

Rate and equilibrium constants for the reaction between N-aryl triazolium N-heterocyclic carbene (NHC) precatalysts and substituted benzaldehyde derivatives to form 3-(hydroxybenzyl)azolium adducts under both catalytic and stoichiometric conditions have been measured. Kinetic analysis and reaction profile fitting of both the forward and reverse reactions, plus onwards reaction to the Breslow intermediate, demonstrate the remarkable effect of the benzaldehyde 2-substituent in these reactions and provide insight into the chemoselectivity of cross-benzoin reactions. PMID:25908493

Rate and Equilibrium Constants for the Addition of N‐Heterocyclic Carbenes into Benzaldehydes: A Remarkable 2‐Substituent Effect†

PubMed Central

Collett, Christopher J.; Massey, Richard S.; Taylor, James E.; Maguire, Oliver R.

2015-01-01

Abstract Rate and equilibrium constants for the reaction between N‐aryl triazolium N‐heterocyclic carbene (NHC) precatalysts and substituted benzaldehyde derivatives to form 3‐(hydroxybenzyl)azolium adducts under both catalytic and stoichiometric conditions have been measured. Kinetic analysis and reaction profile fitting of both the forward and reverse reactions, plus onwards reaction to the Breslow intermediate, demonstrate the remarkable effect of the benzaldehyde 2‐substituent in these reactions and provide insight into the chemoselectivity of cross‐benzoin reactions. PMID:27478264

Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

PubMed

Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

2013-12-13

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

Apo and InsP[subscript 3]-bound crystal structures of the ligand-binding domain of an InsP[subscript 3] receptor

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

Lin, Chun-Chi; Baek, Kyuwon; Lu, Zhe

2012-05-08

We report the crystal structures of the ligand-binding domain (LBD) of a rat inositol 1,4,5-trisphosphate receptor (InsP{sub 3}R) in its apo and InsP{sub 3}-bound conformations. Comparison of these two conformations reveals that LBD's first {beta}-trefoil fold ({beta}-TF1) and armadillo repeat fold (ARF) move together as a unit relative to its second {beta}-trefoil fold ({beta}-TF2). Whereas apo LBD may spontaneously transition between gating conformations, InsP{sub 3} binding shifts this equilibrium toward the active state.

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.

A partially folded structure of amyloid-beta(1-40) in an aqueous environment.

PubMed

Vivekanandan, Subramanian; Brender, Jeffrey R; Lee, Shirley Y; Ramamoorthy, Ayyalusamy

2011-07-29

Aggregation of the Aβ(1-40) peptide is linked to the development of extracellular plaques characteristic of Alzheimer's disease. While previous studies commonly show the Aβ(1-40) is largely unstructured in solution, we show that Aβ(1-40) can adopt a compact, partially folded structure. In this structure (PDB ID: 2LFM), the central hydrophobic region of the peptide forms a 3(10) helix from H13 to D23 and the N- and C-termini collapse against the helix due to the clustering of hydrophobic residues. Helical intermediates have been predicted to be crucial on-pathway intermediates in amyloid fibrillogenesis, and the structure presented here presents a new target for investigation of early events in Aβ(1-40) fibrillogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

Mitochondrial metabolic regulation by GRP78

PubMed Central

Prasad, Manoj; Pawlak, Kevin J.; Burak, William E.; Perry, Elizabeth E.; Marshall, Brendan; Whittal, Randy M.; Bose, Himangshu S.

2017-01-01

Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity. PMID:28275724

tRNA Shifts the G-quadruplex-Hairpin Conformational Equilibrium in RNA towards the Hairpin Conformer.

PubMed

Rode, Ambadas B; Endoh, Tamaki; Sugimoto, Naoki

2016-11-07

Non-coding RNAs play important roles in cellular homeostasis and are involved in many human diseases including cancer. Intermolecular RNA-RNA interactions are the basis for the diverse functions of many non-coding RNAs. Herein, we show how the presence of tRNA influences the equilibrium between hairpin and G-quadruplex conformations in the 5' untranslated regions of oncogenes and model sequences. Kinetic and equilibrium analyses of the hairpin to G-quadruplex conformational transition of purified RNA as well as during co-transcriptional folding indicate that tRNA significantly shifts the equilibrium toward the hairpin conformer. The enhancement of relative translation efficiency in a reporter gene assay is shown to be due to the tRNA-mediated shift in hairpin-G-quadruplex equilibrium of oncogenic mRNAs. Our findings suggest that tRNA is a possible therapeutic target in diseases in which RNA conformational equilibria is dysregulated. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Absolute comparison of simulated and experimental protein-folding dynamics

NASA Astrophysics Data System (ADS)

Snow, Christopher D.; Nguyen, Houbi; Pande, Vijay S.; Gruebele, Martin

2002-11-01

Protein folding is difficult to simulate with classical molecular dynamics. Secondary structure motifs such as α-helices and β-hairpins can form in 0.1-10µs (ref. 1), whereas small proteins have been shown to fold completely in tens of microseconds. The longest folding simulation to date is a single 1-µs simulation of the villin headpiece; however, such single runs may miss many features of the folding process as it is a heterogeneous reaction involving an ensemble of transition states. Here, we have used a distributed computing implementation to produce tens of thousands of 5-20-ns trajectories (700µs) to simulate mutants of the designed mini-protein BBA5. The fast relaxation dynamics these predict were compared with the results of laser temperature-jump experiments. Our computational predictions are in excellent agreement with the experimentally determined mean folding times and equilibrium constants. The rapid folding of BBA5 is due to the swift formation of secondary structure. The convergence of experimentally and computationally accessible timescales will allow the comparison of absolute quantities characterizing in vitro and in silico (computed) protein folding.

Platelet-derived-growth-factor-stimulated heterogeneous polyphosphoinositide metabolism and phosphate uptake in C3H fibroblasts.

PubMed Central

Holmsen, H; Male, R; Rongved, S; Langeland, N; Lillehaug, J

1989-01-01

Pig platelet-derived growth factor (PDGF) increased the rate of [32P]Pi uptake by murine fibroblasts, resulting in a 3-9-fold elevation of the specific radioactivity of ATP, PtdInsP, PtdInsP2, PtdIns and phosphatidic acid. The specific radioactivity was 10-60-fold higher in ATP than in the four phospholipids. These substances are therefore not in metabolic equilibrium, which complicates determination of inositol phospholipid turnover. PMID:2548480

Electronic properties of solids excited with intermediate laser power densities

NASA Astrophysics Data System (ADS)

Sirotti, Fausto; Tempo Beamline Team

Intermediate laser power density up to about 100 GW/cm2 is below the surface damage threshold is currently used to induce modification in the physical properties on short time scales. The absorption of a short laser pulse induces non-equilibrium electronic distributions followed by lattice-mediated equilibrium taking place only in the picosecond range. The role of the hot electrons is particularly important in several domains as for example fast magnetization and demagnetization processes, laser induced phase transitions, charge density waves. Angular resolved photoelectron spectroscopy measuring directly energy and momentum of electrons is the most adapted tool to study the electronic excitations at short time scales during and after fast laser excitations. The main technical problem is the space charge created by the pumping laser pulse. I will present angular resolved multiphoton photoemission results obtained with 800 nm laser pulses showing how space charge electrons emitted during fast demagnetization processes can be measured. Unable enter Affiliation: CNRS-SOLEIL Synchrotron L'Orme des Merisiers , Saint Aubin 91192 Gif sur Yvette France.

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

DOE PAGES

Shi, Jade; Nobrega, R. Paul; Schwantes, Christian; ...

2017-03-08

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. We report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structuremore » of the excited state ensemble. The resulting prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. We then predict incisive single molecule FRET experiments, using these results, as a means of model validation. Our study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.« less

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

NASA Astrophysics Data System (ADS)

Shi, Jade; Nobrega, R. Paul; Schwantes, Christian; Kathuria, Sagar V.; Bilsel, Osman; Matthews, C. Robert; Lane, T. J.; Pande, Vijay S.

2017-03-01

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. Here, we report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. This prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. Using these results, we then predict incisive single molecule FRET experiments as a means of model validation. This study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.

In Vivo Regulation of Hepatitis B Virus Replication by Peroxisome Proliferators†

PubMed Central

Guidotti, Luca G.; Eggers, Carrie M.; Raney, Anneke K.; Chi, Susan Y.; Peters, Jeffrey M.; Gonzalez, Frank J.; McLachlan, Alan

1999-01-01

The role of the peroxisome proliferator-activated receptor α (PPARα) in regulating hepatitis B virus (HBV) transcription and replication in vivo was investigated in an HBV transgenic mouse model. Treatment of HBV transgenic mice with the peroxisome proliferators Wy-14,643 and clofibric acid resulted in a less than twofold increase in HBV transcription rates and steady-state levels of HBV RNAs in the livers of these mice. In male mice, this increase in transcription was associated with a 2- to 3-fold increase in replication intermediates, whereas in female mice it was associated with a 7- to 14-fold increase in replication intermediates. The observed increases in transcription and replication were dependent on PPARα. HBV transgenic mice lacking this nuclear hormone receptor showed similar levels of HBV transcripts and replication intermediates as untreated HBV transgenic mice expressing PPARα but failed to demonstrate alterations in either RNA or DNA synthesis in response to peroxisome proliferators. Therefore, it appears that very modest alterations in transcription can, under certain circumstances, result in relatively large increases in HBV replication in HBV transgenic mice. PMID:10559356

Life prediction of expulsion bladders through fatigue test and fold strain analysis

NASA Technical Reports Server (NTRS)

Chu, H. N.; Unterberg, W.

1972-01-01

Cycle life data are presented in terms of true maximum strain for four metals, two plastics, and two elastomers. The Coffin-Manson fatigue theory was applied for metals and plastics, and cut-growth fatigue theory for elastomers. The data are based on measurements made at room and elevated temperatures. It was found that double folds give rise to far severer folding strains than do simple folds. It was also found that, except for the elastomers, all the bladder materials develop surface cracks due to double folds after only one cycle. The findings indicate that metals, which are bets for premeation resistance, are worst for fatigue resistance, and vice versa for elastomers. The intermediate plastics were found to be unsatisfactory for both permeation and fatigue resistance for missions of extended duration.

Comparative analysis of nitrite uptake and hemoglobin-nitrite reactions in erythrocytes: sorting out uptake mechanisms and oxygenation dependencies.

PubMed

Jensen, Frank B; Rohde, Sabina

2010-04-01

Nitrite uptake into red blood cells (RBCs) precedes its intracellular reactions with hemoglobin (Hb) that forms nitric oxide (NO) during hypoxia. We investigated the uptake of nitrite and its reactions with Hb at different oxygen saturations (So(2)), using RBCs with (carp and rabbit) and without (hagfish and lamprey) anion exchanger-1 (AE1) in the membrane, with the aim to unravel the mechanisms and oxygenation dependencies of nitrite transport. Added nitrite rapidly diffused into the RBCs until equilibrium. The distribution ratio of nitrite across the membrane agreed with that expected from HNO(2) diffusion and AE1-mediated facilitated NO(2)(-) diffusion. Participation of HNO(2) diffusion was emphasized by rapid transmembrane nitrite equilibration also in the natural AE1 knockouts. Following the equilibration, nitrite was consumed by reacting with Hb, which created a continued inward diffusion controlled by intracellular reaction rates. Changes in nitrite uptake with So(2), pH, or species were accordingly explained by corresponding changes in reaction rates. In carp, nitrite uptake rates increased linearly with decreasing So(2) over the entire So(2) range. In rabbit, nitrite uptake rates were highest at intermediate So(2), producing a bell-shaped relationship with So(2). Nitrite consumption increased approximately 10-fold with a 1 unit decrease in pH, as expected from the involvement of protons in the reactions with Hb. The reaction of nitrite with deoxyhemoglobin was favored over that with oxyhemoglobin at intermediate So(2). We propose a model for RBC nitrite uptake that involves both HNO(2) diffusion and AE1-mediated transport and that explains both the present and previous (sometimes puzzling) results.

3D visualization of sheath folds in Ancient Roman marble wall coverings from Ephesos, Turkey

NASA Astrophysics Data System (ADS)

Wex, Sebastian; Passchier, Cees W.; de Kemp, Eric A.; İlhan, Sinan

2014-10-01

Archaeological excavations and restoration of a palatial Roman housing complex in Ephesos, Turkey yielded 40 wall-decorating plates of folded mylonitic marble (Cipollino verde), derived from the internal Hellenides near Karystos, Greece. Cipollino verde was commonly used for decoration purposes in Roman buildings. The plates were serial-sectioned from a single quarried block of 1,25 m3 and provided a research opportunity for detailed reconstruction of the 3D geometry of meterscale folds in mylonitized marble. A GOCAD model is used to visualize the internal fold structures of the marble, comprising curtain folds and multilayered sheath folds. The sheath folds are unusual in that they have their intermediate axis normal to the parent layering. This agrees with regional tectonic studies, which suggest that Cipollino verde structures formed by local constrictional non-coaxial flow. Sheath fold cross-section geometry, exposed on the surface of a plate or outcrop, is found to be independent of the intersection angle of the fold structure with the studied plane. Consequently, a single surface cannot be used as an indicator of the three-dimensional geometry of transected sheath folds.

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

Prion disease susceptibility is affected by β-structure folding propensity and local side-chain interactions in PrP

PubMed Central

Khan, M. Qasim; Sweeting, Braden; Mulligan, Vikram Khipple; Arslan, Pharhad Eli; Cashman, Neil R.; Pai, Emil F.; Chakrabartty, Avijit

2010-01-01

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrPSc). Exposure to PrPSc from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer–octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease. PMID:21041683

Prion disease susceptibility is affected by beta-structure folding propensity and local side-chain interactions in PrP.

PubMed

Khan, M Qasim; Sweeting, Braden; Mulligan, Vikram Khipple; Arslan, Pharhad Eli; Cashman, Neil R; Pai, Emil F; Chakrabartty, Avijit

2010-11-16

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrP(Sc)). Exposure to PrP(Sc) from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer-octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

PubMed

Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M S; Engel, Paul C

2008-08-01

Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

Polyanion-Induced Self Association of Complement Factor H1

PubMed Central

Pangburn, Michael K.; Rawal, Nenoo; Cortes, Claudio; Alam, M. Nurul; Ferreira, Viviana P.; Atkinson, Mark A. L.

2008-01-01

Factor H is the primary soluble regulator of activation of the alternative pathway of complement. It prevents activation of complement on host cells and tissues upon association with C3b and surface polyanions such as sialic acids, heparin and other glycosaminoglycans. Here we show that interaction with polyanions causes self-association forming tetramers of the 155,000 Da glycosylated protein. Monomeric human factor H is an extended flexible protein that exhibits an apparent size of 330,000 Da, relative to globular standards, during gel filtration chromatography in the absence of polyanions. In the presence of dextran sulfate (5,000 Da) or heparin an intermediate species of apparent m.w. 700,000 and a limit species of m.w. 1,400,000 were observed by gel filtration. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a Mr of 607,000, corresponding to a tetramer, in the presence of less than a 2-fold molar excess of dextran sulfate. Increasing concentrations of dextran sulfate increased binding of factor H to zymosan-C3b 4.5-fold. This was accompanied by an increase in both the decay accelerating and cofactor activity of factor H on these cells. An expressed fragment encompassing the C-terminal polyanion binding site (complement control protein domains 18–20) also exhibited polyanion-induced self association, suggesting that the C-terminal ends of factor H mediate self-association. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H, and the resulting regulation of complement activation, may involve formation of dimers and tetramers of factor H. PMID:19124749

A density functional theory study on peptide bond cleavage at aspartic residues: direct vs cyclic intermediate hydrolysis.

PubMed

Sang-aroon, Wichien; Amornkitbamrung, Vittaya; Ruangpornvisuti, Vithaya

2013-12-01

In this work, peptide bond cleavages at carboxy- and amino-sides of the aspartic residue in a peptide model via direct (concerted and step-wise) and cyclic intermediate hydrolysis reaction pathways were explored computationally. The energetics, thermodynamic properties, rate constants, and equilibrium constants of all hydrolysis reactions, as well as their energy profiles were computed at the B3LYP/6-311++G(d,p) level of theory. The result indicated that peptide bond cleavage of the Asp residue occurred most preferentially via the cyclic intermediate hydrolysis pathway. In all reaction pathways, cleavage of the peptide bond at the amino-side occurred less preferentially than at the carboxy-side. The overall reaction rate constants of peptide bond cleavage of the Asp residue at the carboxy-side for the assisted system were, in increasing order: concerted < step-wise < cyclic intermediate.

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.

Cooperative Tertiary Interaction Network Guides RNA Folding

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

Behrouzi, Reza; Roh, Joon Ho; Kilburn, Duncan

2013-04-08

Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends onmore » the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold.« less

Forces Driving Chaperone Action

PubMed Central

Koldewey, Philipp; Stull, Frederick; Horowitz, Scott; Martin, Raoul; Bardwell, James C. A.

2016-01-01

SUMMARY It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client’s affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins. PMID:27293188

Exploring the folding free energy landscape of insulin using bias exchange metadynamics.

PubMed

Todorova, Nevena; Marinelli, Fabrizio; Piana, Stefano; Yarovsky, Irene

2009-03-19

The bias exchange metadynamics (BE-META) technique was applied to investigate the folding mechanism of insulin, one of the most studied and biologically important proteins. The BE-META simulations were performed starting from an extended conformation of chain B of insulin, using only eight replicas and seven reaction coordinates. The folded state, together with the intermediate states along the folding pathway were identified and their free energy was determined. Three main basins were found separated from one another by a large free energy barrier. The characteristic native fold of chain B was observed in one basin, while the other two most populated basins contained "molten-globule" conformations stabilized by electrostatic and hydrophobic interactions, respectively. Transitions between the three basins occur on the microsecond time scale. The implications and relevance of this finding to the folding mechanisms of insulin were investigated.

Coexistence of Native and Denatured Phases in a Single Proteinlike Molecule

NASA Astrophysics Data System (ADS)

Du, Rose; Grosberg, Alexander Yu.; Tanaka, Toyoichi

1999-11-01

In order to understand the nuclei which develop during the course of protein folding and unfolding, we examine equilibrium coexistence of phases within a single heteropolymer chain. We computationally generate the phase segregation by applying a ``folding pressure,'' or adding an energetic bonus for native monomer-monomer contacts. The computer models reveal that in a polymer system some nuclei hinder folding via topological constraints. Using this insight, we show that the critical nucleus size is of the order of the entire chain and that unfolding time scales as exp\\(cN2/3\\), in the large N limit, N and c being the chain length and a constant, respectively.

Flexural flow folding and the paleomagnetic fold test: An example of strain reorientation of remancence in the Mauch Chunk formation

NASA Astrophysics Data System (ADS)

Stamatakos, J.; Kodama, K. P.

1991-08-01

The relationship between the remanent magnetization and the detailed strain geometry around a first-order fold in the Appalachian Valley and Ridge Province was investigated to examine whether penetrative strains associated with folding can generate a apparent synfolding geometry from a prefolding magnetization. Paleomagnetic results from the Mississippian Mauch Chunk Formation on both limbs of the Frackville Anticline near Lavelle, Pennsylvania, yield two magnetic components, an intermediate unblocking temperature (300°C-600°C) Kiaman remagnetization and a two-polarity high unblocking temperature (650°C-680°C) characteristic magnetization. When the magnetic directions are incrementally corrected for bedding tilt, the intermediate-temperature component is most tightly clustered at 85% unfolding (D=176°, I=3°) and the high-temperature component is most tightly clustered at 75% unfolding (D=184°, I=27°). Mesoscopic and microscopic structural fabric analyses suggest a strain history that includes a significant component of flexural slip/flow folding. In the coarser-grained sandstone units, folding has largely been accommodated by slip on bedding, while in the finer-grained beds, folding has been accommodated by grain-scale deformation mechanisms such as pressure solution and low-temperature plasticity. Finite strain measurements, determined from center-to-center distances between quartz grains, yield strain ellipsoids consistent with this folding model. Inclination of the characteristic component varies as a function of the magnitude of the finite strain. This variation suggests that the characteristic magnetization has been systematically reoriented with respect to bedding during folding. Remanence directions on the south dipping limb have been rotated to shallower inclinations, while those on the north dipping limb have been rotated to steeper directions causing the prefolding magnetization to appear synfolding. These rotations are in agreement with models of rigid particle rotation in deforming viscous media. Unlike the characteristic magnetization, the secondary component appears to be unaffected by the deformation, and its synfolding behavior is interpreted as the acquisition of a secondary magnetization during Alleghenian folding. These results show that it is important to consider penetrative strains when evaluating the significance of apparent synfolding magnetizations.

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.

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen1

PubMed Central

2015-01-01

Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux. PMID:25926480

Improved free-energy landscape reconstruction of bacteriorhodopsin highlights local variations in unfolding energy.

PubMed

Heenan, Patrick R; Yu, Hao; Siewny, Matthew G W; Perkins, Thomas T

2018-03-28

Precisely quantifying the energetics that drive the folding of membrane proteins into a lipid bilayer remains challenging. More than 15 years ago, atomic force microscopy (AFM) emerged as a powerful tool to mechanically extract individual membrane proteins from a lipid bilayer. Concurrently, fluctuation theorems, such as the Jarzynski equality, were applied to deduce equilibrium free energies (ΔG 0 ) from non-equilibrium single-molecule force spectroscopy records. The combination of these two advances in single-molecule studies deduced the free-energy of the model membrane protein bacteriorhodopsin in its native lipid bilayer. To elucidate this free-energy landscape at a higher resolution, we applied two recent developments. First, as an input to the reconstruction, we used force-extension curves acquired with a 100-fold higher time resolution and 10-fold higher force precision than traditional AFM studies of membrane proteins. Next, by using an inverse Weierstrass transform and the Jarzynski equality, we removed the free energy associated with the force probe and determined the molecular free-energy landscape of the molecule under study, bacteriorhodopsin. The resulting landscape yielded an average unfolding free energy per amino acid (aa) of 1.0 ± 0.1 kcal/mol, in agreement with past single-molecule studies. Moreover, on a smaller spatial scale, this high-resolution landscape also agreed with an equilibrium measurement of a particular three-aa transition in bacteriorhodopsin that yielded 2.7 kcal/mol/aa, an unexpectedly high value. Hence, while average unfolding ΔG 0 per aa is a useful metric, the derived high-resolution landscape details significant local variation from the mean. More generally, we demonstrated that, as anticipated, the inverse Weierstrass transform is an efficient means to reconstruct free-energy landscapes from AFM data.

Improved free-energy landscape reconstruction of bacteriorhodopsin highlights local variations in unfolding energy

NASA Astrophysics Data System (ADS)

Heenan, Patrick R.; Yu, Hao; Siewny, Matthew G. W.; Perkins, Thomas T.

2018-03-01

Precisely quantifying the energetics that drive the folding of membrane proteins into a lipid bilayer remains challenging. More than 15 years ago, atomic force microscopy (AFM) emerged as a powerful tool to mechanically extract individual membrane proteins from a lipid bilayer. Concurrently, fluctuation theorems, such as the Jarzynski equality, were applied to deduce equilibrium free energies (ΔG0) from non-equilibrium single-molecule force spectroscopy records. The combination of these two advances in single-molecule studies deduced the free-energy of the model membrane protein bacteriorhodopsin in its native lipid bilayer. To elucidate this free-energy landscape at a higher resolution, we applied two recent developments. First, as an input to the reconstruction, we used force-extension curves acquired with a 100-fold higher time resolution and 10-fold higher force precision than traditional AFM studies of membrane proteins. Next, by using an inverse Weierstrass transform and the Jarzynski equality, we removed the free energy associated with the force probe and determined the molecular free-energy landscape of the molecule under study, bacteriorhodopsin. The resulting landscape yielded an average unfolding free energy per amino acid (aa) of 1.0 ± 0.1 kcal/mol, in agreement with past single-molecule studies. Moreover, on a smaller spatial scale, this high-resolution landscape also agreed with an equilibrium measurement of a particular three-aa transition in bacteriorhodopsin that yielded 2.7 kcal/mol/aa, an unexpectedly high value. Hence, while average unfolding ΔG0 per aa is a useful metric, the derived high-resolution landscape details significant local variation from the mean. More generally, we demonstrated that, as anticipated, the inverse Weierstrass transform is an efficient means to reconstruct free-energy landscapes from AFM data.

NMR at Low and Ultra-Low Temperatures

PubMed Central

Tycko, Robert

2017-01-01

Conspectus Solid state nuclear magnetic resonance (NMR) measurements at low temperatures have been common in physical sciences for many years, and are becoming increasingly important in studies of biomolecular systems. This article reviews a diverse set of projects from my laboratory, dating back to the early 1990s, that illustrate the motivations for low-temperature solid state NMR, the types of information that are available from the measurements, and likely directions for future research. These projects include NMR studies of both physical and biological systems, performed at low (cooled with nitrogen, down to 77 K) and very low (cooled with helium, below 77 K) temperatures, and performed with and without magic-angle spinning (MAS). In NMR studies of physical systems, the main motivation is to study phenomena that occur only at low temperatures. Two examples from my laboratory are studies of molecular rotation and an orientational ordering in solid C60 at low temperatures and studies of unusual electronic states, called skyrmions, in two-dimensionally confined electron systems within semiconductor quantum wells. NMR measurements on quantum wells were facilitated by optical pumping of nuclear spin polarizations, a signal enhancement phenomenon that exists at very low temperatures. In studies of biomolecular systems, motivations for low-temperature NMR include suppression of molecular tumbling (thereby permitting solid state NMR measurements on soluble proteins), suppression of conformational exchange (thereby permitting quantitation of conformational distributions), and trapping of transient intermediate states in a non-equilibrium kinetic process (by rapid freeze-quenching). Solid state NMR measurements on AIDS-related peptide/antibody complexes, chemically denatured states of the model protein HP35, and a transient intermediate in the rapid folding pathway of HP35 illustrate these motivations. NMR sensitivity generally increases with decreasing sample temperature. It is therefore advantageous to go as cold as possible, particularly in studies of biomolecular systems in frozen solutions. However, solid state NMR studies of biomolecular systems generally require rapid MAS. A novel MAS NMR probe design that uses nitrogen gas for sample spinning and cold helium only for sample cooling allows a wide variety of solid state NMR measurements to be performed on biomolecular systems at 20-25 K, where signals are enhanced by factors of 12-15 relative to measurements at room temperature. MAS NMR at very low temperatures also facilitates dynamic nuclear polarization (DNP), allowing sizeable additional signal enhancements and large absolute NMR signal amplitudes to be achieved with relatively low microwave powers. Current research in my laboratory seeks to develop and exploit DNP-enhanced MAS NMR at very low temperatures, for example in studies of transient intermediates in protein folding and aggregation processes and studies of peptide/protein complexes that can be prepared only at low concentrations. PMID:23470028

Vibrational non-equilibrium in the hydrogen-oxygen reaction. Comparison with experiment

NASA Astrophysics Data System (ADS)

Skrebkov, Oleg V.

2015-03-01

A theoretical model is proposed for the chemical and vibrational kinetics of hydrogen oxidation based on consistent accounting of the vibrational non-equilibrium of the HO2 radical that forms as a result of the bimolecular recombination H+O2 → HO2. In the proposed model, the chain branching H+O2 = O+OH and inhibiting H+O2+M = HO2+M formal reactions are treated (in the terms of elementary processes) as a single multi-channel process of forming, intramolecular energy redistribution between modes, relaxation, and unimolecular decay of the comparatively long-lived vibrationally excited HO2 radical, which is able to react and exchange energy with the other components of the mixture. The model takes into account the vibrational non-equilibrium of the starting (primary) H2 and O2 molecules, as well as the most important molecular intermediates HO2, OH, O2(1Δ), and the main reaction product H2O. It is shown that the hydrogen-oxygen reaction proceeds in the absence of vibrational equilibrium, and the vibrationally excited HO2(v) radical acts as a key intermediate in a fundamentally important chain branching process and in the generation of electronically excited species O2(1Δ), O(1D), and OH(2Σ+). The calculated results are compared with the shock tube experimental data for strongly diluted H2-O2 mixtures at 1000 < T < 2500 K, 0.5 < p < 4 atm. It is demonstrated that this approach is promising from the standpoint of reconciling the predictions of the theoretical model with experimental data obtained by different authors for various compositions and conditions using different methods. For T < 1500 K, the nature of the hydrogen-oxygen reaction is especially non-equilibrium, and the vibrational non-equilibrium of the HO2 radical is the essence of this process. The quantitative estimation of the vibrational relaxation characteristic time of the HO2 radical in its collisions with H2 molecules has been obtained as a result of the comparison of different experimental data on induction time measurements with the relevant calculations.

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.

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

Assembly of hard spheres in a cylinder: a computational and experimental study.

PubMed

Fu, Lin; Bian, Ce; Shields, C Wyatt; Cruz, Daniela F; López, Gabriel P; Charbonneau, Patrick

2017-05-14

Hard spheres are an important benchmark of our understanding of natural and synthetic systems. In this work, colloidal experiments and Monte Carlo simulations examine the equilibrium and out-of-equilibrium assembly of hard spheres of diameter σ within cylinders of diameter σ≤D≤ 2.82σ. Although phase transitions formally do not exist in such systems, marked structural crossovers can nonetheless be observed. Over this range of D, we find in simulations that structural crossovers echo the structural changes in the sequence of densest packings. We also observe that the out-of-equilibrium self-assembly depends on the compression rate. Slow compression approximates equilibrium results, while fast compression can skip intermediate structures. Crossovers for which no continuous line-slip exists are found to be dynamically unfavorable, which is the main source of this difference. Results from colloidal sedimentation experiments at low diffusion rate are found to be consistent with the results of fast compressions, as long as appropriate boundary conditions are used.

Higgs-boson production in nucleus-nucleus collisions

NASA Technical Reports Server (NTRS)

Norbury, J. W.; Townsend, L. W. (Principal Investigator)

1990-01-01

Cross-section calculations are presented for the production of intermediate-mass Higgs bosons produced in ultrarelativistic nucleus-nucleus collisions via two-photon fusion. The calculations are performed in position space using Baur's method for folding together the Weizsacker-Williams virtual-photon spectra of the two colliding nuclei. It is found that two-photon fusion in nucleus-nucleus collisions is a plausible way of finding intermediate-mass Higgs bosons at the Superconducting Super Collider or the CERN Large Hadron Collider.

Higgs-Boson Production in Nucleus-Nucleus Collisions

NASA Technical Reports Server (NTRS)

Norbury, John W.

1992-01-01

Cross section calculations are presented for the production of intermediate-mass Higgs bosons produced in ultrarelativistic nucleus-nucleus collisions via two photon fusion. The calculations are performed in position space using Baur's method for folding together the Weizsacker-Williams virtual-photon spectra of the two colliding nuclei. It is found that two photon fusion in nucleus-nucleus collisions is a plausible way of finding intermediate-mass Higgs bosons at the Superconducting Super Collider or the CERN Large Hadron Collider.

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin.

PubMed

Pozzi, Nicola; Zerbetto, Mirco; Acquasaliente, Laura; Tescari, Simone; Frezzato, Diego; Polimeno, Antonino; Gohara, David W; Di Cera, Enrico; De Filippis, Vincenzo

2016-07-19

Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ in their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215-217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high-resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold.

Microscopic modeling of direct pre-equilibrium emission from neutron induced reactions on even and odd actinides

NASA Astrophysics Data System (ADS)

Dupuis, M.; Hilaire, S.; Péru, S.; Bauge, E.; Kerveno, M.; Dessagne, P.; Henning, G.

2017-09-01

Direct inelastic scattering to discrete excitations and pre-equilibrium emission are described within a microscopic model. Nuclear structure information are obtained in the (Quasi) Random Phase Approximation ((Q)RPA) framework implemented with the Gogny force. The relevant optical and transition potentials are build considering the JLM folding model. Various successful applications are shown for (n,n), (n,n'), (n,xn) and (n,xnγ) reactions for spherical and axially deformed even-even or odd targets. The rearrangement corrections to transition potentials and the contribution of unnatural parity excitations to pre-equilibrium emission are discussed. Our model predictions for (n,n'γ) reactions, for intra- and inter-band transitions in 238U, and for the 239Pu(n,2n) cross section are analyzed.

The Oligomeric Nature of Triosephosphate Isomerase. Studies of Monomerization

NASA Astrophysics Data System (ADS)

Zárate-Pérez, Francisco; Vázquez-Contreras, Edgar

2008-02-01

In this work, we report the implications of the monomerization of triosephosphate isomerase (TIM) from T. cruzi (TcTIM). A monomeric mutant (monoTcTIM) of this species was constructed by genetic engineering, shortening the main loop of interdigitation, which is fundamental for its dimerization. The properties of monoTcTIM were compared with those of the other TIM monomeric versions: genetically engineered mutants, or equilibrium intermediates obtained by chemical denaturation. The stability for almost all the monomeric variants so far reported appears in the same range, and also presents similar structural characteristics. Regarding the catalytic activity of monomeric mutants, when it is present is in several orders of magnitude lower than those observed in their respective wild-type enzyme. The change in the hydrophobic surface of TcTIM after and before monomerization was also studied and corresponds to an extensive area of the interface region, which becomes exposed when monomers are dissociated. This fact could be related with conformational changes in the local environment of the catalytic amino acids responsible for the isomerization of the substrate and consequently the inactivation of its catalytic properties. Comparison of the structural, folding and unfolding properties, as well as stability studies could give answers on why this enzyme is an obligate oligomer.

Predictors of natively unfolded proteins: unanimous consensus score to detect a twilight zone between order and disorder in generic datasets.

PubMed

Deiana, Antonio; Giansanti, Andrea

2010-04-21

Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and applicative studies. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded ones. In this work we propose an operational method to identify proteins belonging to the twilight zone by combining into a consensus score good performing single predictors of folding. In this methodological paper dichotomic folding indexes are considered: hydrophobicity-charge, mean packing, mean pairwise energy, Poodle-W and a new global index, that is called here gVSL2, based on the local disorder predictor VSL2. The performance of these indexes is evaluated on different datasets, in particular on a new dataset composed by 2369 folded and 81 natively unfolded proteins. Poodle-W, gVSL2 and mean pairwise energy have good performance and stability in all the datasets considered and are combined into a strictly unanimous combination score SSU, that leaves proteins unclassified when the consensus of all combined indexes is not reached. The unclassified proteins: i) belong to an overlap region in the vector space of amino acidic compositions occupied by both folded and unfolded proteins; ii) are composed by approximately the same number of order-promoting and disorder-promoting amino acids; iii) have a mean flexibility intermediate between that of folded and that of unfolded proteins. Our results show that proteins unclassified by SSU belong to a twilight zone. Proteins left unclassified by the consensus score SSU have physical properties intermediate between those of folded and those of natively unfolded proteins and their structural properties and evolutionary history are worth to be investigated.

Predictors of natively unfolded proteins: unanimous consensus score to detect a twilight zone between order and disorder in generic datasets

PubMed Central

2010-01-01

Background Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and applicative studies. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded ones. In this work we propose an operational method to identify proteins belonging to the twilight zone by combining into a consensus score good performing single predictors of folding. Results In this methodological paper dichotomic folding indexes are considered: hydrophobicity-charge, mean packing, mean pairwise energy, Poodle-W and a new global index, that is called here gVSL2, based on the local disorder predictor VSL2. The performance of these indexes is evaluated on different datasets, in particular on a new dataset composed by 2369 folded and 81 natively unfolded proteins. Poodle-W, gVSL2 and mean pairwise energy have good performance and stability in all the datasets considered and are combined into a strictly unanimous combination score SSU, that leaves proteins unclassified when the consensus of all combined indexes is not reached. The unclassified proteins: i) belong to an overlap region in the vector space of amino acidic compositions occupied by both folded and unfolded proteins; ii) are composed by approximately the same number of order-promoting and disorder-promoting amino acids; iii) have a mean flexibility intermediate between that of folded and that of unfolded proteins. Conclusions Our results show that proteins unclassified by SSU belong to a twilight zone. Proteins left unclassified by the consensus score SSU have physical properties intermediate between those of folded and those of natively unfolded proteins and their structural properties and evolutionary history are worth to be investigated. PMID:20409339

G-triplex structure and formation propensity

PubMed Central

Cerofolini, Linda; Amato, Jussara; Giachetti, Andrea; Limongelli, Vittorio; Novellino, Ettore; Parrinello, Michele; Fragai, Marco; Randazzo, Antonio; Luchinat, Claudio

2014-01-01

The occurrence of a G-triplex folding intermediate of thrombin binding aptamer (TBA) has been recently predicted by metadynamics calculations, and experimentally supported by Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) data collected on a 3′ end TBA-truncated 11-mer oligonucleotide (11-mer-3′-t-TBA). Here we present the solution structure of 11-mer-3′-t-TBA in the presence of potassium ions. This structure is the first experimental example of a G-triplex folding, where a network of Hoogsteen-like hydrogen bonds stabilizes six guanines to form two G:G:G triad planes. The G-triplex folding of 11-mer-3′-t-TBA is stabilized by the potassium ion and destabilized by increasing the temperature. The superimposition of the experimental structure with that predicted by metadynamics shows a great similarity, with only significant differences involving two loops. These new structural data show that 11-mer-3′-t-TBA assumes a G-triplex DNA conformation as its stable form, reinforcing the idea that G-triplex folding intermediates may occur in vivo in human guanine-rich sequences. NMR and CD screening of eight different constructs obtained by removing from one to four bases at either the 3′ and the 5′ ends show that only the 11-mer-3′-t-TBA yields a relatively stable G-triplex. PMID:25378342

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

Site-Specific Imaging of Elemental Steps in Dehydration of Diols on TiO 2(110)

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

Acharya, Danda P.; Yoon, Yeohoon; Li, Zhenjun

2013-11-26

The conversion of diols on partially reduced TiO 2(110) at low coverage was studied using variable-temperature scanning tunneling microscopy, temperature programmed desorption and density functional theory calculations. We find, that below ~230 K, ethane-1,2-diol and propane-1,3-diol molecules adsorb predominantly on five-fold coordinated Ti5c atoms. The dynamic equilibrium between molecularly bound and dissociated species resulting from O-H bond scission and reformation is observed. As the diols start to diffuse on the Ti5c rows above ~230 K, they dissociate irreversibly upon encountering bridging oxygen (O b) vacancy (VO’s) defects. Two dissociation pathways, one via O-H and the other via C-O bond scissionmore » leading to identical surface intermediates, hydroxyalkoxy, O b-(CH 2)n-OH (n = 2, 3) and bridging hydroxyl, HO b, are seen. For O-H bond scission, the O b-(CH 2)n-OH is found on the position of the original VO, while for C-O scission it is found on the adjacent Ob site. Theoretical calculations suggest that the observed mixture of C-O/O-H bond breaking processes are a result of the steric factors enforced upon the diols by the second OH group that is bound to a Ti5c site. At room temperature, rich dissociation/reformation dynamics of the second, Ti5c-bound O-H leads to the formation of dioxo, Ob-(CH 2)n-OTi, species. Above ~400 K, both O b-(CH 2)n-OH and Ob-(CH 2)n-OTi species convert into a new intermediate, that is centered on Ob row. Combined experimental and theoretical evidence shows that this intermediate is most likely a new dioxo, O b-(CH 2) 2-Ob, species. Further annealing leads to sequential C-Ob bond cleavage and alkene desorption above ~ 500 K. Simulations find that the sequential C-O bond breaking process follows a homolytic diradical pathway with the first C-O bond breaking event accompanied by a non-adiabatic electron transfer within the TiO 2(110) substrate.« less

2-propen-1-amine derivatives and their synthetic intermediates: activity against pathogenic trypanosomatids.

PubMed

de Souza, A O; Hemerly, F P; Gomes-Cardoso, L; Santa-Rita, R M; Leon, L L; de Castro, S L; Durán, N

2004-12-01

The potential activity of three new derivatives of 3-(4'-Y-[1,1'-biphenyl]-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine (2-PAMs) was assayed against Trypanosoma cruzi and Leishmania amazonensis. They showed higher activity against trypomastigotes and epimastigotes of T. cruzi than the standard drugs, crystal violet and nifurtimox. Besides these derivatives, a series of eleven 2-PAMs derivatives and the corresponding intermediates, biphenyl methanones (BPMs) were assayed against promastigotes of L. amazonensis, showing that the 2-PAMs were remarkably more active than the BPMs. The PAMs 2c, 2e and 2j were about 2-fold more active that pentamidine isothionate and between 27.2- and 46.4-fold less toxic to V79 mammalian cells. The present results encourage further studies, especially against intracellular parasites and in experimental animals.

Mineral Ion Contents and Cell Transmembrane Electropotentials of Pea and Oat Seedling Tissue 1

PubMed Central

Higinbotham, N.; Etherton, Bud; Foster, R. J.

1967-01-01

The relationships of concentration gradients to electropotential gradients resulting from passive diffusion processes, after equilibration, are described by the Nernst equation. The primary criterion for the hypothesis that any given ion is actively transported is to establish that it is not diffusing passively. A test was made of how closely the Nernst equation describes the electrochemical equilibrium in seedling tissues. Segments of roots and epicotyl internodes of pea (Pisum sativum var. Alaska) and of roots and coleoptiles of oat (Avena sativa var. Victory) seedlings were immersed and shaken in defined nutrient solutions containing eight major nutrients (K+, Na+, Ca2+, Mg2+, Cl−, NO3−, H2PO4− and SO42−) at 1-fold and 10-fold concentrations. The tissue content of each ion was assayed at 0, 8, 24, and 48 hours. A near-equilibrium condition was approached by roots for most ions; however, the segments of shoot tissue generally continued to show a net accumulation of some ions, mainly K+ and NO3−. Only K+ approached a reasonable fit to the Nernst equation and this was true for the 1-fold concentration but not the 10-fold. The data suggest that for Na+, Mg2+, and Ca2+ the electrochemical gradient is from the external solution to the cell interior; thus passive diffusion should be in an inward direction. Consequently, some mechanism must exist in plant tissue either to exclude these cations or to extrude them (e.g., by an active efflux pump). For each of the anions the electrochemical gradient is from the tissue to the solution; thus an active influx pump for anions seems required. Root segments approach ionic equilibrium with the solution concentration in which the seedlings were grown. Segments of shoot tissue, however, are far removed from such equilibration. Thus in the intact seedling the extracellular (wall space) fluid must be very different from that of the nutrient solution bathing the segments; it would appear that the root is the site of regulation of ion uptake in the intact plant although other correlative mechanisms may be involved. PMID:16656483

Non-Native α-Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the β-Barrel in β-Lactoglobulin.

PubMed

Sakurai, Kazumasa; Yagi, Masanori; Konuma, Tsuyoshi; Takahashi, Satoshi; Nishimura, Chiaki; Goto, Yuji

2017-09-12

The roles of non-native α-helices frequently observed in the initial folding stage of β-sheet proteins have been examined for many years. We herein investigated the residue-level structures of several mutants of bovine β-lactoglobulin (βLG) in quenched-flow pH-pulse labeling experiments. βLG assumes a collapsed intermediate with a non-native α-helical structure (I 0 ) in the early stage of folding, although its native form is predominantly composed of β-structures. The protection profile in I 0 of pseudo-wild type (WT*) βLG was found to deviate from the pattern of the "average area buried upon folding" (AABUF). In particular, the level of protection at the region of strand A, at which non-native α-helices form in the I 0 state, was significantly low compared to AABUF. G17E, the mutant with an increased helical propensity, showed a similar protection pattern. In contrast, the protection pattern for I 0 of E44L, the mutant with an increased β-sheet propensity, was distinct from that of WT* and resembled the AABUF pattern. Transverse relaxation measurements demonstrated that the positions of the residual structures in the unfolded states of these mutants were consistent with those of the protected residues in the respective I 0 states. On the basis of the slower conversion of I 0 to the native state for E44L to that for WT*, non-native α-helices facilitate the ordered assembly of the β-barrel by preventing interactions that trap folding.

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

PubMed Central

1992-01-01

Mutant V.24.1, a member of the End4 complementation group of temperature-sensitive CHO cells, is defective in secretion at the restrictive temperature (Wang, R.-H., P. A. Colbaugh, C.-Y. Kao, E. A. Rutledge, and R. K. Draper. 1990. J. Biol. Chem. 265:20179-20187; Presley, J. F., R. K. Draper, and D. T. Brown. 1991. J. Virol. 65:1332- 1339). We have further investigated the secretory lesion and report three main findings. First, the block in secretion is not due to aberrant folding or oligomerization of secretory proteins in the endoplasmic reticulum because the hemagglutinin of influenza virus folded and oligomerized at the same rate in mutant and parental cells at the restrictive temperature. Second, secretory proteins accumulated in a compartment intermediate between the ER and the Golgi. Several lines of evidence support this conclusion, the most direct being the colocalization by immunofluorescence microscopy of influenza virus hemagglutinin with a 58-kD protein that is known to reside in an intermediate compartment. Third, at the resolution of fluorescence microscopy, the Golgi complex in the mutant cells vanished at the restrictive temperature. PMID:1577851

A Review of Energy Models with Particular Reference to Employment and Manpower Analysis.

ERIC Educational Resources Information Center

Eckstein, Albert J.; Heien, Dale M.

To analyze the application of quantitative models to energy-employment issues, the energy problem was viewed in three distinct, but related, phases: the post-embargo shock effects, the intermediate-term process of adjustment, and the long-run equilibrium. Against this background eighteen existing energy models (government supported as well as…

Modulation of a protein free-energy landscape by circular permutation.

PubMed

Radou, Gaël; Enciso, Marta; Krivov, Sergei; Paci, Emanuele

2013-11-07

Circular permutations usually retain the native structure and function of a protein while inevitably perturbing its folding dynamics. By using simulations with a structure-based model and a rigorous methodology to determine free-energy surfaces from trajectories, we evaluate the effect of a circular permutation on the free-energy landscape of the protein T4 lysozyme. We observe changes which, although subtle, largely affect the cooperativity between the two subdomains. Such a change in cooperativity has been previously experimentally observed and recently also characterized using single molecule optical tweezers and the Crooks relation. The free-energy landscapes show that both the wild type and circular permutant have an on-pathway intermediate, previously experimentally characterized, in which one of the subdomains is completely formed. The landscapes, however, differ in the position of the rate-limiting step for folding, which occurs before the intermediate in the wild type and after in the circular permutant. This shift of transition state explains the observed change in the cooperativity. The underlying free-energy landscape thus provides a microscopic description of the folding dynamics and the connection between circular permutation and the loss of cooperativity experimentally observed.

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

Mechanism of 1,4,5,8-naphthalene tetracarboxylic acid dianhydride hydrolysis and formation in aqueous solution.

PubMed

Barros, T C; Cuccovia, I M; Farah, J P S; Masini, J C; Chaimovich, H; Politi, M J

2006-01-07

The study of highly conjugated, carbonyl-containing molecules such as 1,4,5,8-naphthalene tetracarboxylic dianhydride, III, is of interest since reactivity differences and transmission of electronic effects through the conjugated framework can be evidenced. The kinetics of hydrolysis of III in aqueous solution were determined from 5 M acid to pH 10. In basic solution hydrolysis of III yields, sequentially, 1,4,5,8-naphthalene diacid monoanhydride, II, and 1,4,5,8-naphthalene tetracarboxylic acid, I. The second order rate constant for alkaline hydrolysis is 200 fold higher for the first ring opening. The water-catalyzed hydrolysis of III yields a pH-dependent mixture of ionic forms of I and II. The rate constant for water-catalyzed hydrolysis of III is 25 fold higher than that for II. In concentrated acid the rates for reaching equilibrium (I, II and III) increase and III is the major product. The pK(a)s of I (3.24, 5.13 and 6.25) and II (3.05, 5.90) were determined by potentiometric, fluorescence and UV spectroscopy titrations and by quantitative fit of the kinetic and equilibrium data. The apparent, pH-dependent, equilibrium constants, K(EqII), for anhydride formation between I and II were obtained from the UV spectra. The quantitative fit of kinetic and equilibrium data are consistent with the assumption that anhydride formation only proceeds with the fully protonated species for both I and II and permitted the estimation of the equilibrium constants for anhydride formation, K(EqII). The value of K(EqII) (I <==> II) between pH 1 and 6 was ca. 5. Geometry optimization calculations in the gas phase of the reactions of III in alkaline, neutral and acid conditions, at the DFT level of theory, gave electronic distributions that were qualitatively consistent with the experimental results.

The Metallicity Evolution of Low Mass Galaxies: New Contraints at Intermediate Redshift

NASA Technical Reports Server (NTRS)

Henry, Alaina; Martin, Crystal L.; Finlator, Kristian; Dressler, Alan

2013-01-01

We present abundance measurements from 26 emission-line-selected galaxies at z approx. 0.6-0.7. By reaching stellar masses as low as 10(exp 8) M stellar mass, these observations provide the first measurement of the intermediate-redshift mass-metallicity (MZ) relation below 10(exp 9)M stellar mass. For the portion of our sample above M is greater than 10(exp 9)M (8/26 galaxies), we find good agreement with previous measurements of the intermediate-redshift MZ relation. Compared to the local relation, we measure an evolution that corresponds to a 0.12 dex decrease in oxygen abundances at intermediate redshifts. This result confirms the trend that metallicity evolution becomes more significant toward lower stellar masses, in keeping with a downsizing scenario where low-mass galaxies evolve onto the local MZ relation at later cosmic times. We show that these galaxies follow the local fundamental metallicity relation, where objects with higher specific (mass-normalized) star formation rates (SFRs) have lower metallicities. Furthermore, we show that the galaxies in our sample lie on an extrapolation of the SFR-M* relation (the star-forming main sequence). Leveraging the MZ relation and star-forming main sequence (and combining our data with higher-mass measurements from the literature), we test models that assume an equilibrium between mass inflow, outflow, and star formation.We find that outflows are required to describe the data. By comparing different outflow prescriptions, we show that momentum, driven winds can describe the MZ relation; however, this model underpredicts the amount of star formation in low-mass galaxies. This disagreement may indicate that preventive feedback from gas heating has been overestimated, or it may signify a more fundamental deviation from the equilibrium assumption.

Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates.

PubMed

Moparthi, Satish Babu; Fristedt, Rikard; Mishra, Rajesh; Almstedt, Karin; Karlsson, Martin; Hammarström, Per; Carlsson, Uno

2010-02-16

The single-domain cyclophilin 18 (Cyp18) has long been known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and was proposed by us to also function as a chaperone [Freskgard, P.-O., Bergenhem, N., Jonsson, B.-H., Svensson, M., and Carlsson, U. (1992) Science 258, 466-468]. Later several multidomain PPIs were demonstrated to work as both a peptidyl-prolyl cis/trans isomerase and a chaperone. However, the chaperone ability of Cyp18 has been debated. In this work, we add additional results that show that Cyp18 can both accelerate the rate of refolding and increase the yield of native protein during the folding reaction, i.e., function as both a folding catalyst and a chaperone. Refolding experiments were performed using severely destabilized mutants of human carbonic anhydrase II under conditions where the unfolding reaction is significant and a larger fraction of a more destabilized variant populates molten globule-like intermediates during refolding. A correlation of native state protein stability of the substrate protein versus Cyp18 chaperone activity was demonstrated. The induced correction of misfolded conformations by Cyp18 likely functions through rescue from misfolding of transient molten globule intermediates. ANS binding data suggest that the interaction by Cyp18 leads to an early stage condensation of accessible hydrophobic portions of the misfolding-prone protein substrate during folding. The opposite effect was observed for GroEL known as an unfoldase at early stages of refolding. The chaperone effect of Cyp18 was also demonstrated for citrate synthase, suggesting a general chaperone effect of this PPI.

Role of the Disulfide Bond in Prion Protein Amyloid Formation: A Thermodynamic and Kinetic Analysis.

PubMed

Honda, Ryo

2018-02-27

Prion diseases are associated with the structural conversion of prion protein (PrP) to a β-sheet-rich aggregate, PrP Sc . Previous studies have indicated that a reduction of the disulfide bond linking C179 and C214 of PrP yields an amyloidlike β-rich aggregate in vitro. To gain mechanistic insights into the reduction-induced aggregation, here I characterized how disulfide bond reduction modulates the protein folding/misfolding landscape of PrP, by examining 1) the equilibrium stabilities of the native (N) and aggregated states relative to the unfolded (U) state, 2) the transition barrier separating the U and aggregated states, and 3) the final structure of amyloidlike misfolded aggregates. Kinetic and thermodynamic experiments revealed that disulfide bond reduction decreases the equilibrium stabilities of both the N and aggregated states by ∼3 kcal/mol, without changing either the amyloidlike aggregate structure, at least at the secondary structural level, or the transition barrier of aggregation. Therefore, disulfide bond reduction modulates the protein folding/misfolding landscape by entropically stabilizing disordered states, including the U and transition state of aggregation. This also indicates that the equilibrium stability of the N state, but not the transition barrier of aggregation, is the dominant factor determining the reduction-induced aggregation of PrP. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Quasi-equilibrium analysis of the ion-pair mediated membrane transport of low-permeability drugs.

PubMed

Miller, Jonathan M; Dahan, Arik; Gupta, Deepak; Varghese, Sheeba; Amidon, Gordon L

2009-07-01

The aim of this research was to gain a mechanistic understanding of ion-pair mediated membrane transport of low-permeability drugs. Quasi-equilibrium mass transport analyses were developed to describe the ion-pair mediated octanol-buffer partitioning and hydrophobic membrane permeation of the model basic drug phenformin. Three lipophilic counterions were employed: p-toluenesulfonic acid, 2-naphthalenesulfonic acid, and 1-hydroxy-2-naphthoic acid (HNAP). Association constants and intrinsic octanol-buffer partition coefficients (Log P(AB)) of the ion-pairs were obtained by fitting a transport model to double reciprocal plots of apparent octanol-buffer distribution coefficients versus counterion concentration. All three counterions enhanced the lipophilicity of phenformin, with HNAP providing the greatest increase in Log P(AB), 3.7 units over phenformin alone. HNAP also enhanced the apparent membrane permeability of phenformin, 27-fold in the PAMPA model, and 4.9-fold across Caco-2 cell monolayers. As predicted from a quasi-equilibrium analysis of ion-pair mediated membrane transport, an order of magnitude increase in phenformin flux was observed per log increase in counterion concentration, such that log-log plots of phenformin flux versus HNAP concentration gave linear relationships. These results provide increased understanding of the underlying mechanisms of ion-pair mediated membrane transport, emphasizing the potential of this approach to enable oral delivery of low-permeability drugs.

Effects of energy conservation on equilibrium properties of hot asymmetric nuclear matter

NASA Astrophysics Data System (ADS)

Zhang, Zhen; Ko, Che Ming

2018-01-01

Based on the relativistic Vlasov-Uehling-Uhlenbeck transport model, which includes relativistic scalar and vector potentials on baryons, we consider an N -Δ -π system in a box with periodic boundary conditions to study the effects of energy conservation in particle production and absorption processes on the equilibrium properties of the system. The density and temperature of the matter in the box are taken to be similar to the hot dense matter formed in heavy ion collisions at intermediate energies. We find that to maintain the equilibrium numbers of N ,Δ , and π , which depend on the mean-field potentials of N and Δ , we must include these potentials in the energy conservation condition that determines the momenta of outgoing particles after a scattering or decay process. We further find that the baryon scalar potentials mainly affect the Δ and pion equilibrium numbers, while the baryon vector potentials have considerable effect on the effective charged pion ratio at equilibrium. Our results thus indicate that it is essential to include in the transport model the effect of potentials in the energy conservation of a scattering or decay process, which is ignored in most transport models, for studying pion production in heavy ion collisions.

Accelerated molecular dynamics simulations of protein folding.

PubMed

Miao, Yinglong; Feixas, Ferran; Eun, Changsun; McCammon, J Andrew

2015-07-30

Folding of four fast-folding proteins, including chignolin, Trp-cage, villin headpiece and WW domain, was simulated via accelerated molecular dynamics (aMD). In comparison with hundred-of-microsecond timescale conventional molecular dynamics (cMD) simulations performed on the Anton supercomputer, aMD captured complete folding of the four proteins in significantly shorter simulation time. The folded protein conformations were found within 0.2-2.1 Å of the native NMR or X-ray crystal structures. Free energy profiles calculated through improved reweighting of the aMD simulations using cumulant expansion to the second-order are in good agreement with those obtained from cMD simulations. This allows us to identify distinct conformational states (e.g., unfolded and intermediate) other than the native structure and the protein folding energy barriers. Detailed analysis of protein secondary structures and local key residue interactions provided important insights into the protein folding pathways. Furthermore, the selections of force fields and aMD simulation parameters are discussed in detail. Our work shows usefulness and accuracy of aMD in studying protein folding, providing basic references in using aMD in future protein-folding studies. © 2015 Wiley Periodicals, Inc.

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

Light-activated control of protein channel assembly mediated by membrane mechanics

NASA Astrophysics Data System (ADS)

Miller, David M.; Findlay, Heather E.; Ces, Oscar; Templer, Richard H.; Booth, Paula J.

2016-12-01

Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump-dump-probe spectroscopy.

PubMed

Larsen, Delmar S; van Stokkum, Ivo H M; Vengris, Mikas; van Der Horst, Michael A; de Weerd, Frank L; Hellingwerf, Klaas J; van Grondelle, Rienk

2004-09-01

Photoactive yellow protein is the protein responsible for initiating the "blue-light vision" of Halorhodospira halophila. The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses. This "incoherent" manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model. This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I(0) and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state. Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment. This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates.

Incoherent Manipulation of the Photoactive Yellow Protein Photocycle with Dispersed Pump-Dump-Probe Spectroscopy

PubMed Central

Larsen, Delmar S.; van Stokkum, Ivo H. M.; Vengris, Mikas; van der Horst, Michael A.; de Weerd, Frank L.; Hellingwerf, Klaas J.; van Grondelle, Rienk

2004-01-01

Photoactive yellow protein is the protein responsible for initiating the “blue-light vision” of Halorhodospira halophila. The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses. This “incoherent” manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model. This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I0 and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state. Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment. This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates. PMID:15345564

PROTERAN: animated terrain evolution for visual analysis of patterns in protein folding trajectory.

PubMed

Zhou, Ruhong; Parida, Laxmi; Kapila, Kush; Mudur, Sudhir

2007-01-01

The mechanism of protein folding remains largely a mystery in molecular biology, despite the enormous effort from many groups in the past decades. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates such as the fraction of native contacts, the radius of gyration and so on. In this paper, we present an integrated approach towards understanding the folding process via visual analysis of patterns of these reaction coordinates. The three disparate processes (1) protein folding simulation, (2) pattern elicitation and (3) visualization of patterns, work in tandem. Thus as the protein folds, the changing landscape in the pattern space can be viewed via the visualization tool, PROTERAN, a program we developed for this purpose. We first present an incremental (on-line) trie-based pattern discovery algorithm to elicit the patterns and then describe the terrain metaphor based visualization tool. Using two example small proteins, a beta-hairpin and a designed protein Trp-cage, we next demonstrate that this combined pattern discovery and visualization approach extracts crucial information about protein folding intermediates and mechanism.

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.

Retarded protein folding of deficient human α1-antitrypsin D256V and L41P variants

PubMed Central

Jung, Chan-Hun; Na, Yu-Ran; Im, Hana

2004-01-01

α1-Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human α1-antitrypsin are associated with early-onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant α1-antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient α1-antitrypsin variants. Unlike most α1-antitrypsin variants, which were unstable, D256V and L41P variants exhibited extremely retarded protein folding as compared with the wild-type molecule. Once folded, however, the stability and inhibitory activity of these variant proteins were comparable to those of the wild-type molecule. Retarded protein folding may promote protein aggregation by allowing the accumulation of aggregation-prone folding intermediates. Repeated observations of retarded protein folding indicate that it is an important mechanism causing α1-antitrypsin deficiency by variant molecules, which have to fold into the metastable native form to be functional. PMID:14767073

The mechanism and thermodynamics of transesterification of acetate-ester enolates in the gas phase

NASA Astrophysics Data System (ADS)

Haas, George W.; Giblin, Daryl E.; Gross, Michael L.

1998-01-01

In solution, base-catalyzed hydrolysis and transesterification of esters are initiated by hydroxide- or alkoxide-ion attack at the carbonyl carbon. At low pressures in the gas phase, however, transesterification proceeds by an attack of the enolate anion of an acetate ester on an alcohol. Fourier transform mass spectrometry (FTMS) indicates that the reaction is the second-order process: -CH2-CO2-R + R'-OH --> - CH2-CO2-R' + R-OH and there is little to no detectable production of either alkoxide anion. Labeling studies show that the product and reactant enolate anion esters undergo exchange of hydrogens located [alpha] to the carbonyl carbon with the deuterium of R'-OD. The extent of the H/D exchange increases with reaction time, pointing to a short-lived intermediate. The alcoholysis reaction rate constants increase with increasing acidity of the primary, straight-chained alkyl alcohols, whereas steric effects associated with branched alcohols cause the rate constants to decrease. Equilibrium constants, which were determined directly from measurements at equilibrium and which were calculated from the forward and reverse rate constants, are near unity and show internal consistency. In the absence of steric effects, the larger enolate is always the favored product at equilibrium. The intermediate for the transesterification reaction, which can be generated at a few tenths of a torr in a tandem mass spectrometer, is tetrahedral, but other adducts that are collisionally stabilized under these conditions are principally loosely bound complexes.

Excited atoms in the free-burning Ar arc: treatment of the resonance radiation

NASA Astrophysics Data System (ADS)

Golubovskii, Yu; Kalanov, D.; Gortschakow, S.; Baeva, M.; Uhrlandt, D.

2016-11-01

The collisional-radiative model with an emphasis on the accurate treatment of the resonance radiation transport is developed and applied to the free-burning Ar arc plasma. This model allows for analysis of the influence of resonance radiation on the spatial density profiles of the atoms in different excited states. The comparison of the radial density profiles obtained using an effective transition probability approximation with the results of the accurate solution demonstrates the distinct impact of transport on the profiles and absolute densities of the excited atoms, especially in the arc fringes. The departures from the Saha-Boltzmann equilibrium distributions, caused by different radiative transitions, are analyzed. For the case of the DC arc, the local thermodynamic equilibrium (LTE) state holds close to the arc axis, while strong deviations from the equilibrium state on the periphery occur. In the intermediate radial positions the conditions of partial LTE are fulfilled.

Two-phase vesicles: a study on evolutionary and stationary models.

PubMed

Sahebifard, MohammadMahdi; Shahidi, Alireza; Ziaei-Rad, Saeed

2017-05-01

In the current article, the dynamic evolution of two-phase vesicles is presented as an extension to a previous stationary model and based on an equilibrium of local forces. In the simplified model, ignoring the effects of membrane inertia, a dynamic equilibrium between the membrane bending potential and local fluid friction is considered in each phase. The equilibrium equations at the domain borders are completed by extended introduction of membrane section reactions. We show that in some cases, the results of stationary and evolutionary models are in agreement with each other and also with experimental observations, while in others the two models differ markedly. The value of our approach is that we can account for unresponsive points of uncertainty using our equations with the local velocity of the lipid membranes and calculating the intermediate states (shapes) in the consequent evolutionary, or response, path.

Analytical Phase Equilibrium Function for Mixtures Obeying Raoult's and Henry's Laws

NASA Astrophysics Data System (ADS)

Hayes, Robert

When a mixture of two substances exists in both the liquid and gas phase at equilibrium, Raoults and Henry's laws (ideal solution and ideal dilute solution approximations) can be used to estimate the gas and liquid mole fractions at the extremes of either very little solute or solvent. By assuming that a cubic polynomial can reasonably approximate the intermediate values to these extremes as a function of mole fraction, the cubic polynomial is solved and presented. A closed form equation approximating the pressure dependence on mole fraction of the constituents is thereby obtained. As a first approximation, this is a very simple and potentially useful means to estimate gas and liquid mole fractions of equilibrium mixtures. Mixtures with an azeotrope require additional attention if this type of approach is to be utilized. This work supported in part by federal Grant NRC-HQ-84-14-G-0059.

Non-canonical distribution and non-equilibrium transport beyond weak system-bath coupling regime: A polaron transformation approach

NASA Astrophysics Data System (ADS)

Xu, Dazhi; Cao, Jianshu

2016-08-01

The concept of polaron, emerged from condense matter physics, describes the dynamical interaction of moving particle with its surrounding bosonic modes. This concept has been developed into a useful method to treat open quantum systems with a complete range of system-bath coupling strength. Especially, the polaron transformation approach shows its validity in the intermediate coupling regime, in which the Redfield equation or Fermi's golden rule will fail. In the polaron frame, the equilibrium distribution carried out by perturbative expansion presents a deviation from the canonical distribution, which is beyond the usual weak coupling assumption in thermodynamics. A polaron transformed Redfield equation (PTRE) not only reproduces the dissipative quantum dynamics but also provides an accurate and efficient way to calculate the non-equilibrium steady states. Applications of the PTRE approach to problems such as exciton diffusion, heat transport and light-harvesting energy transfer are presented.

General equilibrium incidence of energy taxation

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

Solow, J.L.

1984-04-01

The pervasiveness of energy in the economy and the numerous ways in which energy taxation can distort resource allocations implies the necessity of a general equilibrium approach to the question of energy tax incidence. The author presents a general equilibrium model that accounts for domestic energy production and energy imports, direct energy consumption and use of energy as an intermediate good, and production of energy intensive and nonenergy intensive goods. He examines the incidence of three broadly-based energy taxation policies: a tax on all energy consumption, a subsidy to domestic energy production, and a tariff on energy imports. The subsidy,more » although not a revenue raising measure, is included because, like the other policies, it acts to reduce energy imports. Such a measure may be of interest by reducing reliance on unstable foreign sources of supply. 12 references, 3 tables.« less

Exploring Fiscal Policy at Zero Interest Rates in Intermediate Macroeconomics

ERIC Educational Resources Information Center

Ramamurthy, Srikanth; Sedgley, Norman

2013-01-01

Since the financial meltdown of 2007, advanced macroeconomic theory has delved more deeply into the question of the appropriate fiscal policy when the nominal interest rate is close to or at zero percent. Such analysis is typically conducted with the aid of New Keynesian Dynamic Stochastic General Equilibrium models. The policy implications are,…

The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers

PubMed Central

Chadda, Rahul; Krishnamani, Venkatramanan; Mersch, Kacey; Wong, Jason; Brimberry, Marley; Chadda, Ankita; Kolmakova-Partensky, Ludmila; Friedman, Larry J; Gelles, Jeff; Robertson, Janice L

2016-01-01

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states – monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes. DOI: http://dx.doi.org/10.7554/eLife.17438.001 PMID:27484630

Fungal melanins and their interactions with metals.

PubMed

Fogarty, R V; Tobin, J M

1996-09-01

Fungal melanins are dark brown or black pigments located in cell walls. They also exist as extracellular polymers. Melanized fungi possess increased virulence and resistance to microbial attack as well as enhanced survival while under environmental stress. Melanins contain various functional groups which provide an array of multiple nonequivalent binding sites for metal ions. Pigmented Cladosporium cladosporoides was shown to biosorb 2.5- to four-fold more Ni, Cu, Zn, Cd, and Pb than albino Penicillium digitatum and at four- to six-fold higher rates. Metal desorption was significantly lower for extracellular melanin than from pigmented or albino biomass which indicated the strength of the melanin-metal bond. At equilibrium, tributyltin chloride (TBTC) concentrations of 2.5 mM, pigmented and albino Aureobasidium pullulans absorbed approximately 0.9 and 0.7 mumol TBTC mg -1 dry wt, respectively, whereas purified extracellular melanin exhibited uptake levels of approximately 22 mumol TBTC mg-1 dry wt at an equilibrium concentration of only 0.4 mM. Addition of melanin to the growth medium reduced the toxic effect of CuSO4 and TBTC due to melanin metal binding and sequestration.

Electronic polarization stabilizes tertiary structure prediction of HP-36.

PubMed

Duan, Li L; Zhu, Tong; Zhang, Qing G; Tang, Bo; Zhang, John Z H

2014-04-01

Molecular dynamic (MD) simulations with both implicit and explicit solvent models have been carried out to study the folding dynamics of HP-36 protein. Starting from the extended conformation, the secondary structure of all three helices in HP-36 was formed in about 50 ns and remained stable in the remaining simulation. However, the formation of the tertiary structure was difficult. Although some intermediates were close to the native structure, the overall conformation was not stable. Further analysis revealed that the large structure fluctuation of loop and hydrophobic core regions was devoted mostly to the instability of the structure during MD simulation. The backbone root-mean-square deviation (RMSD) of the loop and hydrophobic core regions showed strong correlation with the backbone RMSD of the whole protein. The free energy landscape indicated that the distribution of main chain torsions in loop and turn regions was far away from the native state. Starting from an intermediate structure extracted from the initial AMBER simulation, HP-36 was found to generally fold to the native state under the dynamically adjusted polarized protein-specific charge (DPPC) simulation, while the peptide did not fold into the native structure when AMBER force filed was used. The two best folded structures were extracted and taken into further simulations in water employing AMBER03 charge and DPPC for 25 ns. Result showed that introducing polarization effect into interacting potential could stabilize the near-native protein structure.

Trp-cage: folding free energy landscape in explicit water.

PubMed

Zhou, Ruhong

2003-11-11

Trp-cage is a 20-residue miniprotein, which is believed to be the fastest folder known so far. In this study, the folding free energy landscape of Trp-cage has been explored in explicit solvent by using an OPLSAA force field with periodic boundary condition. A highly parallel replica exchange molecular dynamics method is used for the conformation space sampling, with the help of a recently developed efficient molecular dynamics algorithm P3ME/RESPA (particle-particle particle-mesh Ewald/reference system propagator algorithm). A two-step folding mechanism is proposed that involves an intermediate state where two correctly formed partial hydrophobic cores are separated by an essential salt-bridge between residues Asp-9 and Arg-16 near the center of the peptide. This metastable intermediate state provides an explanation for the superfast folding process. The free energy landscape is found to be rugged at low temperatures, and then becomes smooth and funnel-like above 340 K. The lowest free energy structure at 300 K is only 1.50 A Calpha-RMSD (Calpha-rms deviation) from the NMR structures. The simulated nuclear Overhauser effect pair distances are in excellent agreement with the raw NMR data. The temperature dependence of the Trp-cage population, however, is found to be significantly different from experiment, with a much higher melting transition temperature above 400 K (experimental 315 K), indicating that the current force fields, parameterized at room temperature, need to be improved to correctly predict the temperature dependence.

Trp-cage: Folding free energy landscape in explicit water

NASA Astrophysics Data System (ADS)

Zhou, Ruhong

2003-11-01

Trp-cage is a 20-residue miniprotein, which is believed to be the fastest folder known so far. In this study, the folding free energy landscape of Trp-cage has been explored in explicit solvent by using an OPLSAA force field with periodic boundary condition. A highly parallel replica exchange molecular dynamics method is used for the conformation space sampling, with the help of a recently developed efficient molecular dynamics algorithm P3ME/RESPA (particle-particle particle-mesh Ewald/reference system propagator algorithm). A two-step folding mechanism is proposed that involves an intermediate state where two correctly formed partial hydrophobic cores are separated by an essential salt-bridge between residues Asp-9 and Arg-16 near the center of the peptide. This metastable intermediate state provides an explanation for the superfast folding process. The free energy landscape is found to be rugged at low temperatures, and then becomes smooth and funnel-like above 340 K. The lowest free energy structure at 300 K is only 1.50 Å C-RMSD (C-rms deviation) from the NMR structures. The simulated nuclear Overhauser effect pair distances are in excellent agreement with the raw NMR data. The temperature dependence of the Trp-cage population, however, is found to be significantly different from experiment, with a much higher melting transition temperature above 400 K (experimental 315 K), indicating that the current force fields, parameterized at room temperature, need to be improved to correctly predict the temperature dependence.

Trp-cage: Folding free energy landscape in explicit water

PubMed Central

Zhou, Ruhong

2003-01-01

Trp-cage is a 20-residue miniprotein, which is believed to be the fastest folder known so far. In this study, the folding free energy landscape of Trp-cage has been explored in explicit solvent by using an OPLSAA force field with periodic boundary condition. A highly parallel replica exchange molecular dynamics method is used for the conformation space sampling, with the help of a recently developed efficient molecular dynamics algorithm P3ME/RESPA (particle–particle particle–mesh Ewald/reference system propagator algorithm). A two-step folding mechanism is proposed that involves an intermediate state where two correctly formed partial hydrophobic cores are separated by an essential salt-bridge between residues Asp-9 and Arg-16 near the center of the peptide. This metastable intermediate state provides an explanation for the superfast folding process. The free energy landscape is found to be rugged at low temperatures, and then becomes smooth and funnel-like above 340 K. The lowest free energy structure at 300 K is only 1.50 Å Cα-RMSD (Cα-rms deviation) from the NMR structures. The simulated nuclear Overhauser effect pair distances are in excellent agreement with the raw NMR data. The temperature dependence of the Trp-cage population, however, is found to be significantly different from experiment, with a much higher melting transition temperature above 400 K (experimental 315 K), indicating that the current force fields, parameterized at room temperature, need to be improved to correctly predict the temperature dependence. PMID:14581616

Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins

PubMed Central

Calosci, Nicoletta; Chi, Celestine N.; Richter, Barbara; Camilloni, Carlo; Engström, Åke; Eklund, Lars; Travaglini-Allocatelli, Carlo; Gianni, Stefano; Vendruscolo, Michele; Jemth, Per

2008-01-01

The energy landscape theory provides a general framework for describing protein folding reactions. Because a large number of studies, however, have focused on two-state proteins with single well-defined folding pathways and without detectable intermediates, the extent to which free energy landscapes are shaped up by the native topology at the early stages of the folding process has not been fully characterized experimentally. To this end, we have investigated the folding mechanisms of two homologous three-state proteins, PTP-BL PDZ2 and PSD-95 PDZ3, and compared the early and late transition states on their folding pathways. Through a combination of Φ value analysis and molecular dynamics simulations we obtained atomic-level structures of the transition states of these homologous three-state proteins and found that the late transition states are much more structurally similar than the early ones. Our findings thus reveal that, while the native state topology defines essentially in a unique way the late stages of folding, it leaves significant freedom to the early events, a result that reflects the funneling of the free energy landscape toward the native state. PMID:19033470

Folding Free Energy Landscape of the Decapeptide Chignolin

NASA Astrophysics Data System (ADS)

Dou, Xianghua; Wang, Jihua

Chignolin is an artificially designed ten-residue (GYDPETGTWG) folded peptide, which is the smallest protein and provides a good template for protein folding. In this work, we completed four explicit water molecular dynamics simulations of Chignolin folding using GROMOS and OPLS-AA force fields from extended initial states without any experiment informations. The four-folding free energy landscapes of the peptide has been drawn. The folded state of Chignolin has been successfully predicated based on the free energy landscapes. The four independent simulations gave similar results. (i) The four free energy landscapes have common characters. They are fairly smooth, barrierless, funnel-like and downhill without intermediate state, which consists with the experiment. (ii) The different extended initial structures converge at similar folded structures with the lowest free energy under GROMOS and OPLS-AA force fields. In the GROMOS force field, the backbone RMSD of the folded structures from the NMR native structure of Chignolin is only 0.114 nm, which is a stable structure in this force field. In the OPLS-AA force field, the similar results have been obtained. In addition, the smallest RMSD structure is in better agreement with the NMR native structure but unlikely stable in the force field.

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

Microcanonical thermostatistics of coarse-grained proteins with amyloidogenic propensity

NASA Astrophysics Data System (ADS)

Frigori, Rafael B.; Rizzi, Leandro G.; Alves, Nelson A.

2013-01-01

The formation of fibrillar aggregates seems to be a common characteristic of polypeptide chains, although the observation of these aggregates may depend on appropriate experimental conditions. Partially folded intermediates seem to have an important role in the generation of protein aggregates, and a mechanism for this fibril formation considers that these intermediates also correspond to metastable states with respect to the fibrillar ones. Here, using a coarse-grained (CG) off-lattice model, we carry out a comparative analysis of the thermodynamic aspects characterizing the folding transition with respect to the propensity for aggregation of four different systems: two isoforms of the amyloid β-protein, the Src SH3 domain, and the human prion proteins (hPrP). Microcanonical analysis of the data obtained from replica exchange method is conducted to evaluate the free-energy barrier and latent heat in these models. The simulations of the amyloid β isoforms and Src SH3 domain indicated that the folding process described by this CG model is related to a negative specific heat, a phenomenon that can only be verified in the microcanonical ensemble in first-order phase transitions. The CG simulation of the hPrP heteropolymer yielded a continuous folding transition. The absence of a free-energy barrier and latent heat favors the presence of partially unfolded conformations, and in this context, this thermodynamic aspect could explain the reason why the hPrP heteropolymer is more aggregation-prone than the other heteropolymers considered in this study. We introduced the hydrophobic radius of gyration as an order parameter and found that it can be used to obtain reliable information about the hydrophobic packing and the transition temperatures in the folding process.

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.

Exploring the protein folding free energy landscape: coupling replica exchange method with P3ME/RESPA algorithm.

PubMed

Zhou, Ruhong

2004-05-01

A highly parallel replica exchange method (REM) that couples with a newly developed molecular dynamics algorithm particle-particle particle-mesh Ewald (P3ME)/RESPA has been proposed for efficient sampling of protein folding free energy landscape. The algorithm is then applied to two separate protein systems, beta-hairpin and a designed protein Trp-cage. The all-atom OPLSAA force field with an explicit solvent model is used for both protein folding simulations. Up to 64 replicas of solvated protein systems are simulated in parallel over a wide range of temperatures. The combined trajectories in temperature and configurational space allow a replica to overcome free energy barriers present at low temperatures. These large scale simulations reveal detailed results on folding mechanisms, intermediate state structures, thermodynamic properties and the temperature dependences for both protein systems.

Denaturant-Dependent Conformational Changes in a [beta]-Trefoil Protein: Global and Residue-Specific Aspects of an Equilibrium Denaturation Process

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

Latypov, Ramil F.; Liu, Dingjiang; Jacob, Jaby

2010-01-12

Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by 1H-15N HSQC NMR. The {beta}-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came frommore » the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the {beta}-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the folded ensembles of other {beta}-trefoil proteins, such as IL-1{beta} and hFGF-1.« less

Temperature dependence of Brillouin light scattering spectra of acoustic phonons in silicon

NASA Astrophysics Data System (ADS)

Olsson, Kevin S.; Klimovich, Nikita; An, Kyongmo; Sullivan, Sean; Weathers, Annie; Shi, Li; Li, Xiaoqin

2015-02-01

Electrons, optical phonons, and acoustic phonons are often driven out of local equilibrium in electronic devices or during laser-material interaction processes. The need for a better understanding of such non-equilibrium transport processes has motivated the development of Raman spectroscopy as a local temperature sensor of optical phonons and intermediate frequency acoustic phonons, whereas Brillouin light scattering (BLS) has recently been explored as a temperature sensor of low-frequency acoustic phonons. Here, we report the measured BLS spectra of silicon at different temperatures. The origins of the observed temperature dependence of the BLS peak position, linewidth, and intensity are examined in order to evaluate their potential use as temperature sensors for acoustic phonons.

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

PubMed Central

Beitlich, Thorsten; Lorenz, Thorsten; Reinstein, Jochen

2013-01-01

The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology. PMID:24205218

Divergent actions of the pyrethroid insecticides S-bioallethrin, tefluthrin, and deltamethrin on rat Na{sub v}1.6 sodium channels

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

Tan Jianguo; Soderlund, David M., E-mail: dms6@cornell.ed

2010-09-15

We expressed rat Na{sub v}1.6 sodium channels in combination with the rat {beta}{sub 1} and {beta}{sub 2} auxiliary subunits in Xenopus laevis oocytes and evaluated the effects of the pyrethroid insecticides S-bioallethrin, deltamethrin, and tefluthrin on expressed sodium currents using the two-electrode voltage clamp technique. S-Bioallethrin, a type I structure, produced transient modification evident in the induction of rapidly decaying sodium tail currents, weak resting modification (5.7% modification at 100 {mu}M), and no further enhancement of modification upon repetitive activation by high-frequency trains of depolarizing pulses. By contrast deltamethrin, a type II structure, produced sodium tail currents that were {approx}more » 9-fold more persistent than those caused by S-bioallethrin, barely detectable resting modification (2.5% modification at 100 {mu}M), and 3.7-fold enhancement of modification upon repetitive activation. Tefluthrin, a type I structure with high mammalian toxicity, exhibited properties intermediate between S-bioallethrin and deltamethrin: intermediate tail current decay kinetics, much greater resting modification (14.1% at 100 {mu}M), and 2.8-fold enhancement of resting modification upon repetitive activation. Comparison of concentration-effect data showed that repetitive depolarization increased the potency of tefluthrin {approx} 15-fold and that tefluthrin was {approx} 10-fold more potent than deltamethrin as a use-dependent modifier of Na{sub v}1.6 sodium channels. Concentration-effect data from parallel experiments with the rat Na{sub v}1.2 sodium channel coexpressed with the rat {beta}{sub 1} and {beta}{sub 2} subunits in oocytes showed that the Na{sub v}1.6 isoform was at least 15-fold more sensitive to tefluthrin and deltamethrin than the Na{sub v}1.2 isoform. These results implicate sodium channels containing the Na{sub v}1.6 isoform as potential targets for the central neurotoxic effects of pyrethroids.« less

Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli DNA Ligase (LigA).

PubMed

Wang, Li Kai; Zhu, Hui; Shuman, Stewart

2009-03-27

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria, where they are essential for growth and present attractive targets for antimicrobial drug discovery. LigA has a distinctive modular structure in which a nucleotidyltransferase catalytic domain is flanked by an upstream NMN-binding module and by downstream OB-fold, zinc finger, helix-hairpin-helix, and BRCT domains. Here we conducted a structure-function analysis of the nucleotidyltransferase domain of Escherichia coli LigA, guided by the crystal structure of the LigA-DNA-adenylate intermediate. We tested the effects of 29 alanine and conservative mutations at 15 amino acids on ligase activity in vitro and in vivo. We thereby identified essential functional groups that coordinate the reactive phosphates (Arg(136)), contact the AMP adenine (Lys(290)), engage the phosphodiester backbone flanking the nick (Arg(218), Arg(308), Arg(97) plus Arg(101)), or stabilize the active domain fold (Arg(171)). Finer analysis of the mutational effects revealed step-specific functions for Arg(136), which is essential for the reaction of LigA with NAD(+) to form the covalent ligase-AMP intermediate (step 1) and for the transfer of AMP to the nick 5'-PO(4) to form the DNA-adenylate intermediate (step 2) but is dispensable for phosphodiester formation at a preadenylylated nick (step 3).

High-Resolution Free-Energy Landscape Analysis of α-Helical Protein Folding: HP35 and Its Double Mutant

PubMed Central

2013-01-01

The free-energy landscape can provide a quantitative description of folding dynamics, if determined as a function of an optimally chosen reaction coordinate. Here, we construct the optimal coordinate and the associated free-energy profile for all-helical proteins HP35 and its norleucine (Nle/Nle) double mutant, based on realistic equilibrium folding simulations [Piana et al. Proc. Natl. Acad. Sci. U.S.A.2012, 109, 17845]. From the obtained profiles, we directly determine such basic properties of folding dynamics as the configurations of the minima and transition states (TS), the formation of secondary structure and hydrophobic core during the folding process, the value of the pre-exponential factor and its relation to the transition path times, the relation between the autocorrelation times in TS and minima. We also present an investigation of the accuracy of the pre-exponential factor estimation based on the transition-path times. Four different estimations of the pre-exponential factor for both proteins give k0–1 values of approximately a few tens of nanoseconds. Our analysis gives detailed information about folding of the proteins and can serve as a rigorous common language for extensive comparison between experiment and simulation. PMID:24348206

High-Resolution Free-Energy Landscape Analysis of α-Helical Protein Folding: HP35 and Its Double Mutant.

PubMed

Banushkina, Polina V; Krivov, Sergei V

2013-12-10

The free-energy landscape can provide a quantitative description of folding dynamics, if determined as a function of an optimally chosen reaction coordinate. Here, we construct the optimal coordinate and the associated free-energy profile for all-helical proteins HP35 and its norleucine (Nle/Nle) double mutant, based on realistic equilibrium folding simulations [Piana et al. Proc. Natl. Acad. Sci. U.S.A. 2012 , 109 , 17845]. From the obtained profiles, we directly determine such basic properties of folding dynamics as the configurations of the minima and transition states (TS), the formation of secondary structure and hydrophobic core during the folding process, the value of the pre-exponential factor and its relation to the transition path times, the relation between the autocorrelation times in TS and minima. We also present an investigation of the accuracy of the pre-exponential factor estimation based on the transition-path times. Four different estimations of the pre-exponential factor for both proteins give k 0 -1 values of approximately a few tens of nanoseconds. Our analysis gives detailed information about folding of the proteins and can serve as a rigorous common language for extensive comparison between experiment and simulation.

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

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 Folding Energy Landscape and Free Energy Excitations of Cytochrome c

PubMed Central

Weinkam, Patrick; Zimmermann, Jörg; Romesberg, Floyd E.

2014-01-01

The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Due to the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case that follows different principles than those used to describe folding of proteins without cofactors. Recent investigations, however, demonstrate that models which are commonly used to describe folding for many proteins work well for cytochrome c when heme is explicitly introduced and generally provide results that agree with experimental observations. We will first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Since attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as “perfectly funneled.” In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are sources of frustration which cause specific transient intermediates to be populated. Native structure-based models do include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold in order for folding to proceed. The ability of native structure-based models to capture these type of motions is in part responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe well the sequence of folding events for cytochrome c inferred from hydrogen exchange experiments thereby justifying their use as a starting point. At low pH, the folding sequence of cytochrome c deviates from that at pH 7 and from those predicted from models with perfectly funneled energy landscapes. Alternate folding pathways are a result of “chemical frustration.” This frustration arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH. We construct more complex models that include chemical frustration, in addition to the native structure-based terms. These more complex models only modestly perturb the energy landscape which remains overall well funneled. These perturbed models can accurately describe how alternative folding pathways are used at low pH. At alkaline pH, cytochrome c populates distinctly different structural ensembles. For instance, lysine residues are deprotonated and compete for the heme ligation site. The same models that can describe folding at low pH also predict well the structures and relative stabilities of intermediates populated at alkaline pH. PMID:20143816

Atmospheric Condensational Properties of Ultrafine Chain and Fractal Aerosol Particles

NASA Technical Reports Server (NTRS)

Marlow, William H.

1997-01-01

The purpose for the research sponsored by this grant was to lay the foundations for qualitative understanding and quantitative description of the equilibrium vapor pressure of water vapor over the irregularly shaped, carbonaceous particles that are present in the atmosphere. This work apparently was the first systematic treatment of the subject. Research was conducted in two complementary components: 1. Calculations were performed of the equilibrium vapor pressure of water over particles comprised of aggregates of spheres in the 50-200 nm radius range. The purposes of this work were two-fold. First, since no systematic treatment of this subject had previously been conducted, its availability would be directly useful for quantitative treatment for a limited range of atmospheric aerosols. Second, it would provide qualitative indications of the effects of highly irregular particle shape on equilibrium vapor pressure of aggregates comprised of smaller spheres.

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

Kim, Sang Beom; Dsilva, Carmeline J.; Debenedetti, Pablo G., E-mail: pdebene@princeton.edu

Understanding the mechanisms by which proteins fold from disordered amino-acid chains to spatially ordered structures remains an area of active inquiry. Molecular simulations can provide atomistic details of the folding dynamics which complement experimental findings. Conventional order parameters, such as root-mean-square deviation and radius of gyration, provide structural information but fail to capture the underlying dynamics of the protein folding process. It is therefore advantageous to adopt a method that can systematically analyze simulation data to extract relevant structural as well as dynamical information. The nonlinear dimensionality reduction technique known as diffusion maps automatically embeds the high-dimensional folding trajectories inmore » a lower-dimensional space from which one can more easily visualize folding pathways, assuming the data lie approximately on a lower-dimensional manifold. The eigenvectors that parametrize the low-dimensional space, furthermore, are determined systematically, rather than chosen heuristically, as is done with phenomenological order parameters. We demonstrate that diffusion maps can effectively characterize the folding process of a Trp-cage miniprotein. By embedding molecular dynamics simulation trajectories of Trp-cage folding in diffusion maps space, we identify two folding pathways and intermediate structures that are consistent with the previous studies, demonstrating that this technique can be employed as an effective way of analyzing and constructing protein folding pathways from molecular simulations.« less

Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds

PubMed Central

Roessler, Christian G.; Hall, Branwen M.; Anderson, William J.; Ingram, Wendy M.; Roberts, Sue A.; Montfort, William R.; Cordes, Matthew H. J.

2008-01-01

Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a “stepping-stone” method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and λ. We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and λ. The domains show 40% sequence identity but differ by switching of α-helix to β-sheet in a C-terminal region spanning ≈25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization. PMID:18227506

Hierarchical folding free energy landscape of HP35 revealed by most probable path clustering.

PubMed

Jain, Abhinav; Stock, Gerhard

2014-07-17

Adopting extensive molecular dynamics simulations of villin headpiece protein (HP35) by Shaw and co-workers, a detailed theoretical analysis of the folding of HP35 is presented. The approach is based on the recently proposed most probable path algorithm which identifies the metastable states of the system, combined with dynamical coring of these states in order to obtain a consistent Markov state model. The method facilitates the construction of a dendrogram associated with the folding free-energy landscape of HP35, which reveals a hierarchical funnel structure and shows that the native state is rather a kinetic trap than a network hub. The energy landscape of HP35 consists of the entropic unfolded basin U, where the prestructuring of the protein takes place, the intermediate basin I, which is connected to U via the rate-limiting U → I transition state reflecting the formation of helix-1, and the native basin N, containing a state close to the NMR structure and a native-like state that exhibits enhanced fluctuations of helix-3. The model is in line with recent experimental observations that the intermediate and native states differ mostly in their dynamics (locked vs unlocked states). Employing dihedral angle principal component analysis, subdiffusive motion on a multidimensional free-energy surface is found.

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.

Analysis of the Free-Energy Surface of Proteins from Reversible Folding Simulations

PubMed Central

Allen, Lucy R.; Krivov, Sergei V.; Paci, Emanuele

2009-01-01

Computer generated trajectories can, in principle, reveal the folding pathways of a protein at atomic resolution and possibly suggest general and simple rules for predicting the folded structure of a given sequence. While such reversible folding trajectories can only be determined ab initio using all-atom transferable force-fields for a few small proteins, they can be determined for a large number of proteins using coarse-grained and structure-based force-fields, in which a known folded structure is by construction the absolute energy and free-energy minimum. Here we use a model of the fast folding helical λ-repressor protein to generate trajectories in which native and non-native states are in equilibrium and transitions are accurately sampled. Yet, representation of the free-energy surface, which underlies the thermodynamic and dynamic properties of the protein model, from such a trajectory remains a challenge. Projections over one or a small number of arbitrarily chosen progress variables often hide the most important features of such surfaces. The results unequivocally show that an unprojected representation of the free-energy surface provides important and unbiased information and allows a simple and meaningful description of many-dimensional, heterogeneous trajectories, providing new insight into the possible mechanisms of fast-folding proteins. PMID:19593364

Analysis of the free-energy surface of proteins from reversible folding simulations.

PubMed

Allen, Lucy R; Krivov, Sergei V; Paci, Emanuele

2009-07-01

Computer generated trajectories can, in principle, reveal the folding pathways of a protein at atomic resolution and possibly suggest general and simple rules for predicting the folded structure of a given sequence. While such reversible folding trajectories can only be determined ab initio using all-atom transferable force-fields for a few small proteins, they can be determined for a large number of proteins using coarse-grained and structure-based force-fields, in which a known folded structure is by construction the absolute energy and free-energy minimum. Here we use a model of the fast folding helical lambda-repressor protein to generate trajectories in which native and non-native states are in equilibrium and transitions are accurately sampled. Yet, representation of the free-energy surface, which underlies the thermodynamic and dynamic properties of the protein model, from such a trajectory remains a challenge. Projections over one or a small number of arbitrarily chosen progress variables often hide the most important features of such surfaces. The results unequivocally show that an unprojected representation of the free-energy surface provides important and unbiased information and allows a simple and meaningful description of many-dimensional, heterogeneous trajectories, providing new insight into the possible mechanisms of fast-folding proteins.

Characterization of two distinct beta2-microglobulin unfolding intermediates that may lead to amyloid fibrils of different morphology.

PubMed

Armen, Roger S; Daggett, Valerie

2005-12-13

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.

Pyrococcus prefoldin stabilizes protein-folding intermediates and transfers them to chaperonins for correct folding.

PubMed

Okochi, Mina; Yoshida, Takao; Maruyama, Tadashi; Kawarabayasi, Yutaka; Kikuchi, Hisashi; Yohda, Masafumi

2002-03-08

A molecular chaperone prefoldin/GimC from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 was characterized. Pyrococcus prefoldin protected porcine heart citrate synthase from thermal aggregation whereas each subunit alone afforded little protection. It also arrested the spontaneous refolding of acid-denatured green fluorescent protein and then transferred it not only to a group II chaperonin from the hyperthermophilic archaeum Thermococcus sp. strain KS-1, but also to a group I chaperonin from the thermophilic bacterium Thermus thermophilus HB8 for subsequent ATP dependent refolding.

A Springloaded Metal-Ligand Mesocate Allows Access to Trapped Intermediates of Self-Assembly.

PubMed

Bogie, Paul M; Holloway, Lauren R; Lyon, Yana; Onishi, Nicole C; Beran, Gregory J O; Julian, Ryan R; Hooley, Richard J

2018-04-02

A strained, "springloaded" Fe 2 L 3 iminopyridine mesocate shows highly variable reactivity upon postassembly reaction with competitive diamines. The strained assembly is reactive toward transimination in minutes at ambient temperature and allows observation of kinetically trapped intermediates in the self-assembly pathway. When diamines are used that can only form less favored cage products upon full equilibration, trapped ML 3 fragments with pendant, "hanging" NH 2 groups are selectively formed instead. Slight variations in diamine structure have large effects on the product outcome: less rigid diamines convert the mesocate to more favored self-assembled cage complexes under mild conditions and allow observation of heterocomplex intermediates in the displacement pathway. The mesocate allows control of equilibrium processes and direction of product outcomes via small, iterative changes in added subcomponent structure and provides a method of accessing metal-ligand cage structures not normally observed in multicomponent Fe-iminopyridine self-assembly.

Interrogating viral capsid assembly with ion mobility-mass spectrometry

NASA Astrophysics Data System (ADS)

Uetrecht, Charlotte; Barbu, Ioana M.; Shoemaker, Glen K.; van Duijn, Esther; Heck, Albert J. R.

2011-02-01

Most proteins fulfil their function as part of large protein complexes. Surprisingly, little is known about the pathways and regulation of protein assembly. Several viral coat proteins can spontaneously assemble into capsids in vitro with morphologies identical to the native virion and thus resemble ideal model systems for studying protein complex formation. Even for these systems, the mechanism for self-assembly is still poorly understood, although it is generally thought that smaller oligomeric structures form key intermediates. This assembly nucleus and larger viral assembly intermediates are typically low abundant and difficult to monitor. Here, we characterised small oligomers of Hepatitis B virus (HBV) and norovirus under equilibrium conditions using native ion mobility mass spectrometry. This data in conjunction with computational modelling enabled us to elucidate structural features of these oligomers. Instead of more globular shapes, the intermediates exhibit sheet-like structures suggesting that they are assembly competent. We propose pathways for the formation of both capsids.

Internal friction and mode relaxation in a simple chain model.

PubMed

Fugmann, S; Sokolov, I M

2009-12-21

We consider the equilibrium relaxation properties of the end-to-end distance and of the principal components in a one-dimensional polymer chain model with nonlinear interaction between the beads. While for the single-well potentials these properties are similar to the ones of a Rouse chain, for the double-well interaction potentials, modeling internal friction, they differ vastly from the ones of the harmonic chain at intermediate times and intermediate temperatures. This minimal description within a one-dimensional model mimics the relaxation properties found in much more complex polymer systems. Thus, the relaxation time of the end-to-end distance may grow by orders of magnitude at intermediate temperatures. The principal components (whose directions are shown to coincide with the normal modes of the harmonic chain, whatever interaction potential is assumed) not only display larger relaxation times but also subdiffusive scaling.

The impacts of NAFTA on U.S. and Canadian forest product exports to Mexico

Treesearch

Jeffrey P. Prestemon; Joseph Buongiorno

1996-01-01

The North American Free Trade Agreement (NAFTA) will lower barriers to trade and investment across the continent. This paper presents predictions of the effects of NAFTA on Mexico's imports of intermediate wood products, scrap and waste paper, pulp, and newsprint from the United States and Canada. Predictions were made with a partial equilibrium model. Model...

Interaction of Alpha-Chymotrypsin with a Strained Cyclic Ester: An Undergraduate Experiment in Transient State Kinetics on a Slow Time Scale.

ERIC Educational Resources Information Center

Nitta, Yasunori; And Others

1984-01-01

Describes a set of experiments (for senior-level biochemistry students) which permit evaluation and estimation of rate and equilibrium constants involving an intermediate in the alpha-chymotrypsin mediated hydrolysis of ortho-hydroxy-alpha-toluenesulfonic acid (I). The only equipment required for the experiments is a well-thermostated double beam…

Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence

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

Jensen, P.K.; Lee, Cheng S.; King, J.A.

1997-12-31

The use of capillary electrophoresis equipped with laser-induced tryptophan fluorescence detection is presented for monitoring the refolding pathway of phage P22 tailspike endorhamnosidase. Upon initiation of refolding, tailspike polypeptides rapidly fold into structured monomeric intermediates with a high content of secondary structure. These monomeric species associate to form the triple-chain defined folding intermediates, the protrimers. Conversion of the protrimer into the native, sodium dodecyl sulfate (SDS) resistant tailspike protein is the rate-limiting step in the refolding pathway. Refolding kinetics and yield measured by capillary electrophoresis are in good agreement with those obtained via native gel electrophoresis, SDS polyacrylamide gel electrophoresismore » (SDS-PAGE) and fluorescence spectrophotometry. To enhance separation resolution between protrimer and native protein in capillary electrophoresis, the use of poly(ethylene oxide) is investigated for the introduction of a sieving separation mechanism. The increased viscosity of the electrophoresis buffer may also play a role in resolution enhancement.« less

An infrared spectroscopy approach to follow β-sheet formation in peptide amyloid assemblies

NASA Astrophysics Data System (ADS)

Seo, Jongcheol; Hoffmann, Waldemar; Warnke, Stephan; Huang, Xing; Gewinner, Sandy; Schöllkopf, Wieland; Bowers, Michael T.; von Helden, Gert; Pagel, Kevin

2017-01-01

Amyloidogenic peptides and proteins play a crucial role in a variety of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. These proteins undergo a spontaneous transition from a soluble, often partially folded form, into insoluble amyloid fibrils that are rich in β-sheets. Increasing evidence suggests that highly dynamic, polydisperse folding intermediates, which occur during fibril formation, are the toxic species in the amyloid-related diseases. Traditional condensed-phase methods are of limited use for characterizing these states because they typically only provide ensemble averages rather than information about individual oligomers. Here we report the first direct secondary-structure analysis of individual amyloid intermediates using a combination of ion mobility spectrometry-mass spectrometry and gas-phase infrared spectroscopy. Our data reveal that oligomers of the fibril-forming peptide segments VEALYL and YVEALL, which consist of 4-9 peptide strands, can contain a significant amount of β-sheet. In addition, our data show that the more-extended variants of each oligomer generally exhibit increased β-sheet content.

Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

PubMed

Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

2016-09-06

Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature.

PubMed

Zako, Tamotsu; Banba, Shinya; Sahlan, Muhamad; Sakono, Masafumi; Terada, Naofumi; Yohda, Masafumi; Maeda, Mizuo

2010-01-01

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak. Copyright 2009 Elsevier Inc. All rights reserved.

Beyond the Bend: Exploring the Conformational Landscape of Decyl, Undecyl, and Dodecylbenzene

NASA Astrophysics Data System (ADS)

Hewett, Daniel M.; Zwier, Timothy S.

2017-06-01

Alkylbenzenes are important components in the combustion process: they make up 20-30% of petroleum fuels and are intermediates on the pathway to soot formation. Understanding their conformational preferences is a vital step in understanding the processes by which fuels begin their journey from small, simple hydrocarbons into the large, graphitic masses of soot. Previous work done in our group, in collaboration with the Sibert group, found that the smallest alkylbenzene which folds its chain back over the ring is octylbenzene. The population of the lone folded structure in octylbenzene is low; however, theory predicts a rapid stabilization of the folded conformations relative to more extended structures as the chain length is increased, suggesting a likely shift in population towards folded structures. This talk will focus on our exploration of this possibility by discussing the UV excitation and single conformation IR spectra of decyl, undecyl, and dodecylbenzene, where increasing chain length allows for multiple stable folded configurations.

Survival of nature's rarest isotope {sup 180}Ta under stellar conditions

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

Mohr, P.; Kaeppeler, F.; Gallino, R.

2007-01-15

The nucleosynthesis of nature's rarest isotope {sup 180}Ta depends sensitively on the temperature of the astrophysical environment because of depopulation of the long-living isomeric state via intermediate states to the short-living ground state by thermal photons. Reaction rates for this transition have been measured in the laboratory. These ground state rates underestimate the stellar rates dramatically because under stellar conditions intermediate states are mainly populated by excitations from thermally excited states in {sup 180m}Ta. Full thermalization of {sup 180}Ta is already achieved for typical s-process temperatures around kT=25 keV. Consequently, for the survival of {sup 180}Ta in the s-process fastmore » convective mixing is required which has to transport freshly synthesized {sup 180}Ta to cooler regions. In supernova explosions {sup 180}Ta is synthesized by photon- or neutrino-induced reactions at temperatures above T{sub 9}=1 in thermal equilibrium; independent of the production mechanism, freeze-out from thermal equilibrium occurs at kT{approx_equal}40 keV, and only 35{+-}4% of the synthesized {sup 180}Ta survive in the isomeric state.« less

Shockley-Read-Hall recombination in pre-filled and photo-filled intermediate band solar cells

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

Mayani, Maryam Gholami; Reenaas, Turid Worren, E-mail: turid.reenaas@ntnu.no

2014-08-18

In this work, we study how Shockley-Read-Hall (SRH) recombination via energy levels in the bandgap, caused by defects or impurities, affects the performance of both photo-filled and pre-filled intermediate band solar cells (IBSCs). For a pre-filled cell, the IB is half-filled in equilibrium, while it is empty for the photo-filled cell in equilibrium. The energy level, density, and capture cross-sections of the defects/impurities are varied systematically. We find that the photo-filled cells are, in general, less efficient than pre-filled cells, except when the defect level is between the conduction band and the IB. In that case, for a range ofmore » light intensities, the photo-filled cell performs better than the pre-filled. When the defect level is at the same energy as the IB, the efficiency is above 82% of the defect-free case, when less than 50% of the states at the IB lead to SRH recombination. This shows that even if SRH recombination via the IB takes place, high efficiencies can be achieved. We also show that band gap optimization can be used to reduce the SRH recombination.« less

Gauge coupling unification and nonequilibrium thermal dark matter.

PubMed

Mambrini, Yann; Olive, Keith A; Quevillon, Jérémie; Zaldívar, Bryan

2013-06-14

We study a new mechanism for the production of dark matter in the Universe which does not rely on thermal equilibrium. Dark matter is populated from the thermal bath subsequent to inflationary reheating via a massive mediator whose mass is above the reheating scale T(RH). To this end, we consider models with an extra U(1) gauge symmetry broken at some intermediate scale (M(int) ≃ 10(10)-10(12) GeV). We show that not only does the model allow for gauge coupling unification (at a higher scale associated with grand unification) but it can provide a dark matter candidate which is a standard model singlet but charged under the extra U(1). The intermediate scale gauge boson(s) which are predicted in several E6/SO(10) constructions can be a natural mediator between dark matter and the thermal bath. We show that the dark matter abundance, while never having achieved thermal equilibrium, is fixed shortly after the reheating epoch by the relation T(RH)(3)/M(int)(4). As a consequence, we show that the unification of gauge couplings which determines M(int) also fixes the reheating temperature, which can be as high as T(RH) ≃ 10(11) GeV.

Unstable equilibrium behaviour in collapsible tubes.

PubMed

Bertram, C D

1986-01-01

Thick-walled silicone rubber tube connected to rigid pipes upstream and downstream was externally pressurised (pe) to cause collapse while aqueous fluid flowed through propelled by a constant upstream head. Three types of equilibrium were found: stable equilibria (steady flow) at high downstream flow resistance R2, self-excited oscillations at low R2, and 'unattainable' (by varying external pressure) or exponentially unstable equilibria at intermediate R2. The self-excited oscillations were highly non-linear and appeared in four, apparently discrete, frequency bands: 2.7 Hz, 3.8-5.0 Hz, 12-16 Hz and 60-63 Hz, suggesting that the possible oscillation modes may be harmonically related. Stable, intermediate 'two-in-every-three-beats' oscillation was also observed, with a repetition frequency in the 3.8-5.0 Hz band. As pe was increased, self-excited oscillations were eventually suppressed, leaving internal fluid pressure varying with no single dominant frequency as a result of turbulent jet dissipation at the downstream rigid pipe connection. Comparison of pressure-wave velocity calculated from the local pressure-area relation for the tube with fluid velocity indicated that supercritical velocities were attained in the course of the self-excited oscillations.

Influence of chain topology on polymer crystallization: poly(ethylene oxide) (PEO) rings vs. linear chains.

PubMed

Zardalidis, George; Mars, Julian; Allgaier, Jürgen; Mezger, Markus; Richter, Dieter; Floudas, George

2016-10-04

The absence of entanglements, the more compact structure and the faster diffusion in melts of cyclic poly(ethylene oxide) (PEO) chains have consequences on their crystallization behavior at the lamellar and spherulitic length scales. Rings with molecular weight below the entanglement molecular weight (M < M e ), attain the equilibrium configuration composed from twice-folded chains with a lamellar periodicity that is half of the corresponding linear chains. Rings with M > M e undergo distinct step-like conformational changes to a crystalline lamellar with the equilibrium configuration. Rings melt from this configuration in the absence of crystal thickening in sharp contrast to linear chains. In general, rings more easily attain their extended equilibrium configuration due to strained segments and the absence of entanglements. In addition, rings have a higher equilibrium melting temperature. At the level of the spherulitic superstructure, growth rates are much faster for rings reflecting the faster diffusion and more compact structure. With respect to the segmental dynamics in their semi-crystalline state, ring PEOs with a steepness index of ∼34 form some of the "strongest" glasses.

Exact results in nonequilibrium statistical mechanics: Formalism and applications in chemical kinetics and single-molecule free energy estimation

NASA Astrophysics Data System (ADS)

Adib, Artur B.

In the last two decades or so, a collection of results in nonequilibrium statistical mechanics that departs from the traditional near-equilibrium framework introduced by Lars Onsager in 1931 has been derived, yielding new fundamental insights into far-from-equilibrium processes in general. Apart from offering a more quantitative statement of the second law of thermodynamics, some of these results---typified by the so-called "Jarzynski equality"---have also offered novel means of estimating equilibrium quantities from nonequilibrium processes, such as free energy differences from single-molecule "pulling" experiments. This thesis contributes to such efforts by offering three novel results in nonequilibrium statistical mechanics: (a) The entropic analog of the Jarzynski equality; (b) A methodology for estimating free energies from "clamp-and-release" nonequilibrium processes; and (c) A directly measurable symmetry relation in chemical kinetics similar to (but more general than) chemical detailed balance. These results share in common the feature of remaining valid outside Onsager's near-equilibrium regime, and bear direct applicability in protein folding kinetics as well as in single-molecule free energy estimation.

Discriminating trpzip2 and trpzip4 peptides’ folding landscape using the two-dimensional infrared spectroscopy: A simulation study

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

Wu, Tianmin; Zhang, Ruiting; Li, Huanhuan

2014-02-07

We analyzed, based on the theoretical spectroscopic modeling, how the differences in the folding landscapes of two β-hairpin peptides trpzip2 and trpzip4 are reflected in their thermal unfolding infrared measurements. The isotope-edited equilibrium FTIR and two dimensional infrared spectra of the two peptides were calculated, using the nonlinear exciton propagation method, at a series of temperatures. The spectra calculations were based on the configuration distributions generated using the GB{sup OBC} implicit solvent MD simulation and the integrated tempering sampling technique. Conformational analysis revealed the different local thermal stabilities for these two peptides, which suggested the different folding landscapes. Our studymore » further suggested that the ellipticities of the isotope peaks in the coherent IR signals are more sensitive to these local stability differences compared with other spectral features such as the peak intensities. Our technique can thus be combined with the relevant experimental measurements to achieve a better understanding of the peptide folding behaviors.« less

Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance.

PubMed

Okochi, Mina; Kanie, Kei; Kurimoto, Masaki; Yohda, Masafumi; Honda, Hiroyuki

2008-06-01

Prefoldin is a jellyfish-shaped hexameric chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. In this work, we characterized the organic solvent tolerance of Escherichia coli cells that overexpress prefoldin and group II chaperonin from a hyperthermophilic archeaum, Pyrococcus horikoshii OT3. The colony-forming efficiency of E. coli cells overexpressing prefoldin increased by 1,000-fold and decreased the accumulation of intracellular organic solvent. The effect was impaired by deletions of the region responsible for the chaperone function of prefoldin. Therefore, we concluded that prefoldin endows E. coli cells by preventing accumulation of intracellular organic solvent through its molecular chaperone activity.

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.

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.

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.

New insights into structural determinants of prion protein folding and stability.

PubMed

Benetti, Federico; Legname, Giuseppe

2015-01-01

Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

Erratum to "The mechanism and thermodynamics of transesterification of acetate-ester enolates in the gas phase" : [Int. J. Mass Spectrom. Ion Process. 172 (1998) 25

NASA Astrophysics Data System (ADS)

Haas, George W.; Giblin, Daryl E.; Gross, Michael L.

1998-02-01

In solution, base-catalyzed hydrolysis and transesterification of esters are initiated by hydroxide- or alkoxide-ion attack at the carbonyl carbon. At low pressures in the gas phase, however, transesterification proceeds by an attack of the enolate anion of an acetate ester on an alcohol. Fourier transform mass spectrometry (FTMS) indicates that the reaction is the second-order process: -CH2-CO2-R+R'-OH-->-CH2-CO2-R'+R-OH and there is little to no detectable production of either alkoxide anion. Labeling studies show that the product and reactant enolate anion esters undergo exchange of hydrogens located [alpha] to the carbonyl carbon with the deuterium of R'--OD. The extent of the H/D exchange increases with reaction time, pointing to a short-lived intermediate. The alcoholysis reaction rate constants increase with increasing acidity of the primary, straight-chained alkyl alcohols, whereas steric effects associated with branched alcohols cause the rate constants to decrease. Equilibrium constants, which were determined directly from measurements at equilibrium and which were calculated from the forward and reverse rate constants, are near unity and show internal consistency. In the absence of steric effects, the larger enolate is always the favored product at equilibrium. The intermediate for the transesterification reaction, which can be generated at a few tenths of a torr in a tandem mass spectrometer, is tetrahedral, but other adducts that are collisionally stabilized under these conditions are principally loosely bound complexes.

The folding energy landscape and free energy excitations of cytochrome c.

PubMed

Weinkam, Patrick; Zimmermann, Jörg; Romesberg, Floyd E; Wolynes, Peter G

2010-05-18

The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Because of the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case, following principles different from those used to describe the folding of proteins without cofactors. Recent investigations, however, demonstrate that common models describing folding for many proteins work well for cytochrome c when heme is explicitly introduced, generally providing results that agree with experimental observations. In this Account, we first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Because attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as "perfectly funneled". In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are denoted sources of "frustration", which cause specific transient intermediates to be populated. Native structure-based models do, however, include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold so that folding can proceed. The ability of native structure-based models to capture this kind of motion is partly responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe the sequence of folding events for cytochrome c well (as inferred from hydrogen-exchange experiments), thereby justifying their use as a starting point. At low pH, the experimentally observed folding sequence of cytochrome c deviates from that at pH 7 and from models with perfectly funneled energy landscapes. Here, alternate folding pathways are a result of "chemical frustration". This frustration arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH. Beginning with native structure-based terms, we construct more complex models by adding chemical frustration. These more complex models only modestly perturb the energy landscape, which remains, overall, well funneled. These perturbed models can accurately describe how alternative folding pathways are used at low pH. At alkaline pH, cytochrome c populates distinctly different structural ensembles. For instance, lysine residues are deprotonated and compete for the heme ligation site. The same models that can describe folding at low pH also predict well the structures and relative stabilities of intermediates populated at alkaline pH. The success of models based on funneled energy landscapes suggest that cytochrome c folding is driven primarily by native contacts. The presence of heme appears to add chemical complexity to the folding process, but it does not require fundamental modification of the general principles used to describe folding. Moreover, its added complexity provides a valuable means of probing the folding energy landscape in greater detail than is possible with simpler systems.

Proton transfer from C-6 of uridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase: formation and stability of a vinyl carbanion intermediate and the effect of a 5-fluoro substituent.

PubMed

Tsang, Wing-Yin; Wood, B McKay; Wong, Freeman M; Wu, Weiming; Gerlt, John A; Amyes, Tina L; Richard, John P

2012-09-05

The exchange for deuterium of the C-6 protons of uridine 5'-monophosphate (UMP) and 5-fluorouridine 5'-monophosphate (F-UMP) catalyzed by yeast orotidine 5'-monophosphate decarboxylase (ScOMPDC) at pD 6.5-9.3 and 25 °C was monitored by (1)H NMR spectroscopy. Deuterium exchange proceeds by proton transfer from C-6 of the bound nucleotide to the deprotonated side chain of Lys-93 to give the enzyme-bound vinyl carbanion. The pD-rate profiles for k(cat) give turnover numbers for deuterium exchange into enzyme-bound UMP and F-UMP of 1.2 × 10(-5) and 0.041 s(-1), respectively, so that the 5-fluoro substituent results in a 3400-fold increase in the first-order rate constant for deuterium exchange. The binding of UMP and F-UMP to ScOMPDC results in 0.5 and 1.4 unit decreases, respectively, in the pK(a) of the side chain of the catalytic base Lys-93, showing that these nucleotides bind preferentially to the deprotonated enzyme. We also report the first carbon acid pK(a) values for proton transfer from C-6 of uridine (pK(CH) = 28.8) and 5-fluorouridine (pK(CH) = 25.1) in aqueous solution. The stabilizing effects of the 5-fluoro substituent on C-6 carbanion formation in solution (5 kcal/mol) and at ScOMPDC (6 kcal/mol) are similar. The binding of UMP and F-UMP to ScOMPDC results in a greater than 5 × 10(9)-fold increase in the equilibrium constant for proton transfer from C-6, so that ScOMPDC stabilizes the bound vinyl carbanions, relative to the bound nucleotides, by at least 13 kcal/mol. The pD-rate profile for k(cat)/K(m) for deuterium exchange into F-UMP gives the intrinsic second-order rate constant for exchange catalyzed by the deprotonated enzyme as 2300 M(-1) s(-1). This was used to calculate a total rate acceleration for ScOMPDC-catalyzed deuterium exchange of 3 × 10(10) M(-1), which corresponds to a transition-state stabilization for deuterium exchange of 14 kcal/mol. We conclude that a large portion of the total transition-state stabilization for the decarboxylation of orotidine 5'-monophosphate can be accounted for by stabilization of the enzyme-bound vinyl carbanion intermediate of the stepwise reaction.

Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent

PubMed Central

Tsang, Wing-Yin; Wood, B. McKay; Wong, Freeman M.; Wu, Weiming; Gerlt, John A.; Amyes, Tina L.; Richard, John P.

2012-01-01

The exchange for deuterium of the C-6 protons of uridine 5′-monophosphate (UMP) and 5-fluorouridine 5′-monophosphate (F-UMP) catalyzed by yeast orotidine 5′-monophosphate decarboxylase (ScOMPDC) at pD 6.5 – 9.3 and 25 °C was monitored by 1H NMR spectroscopy. Deuterium exchange proceeds by proton transfer from C-6 of the bound nucleotide to the deprotonated side chain of Lys-93 to give the enzyme-bound vinyl carbanion. The pD-rate profiles for kcat give turnover numbers for deuterium exchange into enzyme-bound UMP and F-UMP of 1.2 × 10−5 and 0.041 s−1, respectively, so that the 5-fluoro substituent results in a 3400-fold increase in the first-order rate constant for deuterium exchange. The binding of UMP and F-UMP to ScOMPDC results in 0.5 and 1.4 unit decreases, respectively, in the pKa of the side chain of the catalytic base Lys-93, showing that these nucleotides bind preferentially to the deprotonated enzyme. We also report the first carbon acid pKas for proton transfer from C-6 of uridine (pKCH = 28.8) and 5-fluorouridine (pKCH = 25.1) in aqueous solution. The stabilizing effects of the 5-fluoro substituent on C-6 carbanion formation in solution (5 kcal/mol) and at ScOMPDC (6 kcal/mol) are similar. The binding of UMP and F-UMP to ScOMPDC results in a greater than 5 × 109-fold increase in the equilibrium constant for proton transfer from C-6 so that ScOMPDC stabilizes the bound vinyl carbanions, relative to the bound nucleotides, by at least 13 kcal/mol. The pD-rate profile for kcat/Km for deuterium exchange into F-UMP gives the intrinsic second-order rate constant for exchange catalyzed by the deprotonated enzyme as 2300 M−1 s−1. This was used to calculate a total rate acceleration for ScOMPDC-catalyzed deuterium exchange of 3 × 1010 M−1, which corresponds to a transition state stabilization for deuterium exchange of 14 kcal/mol. We conclude that a large portion of the total transition state stabilization for the decarboxylation of orotidine 5′-monophosphate can be accounted for by stabilization of the enzyme-bound vinyl carbanion intermediate of the stepwise reaction. PMID:22812629

Influence of porewater advection on denitrification in carbonate sands: Evidence from repacked sediment column experiments

NASA Astrophysics Data System (ADS)

Santos, Isaac R.; Eyre, Bradley D.; Glud, Ronnie N.

2012-11-01

Porewater flow enhances mineralization rates in organic-poor permeable sands. Here, a series of sediment column experiments were undertaken to assess the potential effect of advective porewater transport on denitrification in permeable carbonate sands collected from Heron Island (Great Barrier Reef). Experimental conditions (flow path length, advection rate, and temperature) were manipulated to represent conditions similar to near shore tropical environments. HgCl2-poisoned controls were used to assess whether reactions were microbially mediated. Overall, significant correlations were found between oxygen consumption and N2 production. The N:O2 slope of 0.114 implied that about 75% of all the nitrogen mineralized was denitrified. A 4-fold increase in sediment column length (from 10 to 40 cm) resulted in an overall increase in oxygen consumption (1.6-fold), TCO2 production (1.8-fold), and denitrification (1.9-fold). Oxic respiration increased quickly until advection reached 80 L m-2 h-1 and then plateaued at higher advection rates. Interestingly, denitrification peaked (up to 336 μmol N2 m-2 h-1) at intermediate advection rates (30-80 L m-2 h-1). We speculate that intermediate advection rates enhance the development of microniches (i.e., steep oxygen gradients) within porous carbonate sands, perhaps providing optimum conditions for denitrification. The denitrification peak fell within the broad range of advection rates (often on scales of 1-100 L m-2 h-1) typically found on continental shelves implying that carbonate sands may play a major, but as yet unquantified, role in oceanic nitrogen budgets.

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.

Internal friction controls the speed of protein folding from a compact configuration.

PubMed

Pabit, Suzette A; Roder, Heinrich; Hagen, Stephen J

2004-10-05

Several studies have found millisecond protein folding reactions to be controlled by the viscosity of the solvent: Reducing the viscosity allows folding to accelerate. In the limit of very low solvent viscosity, however, one expects a different behavior. Internal interactions, occurring within the solvent-excluded interior of a compact molecule, should impose a solvent-independent upper limit to folding speed once the bulk diffusional motions become sufficiently rapid. Why has this not been observed? We have studied the effect of solvent viscosity on the folding of cytochrome c from a highly compact, late-stage intermediate configuration. Although the folding rate accelerates as the viscosity declines, it tends toward a finite limiting value approximately 10(5) s(-1) as the viscosity tends toward zero. This limiting rate is independent of the cosolutes used to adjust solvent friction. Therefore, interactions within the interior of a compact denatured polypeptide can limit the folding rate, but the limiting time scale is very fast. It is only observable when the solvent-controlled stages of folding are exceedingly rapid or else absent. Interestingly, we find a very strong temperature dependence in these "internal friction"-controlled dynamics, indicating a large energy scale for the interactions that govern reconfiguration within compact, near-native states of a protein.

What is the role of the second “structural” NADP+-binding site in human glucose 6-phosphate dehydrogenase?

PubMed Central

Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M.S.; Engel, Paul C.

2008-01-01

Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable “structural” NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of “stripped” enzyme by gel filtration was ∼100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for “catalytic” NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37°C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4°C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The K d values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of K d constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations. PMID:18493020

Cooperativity and modularity in protein folding

PubMed Central

Sasai, Masaki; Chikenji, George; Terada, Tomoki P.

2016-01-01

A simple statistical mechanical model proposed by Wako and Saitô has explained the aspects of protein folding surprisingly well. This model was systematically applied to multiple proteins by Muñoz and Eaton and has since been referred to as the Wako-Saitô-Muñoz-Eaton (WSME) model. The success of the WSME model in explaining the folding of many proteins has verified the hypothesis that the folding is dominated by native interactions, which makes the energy landscape globally biased toward native conformation. Using the WSME and other related models, Saitô emphasized the importance of the hierarchical pathway in protein folding; folding starts with the creation of contiguous segments having a native-like configuration and proceeds as growth and coalescence of these segments. The Φ-values calculated for barnase with the WSME model suggested that segments contributing to the folding nucleus are similar to the structural modules defined by the pattern of native atomic contacts. The WSME model was extended to explain folding of multi-domain proteins having a complex topology, which opened the way to comprehensively understanding the folding process of multi-domain proteins. The WSME model was also extended to describe allosteric transitions, indicating that the allosteric structural movement does not occur as a deterministic sequential change between two conformations but as a stochastic diffusive motion over the dynamically changing energy landscape. Statistical mechanical viewpoint on folding, as highlighted by the WSME model, has been renovated in the context of modern methods and ideas, and will continue to provide insights on equilibrium and dynamical features of proteins. PMID:28409080

Temperature dependence of Brillouin light scattering spectra of acoustic phonons in silicon

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

Olsson, Kevin S.; Klimovich, Nikita; An, Kyongmo

2015-02-02

Electrons, optical phonons, and acoustic phonons are often driven out of local equilibrium in electronic devices or during laser-material interaction processes. The need for a better understanding of such non-equilibrium transport processes has motivated the development of Raman spectroscopy as a local temperature sensor of optical phonons and intermediate frequency acoustic phonons, whereas Brillouin light scattering (BLS) has recently been explored as a temperature sensor of low-frequency acoustic phonons. Here, we report the measured BLS spectra of silicon at different temperatures. The origins of the observed temperature dependence of the BLS peak position, linewidth, and intensity are examined in ordermore » to evaluate their potential use as temperature sensors for acoustic phonons.« less

Computer codes for the evaluation of thermodynamic and transport properties for equilibrium air to 30000 K

NASA Technical Reports Server (NTRS)

Thompson, Richard A.; Lee, Kam-Pui; Gupta, Roop N.

1991-01-01

The computer codes developed here provide self-consistent thermodynamic and transport properties for equilibrium air for temperatures from 500 to 30000 K over a temperature range of 10 (exp -4) to 10 (exp -2) atm. These properties are computed through the use of temperature dependent curve fits for discrete values of pressure. Interpolation is employed for intermediate values of pressure. The curve fits are based on mixture values calculated from an 11-species air model. Individual species properties used in the mixture relations are obtained from a recent study by the present authors. A review and discussion of the sources and accuracy of the curve fitted data used herein are given in NASA RP 1260.

Chemical composition of donor-acceptor complexes of hydroxyoxo(5,10,15,20-tetraphenylporphinato)molybdenum(V) with 3,5-dimethylpyrazole and equilibrium constants for their formation

NASA Astrophysics Data System (ADS)

Motorina, E. V.; Lomova, T. N.

2017-11-01

The results from a quantitative study of reactions between hydroxyoxo(5,10,15,20-tetraphenylporphinato)molybdenum(V) (O=Mo(OH)TPP) and 3,5-dimethylpyrazole, a biologically active base, in toluene are presented. The chemical structure and key parameters of intermediates and reaction products are determined by spectral means. The equilibrium constant ( K = 51.3 L/mol) is calculated and a full kinetic description of simple reactions that occur in this system during complex transformation is obtained. The prospect of using a mixed porphyrin-containing complex as a receptor for 3,5-dimethylpyrazole, a building block for alkaloids and pharmaceutical preparations, is substantiated.

Direct Detection of a Chemical Equilibrium between a Localized Singlet Diradical and Its σ-Bonded Species by Time-Resolved UV/Vis and IR Spectroscopy.

PubMed

Yoshidomi, Shohei; Mishima, Megumi; Seyama, Shin; Abe, Manabu; Fujiwara, Yoshihisa; Ishibashi, Taka-Aki

2017-03-06

Localized singlet diradicals are key intermediates in bond homolyses. The singlet diradicals are energetically much less stable than the σ-bonded species. In general, only one-way reactions from diradicals to σ-bonded species are observed. In this study, a thermal equilibrium between a singlet 1,2-diazacyclopentane-3,5-diyl diradical and the corresponding σ-bonded species was directly observed. The singlet diradical was more stable than the σ-bonded species. The solvent effect clarified key features, such as the zwitterionic character of the singlet diradical. The effect of the nitrogen atoms is discussed in detail. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cooperativity in self-limiting equilibrium self-associating systems

NASA Astrophysics Data System (ADS)

Freed, Karl F.

2012-11-01

A wide variety of highly cooperative self-assembly processes in biological and synthetic systems involve the assembly of a large number (m) of units into clusters, with m narrowly peaked about a large size m0 ≫ 1 and with a second peak centered about the m = 1 unassembled monomers. While very specific models have been proposed for the assembly of, for example, viral capsids and core-shell micelles of ß-casein, no available theory describes a thermodynamically general mechanism for this double peaked, highly cooperative equilibrium assembly process. This study provides a general mechanism for these cooperative processes by developing a minimal Flory-Huggins type theory. Beginning from the simplest non-cooperative, free association model in which the equilibrium constant for addition of a monomer to a cluster is independent of cluster size, the new model merely allows more favorable growth for clusters of intermediate sizes. The theory is illustrated by computing the phase diagram for cases of self-assembly on cooling or heating and for the mass distribution of the two phases.

Assessment of tautomer distribution using the condensed reaction graph approach

NASA Astrophysics Data System (ADS)

Gimadiev, T. R.; Madzhidov, T. I.; Nugmanov, R. I.; Baskin, I. I.; Antipin, I. S.; Varnek, A.

2018-03-01

We report the first direct QSPR modeling of equilibrium constants of tautomeric transformations (logK T ) in different solvents and at different temperatures, which do not require intermediate assessment of acidity (basicity) constants for all tautomeric forms. The key step of the modeling consisted in the merging of two tautomers in one sole molecular graph ("condensed reaction graph") which enables to compute molecular descriptors characterizing entire equilibrium. The support vector regression method was used to build the models. The training set consisted of 785 transformations belonging to 11 types of tautomeric reactions with equilibrium constants measured in different solvents and at different temperatures. The models obtained perform well both in cross-validation (Q2 = 0.81 RMSE = 0.7 logK T units) and on two external test sets. Benchmarking studies demonstrate that our models outperform results obtained with DFT B3LYP/6-311 ++ G(d,p) and ChemAxon Tautomerizer applicable only in water at room temperature.

Developing Compressed Beginning and Intermediate Algebra Courses

ERIC Educational Resources Information Center

Walker, Sylvia E.

2017-01-01

The purpose of this project was two-fold. First, it would provide an opportunity for students to complete the developmental math course sequence more quickly, thereby enabling students to proceed to a college-level mathematics course sooner. To accomplish this, the classroom was designed with computer-assisted homework courses that blended…

Non-destructive Techniques for Classifying Aircraft Coating Degradation

DTIC Science & Technology

2015-03-26

model is bidirectional reflectance distribution func- tions ( BRDF ) which describes how much radiation is reflected for each solid angle and each...incident angle. An intermediate model between ideal reflectors and BRDF is to assume all reflectance is a combination of diffuse and specular reflectance...19 K-Fold Cross Validation

The Metallicity Evolution of Low-mass Galaxies: New Constraints at Intermediate Redshift

NASA Astrophysics Data System (ADS)

Henry, Alaina; Martin, Crystal L.; Finlator, Kristian; Dressler, Alan

2013-06-01

We present abundance measurements from 26 emission-line-selected galaxies at z ~ 0.6-0.7. By reaching stellar masses as low as 108 M ⊙, these observations provide the first measurement of the intermediate-redshift mass-metallicity (MZ) relation below 109 M ⊙. For the portion of our sample above M > 109 M ⊙ (8/26 galaxies), we find good agreement with previous measurements of the intermediate-redshift MZ relation. Compared to the local relation, we measure an evolution that corresponds to a 0.12 dex decrease in oxygen abundances at intermediate redshifts. This result confirms the trend that metallicity evolution becomes more significant toward lower stellar masses, in keeping with a downsizing scenario where low-mass galaxies evolve onto the local MZ relation at later cosmic times. We show that these galaxies follow the local fundamental metallicity relation, where objects with higher specific (mass-normalized) star formation rates (SFRs) have lower metallicities. Furthermore, we show that the galaxies in our sample lie on an extrapolation of the SFR-M * relation (the star-forming main sequence). Leveraging the MZ relation and star-forming main sequence (and combining our data with higher-mass measurements from the literature), we test models that assume an equilibrium between mass inflow, outflow, and star formation. We find that outflows are required to describe the data. By comparing different outflow prescriptions, we show that momentum, driven winds can describe the MZ relation; however, this model underpredicts the amount of star formation in low-mass galaxies. This disagreement may indicate that preventive feedback from gas heating has been overestimated, or it may signify a more fundamental deviation from the equilibrium assumption. Some of the data presented herein were obtained at the W. M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California, and the National Aeronautics and Space Administration. The Observatory was made possible by the generous financial support of the W. M. Keck Foundation.

The fast-folding HP35 double mutant has a substantially reduced primary folding free energy barrier

NASA Astrophysics Data System (ADS)

Lei, Hongxing; Deng, Xiaojian; Wang, Zhixiang; Duan, Yong

2008-10-01

The LYS24/29NLE double mutant of villin headpiece subdomain (HP35) is the fastest folding protein known so far with a folding time constant of 0.6μs. In this work, the folding mechanism of the mutant has been investigated by both conventional and replica exchange molecular dynamics (CMD and REMD) simulations with AMBER FF03 force field and a generalized-Born solvation model. Direct comparison to the ab initio folding of the wild type HP35 enabled a close examination on the mutational effect on the folding process. The mutant folded to the native state, as demonstrated by the 0.50Å Cα-root mean square deviation (RMSD) sampled in both CMD and REMD simulations and the high population of the folded conformation compared with the denatured conformations. Consistent with experiments, the significantly reduced primary folding free energy barrier makes the mutant closer to a downhill folder than the wild type HP35 that directly leads to the faster transition and higher melting temperature. However, unlike the proposed downhill folding which envisages a smooth shift between unfolded and folded states without transition barrier, we observed a well-defined folding transition that was consistent with experiments. Further examination of the secondary structures revealed that the two mutated residues have higher intrinsic helical preference that facilitated the formation of both helix III and the intermediate state which contains the folded segment helix II/III. Other factors contributing to the faster folding include the more favorable electrostatic interactions in the transition state with the removal of the charged NH3+ groups from LYS. In addition, both transition state ensemble and denatured state ensemble are shifted in the mutant.

Hydrophobic Collapse of Ubiquitin Generates Rapid Protein-Water Motions.

PubMed

Wirtz, Hanna; Schäfer, Sarah; Hoberg, Claudius; Reid, Korey M; Leitner, David M; Havenith, Martina

2018-06-04

We report time-resolved measurements of the coupled protein-water modes of solvated ubiquitin during protein folding. Kinetic terahertz absorption (KITA) spectroscopy serves as a label-free technique for monitoring large scale conformational changes and folding of proteins subsequent to a sudden T-jump. We report here KITA measurements at an unprecedented time resolution of 500 ns, a resolution 2 orders of magnitude better than those of any previous KITA measurements, which reveal the coupled ubiquitin-solvent dynamics even in the initial phase of hydrophobic collapse. Complementary equilibrium experiments and molecular simulations of ubiquitin solutions are performed to clarify non-equilibrium contributions and reveal the molecular picture upon a change in structure, respectively. On the basis of our results, we propose that in the case of ubiquitin a rapid (<500 ns) initial phase of the hydrophobic collapse from the elongated protein to a molten globule structure precedes secondary structure formation. We find that these very first steps, including large-amplitude changes within the unfolded manifold, are accompanied by a rapid (<500 ns) pronounced change of the coupled protein-solvent response. The KITA response upon secondary structure formation exhibits an opposite sign, which indicates a distinct effect on the solvent-exposed surface.

Bifurcation of self-folded polygonal bilayers

NASA Astrophysics Data System (ADS)

Abdullah, Arif M.; Braun, Paul V.; Hsia, K. Jimmy

2017-09-01

Motivated by the self-assembly of natural systems, researchers have investigated the stimulus-responsive curving of thin-shell structures, which is also known as self-folding. Self-folding strategies not only offer possibilities to realize complicated shapes but also promise actuation at small length scales. Biaxial mismatch strain driven self-folding bilayers demonstrate bifurcation of equilibrium shapes (from quasi-axisymmetric doubly curved to approximately singly curved) during their stimulus-responsive morphing behavior. Being a structurally instable, bifurcation could be used to tune the self-folding behavior, and hence, a detailed understanding of this phenomenon is appealing from both fundamental and practical perspectives. In this work, we investigated the bifurcation behavior of self-folding bilayer polygons. For the mechanistic understanding, we developed finite element models of planar bilayers (consisting of a stimulus-responsive and a passive layer of material) that transform into 3D curved configurations. Our experiments with cross-linked Polydimethylsiloxane samples that change shapes in organic solvents confirmed our model predictions. Finally, we explored a design scheme to generate gripper-like architectures by avoiding the bifurcation of stimulus-responsive bilayers. Our research contributes to the broad field of self-assembly as the findings could motivate functional devices across multiple disciplines such as robotics, artificial muscles, therapeutic cargos, and reconfigurable biomedical devices.

Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin.

PubMed

Petrillo, Teodolinda; O'Donohoe, Catrina A; Howe, Nicole; Malthouse, J Paul G

2012-08-07

Two new inhibitors in which the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3-4-fold while a glyoxal group increases the level of binding by 500-2000-fold. We show that at pH 7.2 the effective molarities of the catalytic hydroxyl group of the active site serine are 41000-229000 and 101000-159000 for α-chymotrypsin and subtilisin Carlsberg, respectively. It is estimated that oxyanion stabilization and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low-barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pK(a) of the active site histidine, allowing it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed.

Unfolding pathway of CotA-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state

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

Fernandes, Andre T.; Lopes, Carlos; Martins, Ligia O.

2012-06-08

Highlights: Black-Right-Pointing-Pointer CotA-laccase unfolds with an intermediate state. Black-Right-Pointing-Pointer Copper stabilizes the native and the intermediate state. Black-Right-Pointing-Pointer Copper binding to the unfolded state prevents refolding through protein aggregation. Black-Right-Pointing-Pointer Copper incorporation in CotA-laccase occurs as a later step during folding. -- Abstract: Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediatemore » in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.« less

M≡E and M=E Complexes of Iron and Cobalt that Emphasize Three-fold Symmetry (E = O, N, NR)

PubMed Central

Saouma, Caroline T.; Peters, Jonas C.

2011-01-01

Mid-to-late transition metal complexes that feature terminal, multiply bonded ligands such as oxos, imides, and nitrides have been invoked as intermediates in several catalytic transformations of synthetic and biological significance. Until about ten years ago, isolable examples of such species were virtually unknown. Over the past decade or so, numerous chemically well-defined examples of such species have been discovered. In this context, the presentreview summarizes the development of 4- and 5-coordinate Fe(E) and Co(E) species under local three-fold symmetry. PMID:21625302

Reconstructing Folding Energy Landscapes by Single-Molecule Force Spectroscopy

PubMed Central

Woodside, Michael T.; Block, Steven M.

2015-01-01

Folding may be described conceptually in terms of trajectories over a landscape of free energies corresponding to different molecular configurations. In practice, energy landscapes can be difficult to measure. Single-molecule force spectroscopy (SMFS), whereby structural changes are monitored in molecules subjected to controlled forces, has emerged as a powerful tool for probing energy landscapes. We summarize methods for reconstructing landscapes from force spectroscopy measurements under both equilibrium and nonequilibrium conditions. Other complementary, but technically less demanding, methods provide a model-dependent characterization of key features of the landscape. Once reconstructed, energy landscapes can be used to study critical folding parameters, such as the characteristic transition times required for structural changes and the effective diffusion coefficient setting the timescale for motions over the landscape. We also discuss issues that complicate measurement and interpretation, including the possibility of multiple states or pathways and the effects of projecting multiple dimensions onto a single coordinate. PMID:24895850

Simulation of Peptides at Aqueous Interfaces

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Wilson, M.; Chipot, C.; DeVincenzi, Donald L. (Technical Monitor)

2001-01-01

Behavior of peptides at water-membrane interfaces is of great interest in studies on cellular transport and signaling, membrane fusion, and the action of toxins and antibiotics. Many peptides, which exist in water only as random coils, can form sequence-dependent, ordered structures at aqueous interfaces, incorporate into membranes and self-assembly into functional units, such as simple ion channels. Multi -nanosecond molecular dynamics simulations have been carried out to study the mechanism and energetics of interfacial folding of both non-polar and amphiphilic peptides, their insertion into membranes and association into higher-order structures. The simulations indicate that peptides fold non-sequentially, often through a series of amphiphilic intermediates. They further incorporate into the membrane in a preferred direction as folded monomers, and only then aggregate into dimers and, possibly, further into "dimers of dimers".

Folding dynamics of linear emulsion polymers into 3D architectures

NASA Astrophysics Data System (ADS)

McMullen, Angus; Bargteil, Dylan; Brujic, Jasna

Colloidal polymers have been limited to inflexible, solid colloids. Here we show that the fluidity of emulsion droplets allows for the self-assembly of flexible droplet chains, which can subsequently be folded into 3D structures via secondary interactions. We achieve this using DNA-guided interactions, to initially form the chain, and then program its folding pathways. When two emulsion droplets labeled with complementary DNA meet, the balance of hybridization energy and droplet deformation yields an equilibrium patch size. Therefore, the concentration of DNA on the surface determines the number of droplet-droplet bonds in the assembly. We find that 96 % of bound droplets successfully self-assemble into chains. Droplet binding is a stochastic process, following a Poisson distribution of lengths. Since the fluid droplets can rearrange, we compare the dynamics of emulsion chains to that of polymers. We also trigger secondary interactions along the chain, causing the formation of specific loops or compact clusters. This approach will allow us to fold our emulsion polymers into a wide array of soft structures, giving us a powerful biomimetic colloidal system to investigate protein folding on the mesoscopic scale. This work was supported by the NSF MRSEC Program (DMR-0820341).

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

PubMed Central

Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

2013-01-01

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

Polymorphism in the two-locus Levene model with nonepistatic directional selection.

PubMed

Bürger, Reinhard

2009-11-01

For the Levene model with soft selection in two demes, the maintenance of polymorphism at two diallelic loci is studied. Selection is nonepistatic and dominance is intermediate. Thus, there is directional selection in every deme and at every locus. We assume that selection is in opposite directions in the two demes because otherwise no polymorphism is possible. If at one locus there is no dominance, then a complete analysis of the dynamical and equilibrium properties is performed. In particular, a simple necessary and sufficient condition for the existence of an internal equilibrium and sufficient conditions for global asymptotic stability are obtained. These results are extended to deme-independent degree of dominance at one locus. A perturbation analysis establishes structural stability within the full parameter space. In the absence of genotype-environment interaction, which requires deme-independent dominance at both loci, nongeneric equilibrium behavior occurs, and the introduction of arbitrarily small genotype-environment interaction changes the equilibrium structure and may destroy stable polymorphism. The volume of the parameter space for which a (stable) two-locus polymorphism is maintained is computed numerically. It is investigated how this volume depends on the strength of selection and on the dominance relations. If the favorable allele is (partially) dominant in its deme, more than 20% of all parameter combinations lead to a globally asymptotically stable, fully polymorphic equilibrium.

Impact of rainfall intensity on the transport of two herbicides in undisturbed grassed filter strip soil cores

NASA Astrophysics Data System (ADS)

Pot, V.; Šimůnek, J.; Benoit, P.; Coquet, Y.; Yra, A.; Martínez-Cordón, M.-J.

2005-12-01

Two series of displacement experiments with isoproturon and metribuzin herbicides were performed on two undisturbed grassed filter strip soil cores, under unsaturated steady-state flow conditions. Several rainfall intensities (0.070, 0.147, 0.161, 0.308 and 0.326 cm h - 1 ) were used. A water tracer (bromide) was simultaneously injected in each displacement experiment. A descriptive analysis of experimental breakthrough curves of bromide and herbicides combined with a modeling analysis showed an impact of rainfall intensity on the solute transport. Two contrasting physical non-equilibrium transport processes occurred. Multiple (three) porosity domains contributed to flow at the highest rainfall intensities, including preferential flow through macropore pathways. Macropores were not active any longer at intermediate and lowest velocities, and the observed preferential transport was described using dual-porosity-type models with a zero or low flow in the matrix domain. Chemical non-equilibrium transport of herbicides was found at all rainfall intensities. Significantly higher estimated values of degradation rate parameters as compared to batch data were correlated with the degree of non-equilibrium sorption. Experimental breakthrough curves were analyzed using different physical and chemical equilibrium and non-equilibrium transport models: convective-dispersive model (CDE), dual-porosity model (MIM), dual-permeability model (DP), triple-porosity, dual permeability model (DP-MIM); each combined with both chemical instantaneous and kinetic sorption.

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.

A new intermediate in the Prins reaction

PubMed Central

Fukuda, Takeshi; Yamazaki, Shoko

2013-01-01

Summary Two Prins reactions were investigated by the use of DFT calculations. A model composed of R–CH=CH2 + H3O+(H2O)13 + (H2C=O)2, R = Me and Ph, was adopted to trace reaction paths. For both alkenes, the concerted path forming 1,3-diols was obtained as the rate determining step (TS1). TS stands for a transition state. From the 1,3-diol, a bimolecular elimination (TS2) leads to the allylic alcohol as the first channel. In the second channel, the 1,3-diol was converted via TS3 into an unprecedented hemiacetal intermediate, HO–CH2–O–CH(R)–CH2–CH2–OH. This intermediate undergoes ring closure (TS4), affording the 1,3-dioxane product. The intermediate is of almost the same stability as the product, and two species were suggested to be in a state of equilibrium. While the geometry of TS1 appears to be forwarded to that of a carbocation intermediate, the cation disappeared through the enlargement of the water cluster. Dynamical calculations of a classical trajectory using the atom-centered density matrix propagation molecular dynamics model on the four TSs were carried out, and results of IRC calculations were confirmed by them. PMID:23532354

Transition Pathway and Its Free-Energy Profile: A Protocol for Protein Folding Simulations

PubMed Central

Lee, In-Ho; Kim, Seung-Yeon; Lee, Jooyoung

2013-01-01

We propose a protocol that provides a systematic definition of reaction coordinate and related free-energy profile as the function of temperature for the protein-folding simulation. First, using action-derived molecular dynamics (ADMD), we investigate the dynamic folding pathway model of a protein between a fixed extended conformation and a compact conformation. We choose the pathway model to be the reaction coordinate, and the folding and unfolding processes are characterized by the ADMD step index, in contrast to the common a priori reaction coordinate as used in conventional studies. Second, we calculate free-energy profile as the function of temperature, by employing the replica-exchange molecular dynamics (REMD) method. The current method provides efficient exploration of conformational space and proper characterization of protein folding/unfolding dynamics from/to an arbitrary extended conformation. We demonstrate that combination of the two simulation methods, ADMD and REMD, provides understanding on molecular conformational changes in proteins. The protocol is tested on a small protein, penta-peptide of met-enkephalin. For the neuropeptide met-enkephalin system, folded, extended, and intermediate sates are well-defined through the free-energy profile over the reaction coordinate. Results are consistent with those in the literature. PMID:23917881

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.

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

Factors That Affect Oxygen Activation and Coupling of the Two Redox Cycles in the Aromatization Reaction Catalyzed by NikD, an Unusual Amino Acid Oxidase

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

Kommoju, Phaneeswara-Rao; Bruckner, Robert C.; Ferreira, Patricia

2009-10-21

NikD is a flavoprotein oxidase that catalyzes the oxidation of piperideine-2-carboxylate (P2C) to picolinate in a remarkable aromatization reaction comprising two redox cycles and at least one isomerization step. Tyr258 forms part of an 'aromatic cage' that surrounds the ring in picolinate and its precursors. Mutation of Tyr258 to Phe does not perturb the structure of nikD but does affect the coupling of the two redox cycles and causes a 10-fold decrease in turnover rate. Tyr258Phe catalyzes a quantitative two-electron oxidation of P2C, but only 60% of the resulting dihydropicolinate intermediate undergoes a second redox cycle to produce picolinate. Themore » mutation does not affect product yield with an alternate substrate (3,4-dehydro-l-proline) that is aromatized in a single two-electron oxidation step. Wild-type and mutant enzymes exhibit identical rate constants for oxidation of P2C to dihydropicolinate and isomerization of a reduced enzyme-dihydropicolinate complex. The observed rates are 200- and 10-fold faster, respectively, than the mutant turnover rate. Release of picolinate from Tyr258Phe is 100-fold faster than turnover. The presence of a bound substrate or product is a key factor in oxygen activation by wild-type nikD, as judged by the 10-75-fold faster rates observed for complexes of the reduced enzyme with picolinate, benzoate, or 1-cyclohexenoate, a 1-deaza-P2C analogue. The reduced Tyr258Phe-1-cyclohexenoate complex is 25-fold less reactive with oxygen than the wild-type complex. We postulate that mutation of Tyr258 causes subtle changes in active site dynamics that promote release of the reactive dihydropicolinate intermediate and disrupt the efficient synchronization of oxygen activation observed with wild-type nikD.« less

Magnesium Reduces Hepatic Lipid Accumulation in Yellow Catfish (Pelteobagrus fulvidraco) and Modulates Lipogenesis and Lipolysis via PPARA, JAK-STAT, and AMPK Pathways in Hepatocytes.

PubMed

Wei, Chuan-Chuan; Wu, Kun; Gao, Yan; Zhang, Li-Han; Li, Dan-Dan; Luo, Zhi

2017-06-01

Background: Magnesium influences hepatic lipid deposition in vertebrates, but the underlying mechanism is unknown. Objective: We used yellow catfish and their isolated hepatocytes to test the hypothesis that magnesium influences lipid deposition by modulating lipogenesis and lipolysis. Methods: Juvenile yellow catfish (mean ± SEM weight: 3.43 ± 0.02 g, 3 mo old, mixed sex) were fed a 0.14- (low), 0.87- (intermediate) or 2.11- (high) g Mg/kg diet for 56 d. Primary hepatocytes were incubated for 48 h in control or MgSO 4 -containing medium with or without 2-h pretreatment with an inhibitor (AG490, GW6471, or Compound C). Growth performance, cell viability, triglyceride (TG) concentrations, and expression of enzymes and genes involved in lipid metabolism were measured. Results: Compared with fish fed low magnesium, those fed intermediate or high magnesium had lower hepatic lipids (18%, 22%) and 6-phosphogluconate dehydrogenase (6PGD; 3.7%, 3.8%) and malic enzyme (ME; 35%, 48%) activities and greater mRNA levels of the lipolytic genes adipose triacylglyceride lipase ( atgl ; 82% and 1.7-fold) and peroxisome proliferator-activated receptor ( ppara ; 18% and 1.0-fold), respectively ( P < 0.05). Relative mRNA levels of AMP-activated protein kinase ( ampk ) a1 , ampka2 , ampkb1 , ampkb2 , ampkg1a , ampkg1b , Janus kinase (jak) 2a , jak2b, and signal transducers and activators of transcription ( stat ) 3 in fish fed high magnesium were higher (24% to 3.1-fold, P < 0.05) than in those fed low or intermediate magnesium. Compared with cells incubated with MgSO 4 alone, those incubated with MgSO 4 and pretreated with AG490, GW6471, or Compound C had greater TG concentrations (42%, 31%, or 56%), g6pd (98%, 59%, or 51%), 6pgd (68%, 73%, or 32%) mRNA expression, and activities of G6PD (35%, 45%, or 16%) and ME (1.5-fold, 1.3-fold, or 13%), and reduced upregulation (61%, 25%, or 45%) of the lipolytic gene, atgl ( P < 0.05). Conclusions: Magnesium reduced hepatic lipid accumulation in yellow catfish and the variation might be attributed to inhibited lipogenesis and increased lipolysis. PPARA, JAK-STAT, and AMPK pathways mediated the magnesium-induced changes in lipid deposition and metabolism. These results offer new insight into magnesium nutrition in vertebrates. © 2017 American Society for Nutrition.

Self-organized morphological evolution and dewetting in solvent vapor annealing of spin coated polymer blend nanostructures.

PubMed

Roy, Sudeshna; Sharma, Ashutosh

2015-07-01

Dewetting pathways, kinetics and morphologies of thin films of phase separating polymer blends are governed by the relative mobilities of the two components. We characterize the morphological transformations of the nanostructures of a PS/PMMA blend by annealing in toluene and chloroform vapors. Toluene leads to faster reorganization of PS, whereas chloroform engenders the opposite effect. Spin coating produces a very rough PMMA rich layer that completely wets the substrate and forms a plethora of slender columns protruding through the continuous PS rich layer on top. The nanostructures were stable under long thermal annealing but in the vapor annealing, phase separation and dewetting occurred readily to form the equilibrium structures of dewetted droplets of PS on top of PMMA which also climbed around the PS droplets to form rims. Toluene and chloroform annealing required around 50 h and 1 h respectively to attain the equilibrium. Substantial differences are observed in the intermediate morphologies (heights of nanostructures, roughness and size). PMMA columns remained embedded in the dewetted PS droplets, whereas a high mobility of PMMA in chloroform allowed its rapid evacuation during dewetting to produce an intermediate swiss-cheese like morphology of PS domains. Copyright © 2015 Elsevier Inc. All rights reserved.

Energetic frustrations in protein folding at residue resolution: a homologous simulation study of Im9 proteins.

PubMed

Sun, Yunxiang; Ming, Dengming

2014-01-01

Energetic frustration is becoming an important topic for understanding the mechanisms of protein folding, which is a long-standing big biological problem usually investigated by the free energy landscape theory. Despite the significant advances in probing the effects of folding frustrations on the overall features of protein folding pathways and folding intermediates, detailed characterizations of folding frustrations at an atomic or residue level are still lacking. In addition, how and to what extent folding frustrations interact with protein topology in determining folding mechanisms remains unclear. In this paper, we tried to understand energetic frustrations in the context of protein topology structures or native-contact networks by comparing the energetic frustrations of five homologous Im9 alpha-helix proteins that share very similar topology structures but have a single hydrophilic-to-hydrophobic mutual mutation. The folding simulations were performed using a coarse-grained Gō-like model, while non-native hydrophobic interactions were introduced as energetic frustrations using a Lennard-Jones potential function. Energetic frustrations were then examined at residue level based on φ-value analyses of the transition state ensemble structures and mapped back to native-contact networks. Our calculations show that energetic frustrations have highly heterogeneous influences on the folding of the four helices of the examined structures depending on the local environment of the frustration centers. Also, the closer the introduced frustration is to the center of the native-contact network, the larger the changes in the protein folding. Our findings add a new dimension to the understanding of protein folding the topology determination in that energetic frustrations works closely with native-contact networks to affect the protein folding.

Contingency Table Browser - prediction of early stage protein structure.

PubMed

Kalinowska, Barbara; Krzykalski, Artur; Roterman, Irena

2015-01-01

The Early Stage (ES) intermediate represents the starting structure in protein folding simulations based on the Fuzzy Oil Drop (FOD) model. The accuracy of FOD predictions is greatly dependent on the accuracy of the chosen intermediate. A suitable intermediate can be constructed using the sequence-structure relationship information contained in the so-called contingency table - this table expresses the likelihood of encountering various structural motifs for each tetrapeptide fragment in the amino acid sequence. The limited accuracy with which such structures could previously be predicted provided the motivation for a more indepth study of the contingency table itself. The Contingency Table Browser is a tool which can visualize, search and analyze the table. Our work presents possible applications of Contingency Table Browser, among them - analysis of specific protein sequences from the point of view of their structural ambiguity.

Discovering the electronic circuit diagram of life: structural relationships among transition metal binding sites in oxidoreductases

PubMed Central

Kim, J. Dongun; Senn, Stefan; Harel, Arye; Jelen, Benjamin I.; Falkowski, Paul G.

2013-01-01

Oxidoreductases play a central role in catalysing enzymatic electron-transfer reactions across the tree of life. To first order, the equilibrium thermodynamic properties of these proteins are governed by protein folds associated with specific transition metals and ligands at the active site. A global analysis of holoenzyme structures and functions suggests that there are fewer than approximately 500 fundamental oxidoreductases, which can be further clustered into 35 unique groups. These catalysts evolved in prokaryotes early in the Earth's history and are largely responsible for the emergence of non-equilibrium biogeochemical cycles on the planet's surface. Although the evolutionary history of the amino acid sequences in the oxidoreductases is very difficult to reconstruct due to gene duplication and horizontal gene transfer, the evolution of the folds in the catalytic sites can potentially be used to infer the history of these enzymes. Using a novel, yet simple analysis of the secondary structures associated with the ligands in oxidoreductases, we developed a structural phylogeny of these enzymes. The results of this ‘composome’ analysis suggest an early split from a basal set of a small group of proteins dominated by loop structures into two families of oxidoreductases, one dominated by α-helices and the second by β-sheets. The structural evolutionary patterns in both clades trace redox gradients and increased hydrogen bond energy in the active sites. The overall pattern suggests that the evolution of the oxidoreductases led to decreased entropy in the transition metal folds over approximately 2.5 billion years, allowing the enzymes to use increasingly oxidized substrates with high specificity. PMID:23754810

Stereochemistry and solvent role in protein folding: nuclear magnetic resonance and molecular dynamics studies of poly-L and alternating-L,D homopolypeptides in dimethyl sulfoxide.

PubMed

Srivastava, Kinshuk Raj; Kumar, Anil; Goyal, Bhupesh; Durani, Susheel

2011-05-26

The competing interactions folding and unfolding protein structure remain obscure. Using homopolypeptides, we ask if poly-L structure may have a role. We mutate the structure to alternating-L,D stereochemistry and substitute water as the fold-promoting solvent with methanol and dimethyl sulfoxide (DMSO) as the fold-denaturing solvents. Circular dichroism and molecular dynamics established previously that, while both isomers were folded in water, the poly-L isomer was unfolded and alternating-L,D isomer folded in methanol. Nuclear magnetic resonance and molecular dynamics establish now that both isomers are unfolded in DMSO. We calculated energetics of folding-unfolding equilibrium with water and methanol as solvents. We have now calculated interactions of unfolded polypeptide structures with DMSO as solvent. Methanol was found to unfold and water fold poly-L structure as a dielectric. DMSO has now been found to unfold both poly-L and alternating-L,D structures by strong solvation of peptides to disrupt their hydrogen bonds. Accordingly, we propose that while linked peptides fold protein structure with hydrogen bonds they unfold the structure electrostatically due to the stereochemical effect of the poly-L structure. Protein folding to ordering of peptide hydrogen bonds with water as canonical solvent may thus involve two specific and independent solvent effects-one, strong screening of electrostatics of poly-L linked peptides, and two, weak dipolar solvation of peptides. Correspondingly, protein denaturation may involve two independent solvent effects-one, weak dielectric to unfold poly-L structure electrostatically, and two, strong polarity to disrupt peptide hydrogen bonds by solvation of peptides.

Solvent isotope-induced equilibrium perturbation for isocitrate lyase.

PubMed

Quartararo, Christine E; Hadi, Timin; Cahill, Sean M; Blanchard, John S

2013-12-23

Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacterium's life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage ((D₂O)V = 2.0 ± 0.1, and (D₂O)[V/K(isocitrate)] = 2.2 ± 0.3) arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of the succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate, and succinate prepared in D₂O would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by ¹H NMR spectroscopy shows a clear equilibrium perturbation in D₂O. The final equilibrium isotopic composition of reactants in D₂O revealed dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate, with the transient appearance and disappearance of monodeuterated succinate. A model for the equilibrium perturbation of substrate species and their time-dependent isotopic composition is presented.

Early Events in the Folding of an Amphipathic Peptide A Multi- Nanosecond Molecular Dynamics Study

NASA Technical Reports Server (NTRS)

Chipot, Christophe; Maigret, Bernard; Pohorille, Andrew

1999-01-01

Folding of the capped LQQLLQQLLQL peptide is investigated at the water-hexane interface by molecular dynamics simulations over 161.5 nanoseconds. Initially placed in the aqueous phase as a beta-strand, the peptide rapidly adsorbs to the interface, where it adopts an amphipathic conformation. The marginal presence of non-amphipathic structures throughout the complete trajectory indicate- that the corresponding conformations are strongly disfavored at the interface. It is further suggestive that folding in an interfacial environment proceeds through a pathway of successive amphipathic intermediates. The energetic and entropic penalties involved in the conformational changes along this pathway markedly increase the folding time-scales of LQQLLQQLLQL, explaining why the alpha-helix, the hypothesized lowest free energy structure for a sequence with a hydrophobic periodicity of 3.6, has not been reached yet. The formation of a type I beta-turn at the end of the simulation confirms the importance of such motifs as initiation sites allowing the peptide to coalesce towards a secondary structure.

Evolution of a protein folding nucleus.

PubMed

Xia, Xue; Longo, Liam M; Sutherland, Mason A; Blaber, Michael

2016-07-01

The folding nucleus (FN) is a cryptic element within protein primary structure that enables an efficient folding pathway and is the postulated heritable element in the evolution of protein architecture; however, almost nothing is known regarding how the FN structurally changes as complex protein architecture evolves from simpler peptide motifs. We report characterization of the FN of a designed purely symmetric β-trefoil protein by ϕ-value analysis. We compare the structure and folding properties of key foldable intermediates along the evolutionary trajectory of the β-trefoil. The results show structural acquisition of the FN during gene fusion events, incorporating novel turn structure created by gene fusion. Furthermore, the FN is adjusted by circular permutation in response to destabilizing functional mutation. FN plasticity by way of circular permutation is made possible by the intrinsic C3 cyclic symmetry of the β-trefoil architecture, identifying a possible selective advantage that helps explain the prevalence of cyclic structural symmetry in the proteome. © 2015 The Protein Society.

Foldability of a Natural De Novo Evolved Protein.

PubMed

Bungard, Dixie; Copple, Jacob S; Yan, Jing; Chhun, Jimmy J; Kumirov, Vlad K; Foy, Scott G; Masel, Joanna; Wysocki, Vicki H; Cordes, Matthew H J

2017-11-07

The de novo evolution of protein-coding genes from noncoding DNA is emerging as a source of molecular innovation in biology. Studies of random sequence libraries, however, suggest that young de novo proteins will not fold into compact, specific structures typical of native globular proteins. Here we show that Bsc4, a functional, natural de novo protein encoded by a gene that evolved recently from noncoding DNA in the yeast S. cerevisiae, folds to a partially specific three-dimensional structure. Bsc4 forms soluble, compact oligomers with high β sheet content and a hydrophobic core, and undergoes cooperative, reversible denaturation. Bsc4 lacks a specific quaternary state, however, existing instead as a continuous distribution of oligomer sizes, and binds dyes indicative of amyloid oligomers or molten globules. The combination of native-like and non-native-like properties suggests a rudimentary fold that could potentially act as a functional intermediate in the emergence of new folded proteins de novo. Copyright © 2017 Elsevier Ltd. All rights reserved.

Financial Structure and Economic Welfare: Applied General Equilibrium Development Economics.

PubMed

Townsend, Robert

2010-09-01

This review provides a common framework for researchers thinking about the next generation of micro-founded macro models of growth, inequality, and financial deepening, as well as direction for policy makers targeting microfinance programs to alleviate poverty. Topics include treatment of financial structure general equilibrium models: testing for as-if-complete markets or other financial underpinnings; examining dual-sector models with both a perfectly intermediated sector and a sector in financial autarky, as well as a second generation of these models that embeds information problems and other obstacles to trade; designing surveys to capture measures of income, investment/savings, and flow of funds; and aggregating individuals and households to the level of network, village, or national economy. The review concludes with new directions that overcome conceptual and computational limitations.

Response to a temperature modulation as a signature of chemical mechanisms.

PubMed

Berthoumieux, H; Jullien, L; Lemarchand, A

2007-11-01

We consider n reactive species involved in unimolecular reactions and submitted to a temperature modulation of small amplitude. We determine the conditions on the rate constants for which the deviations from the equilibrium concentrations of each species can be optimized and find the analytical expression of the frequency associated with an extremum of concentration shift in the case n=3. We prove that the frequency dependence of the displacement of equilibrium gives access to the number n of species involved in the mechanism. We apply the results to the case of the transformation of a reactant into a product through a possible reactive intermediate and find the order relation obeyed by the activation energies of the different barriers. The results typically apply to enzymatic catalysis with kinetics of Michaelis-Menten type.

Financial Structure and Economic Welfare: Applied General Equilibrium Development Economics

PubMed Central

Townsend, Robert

2010-01-01

This review provides a common framework for researchers thinking about the next generation of micro-founded macro models of growth, inequality, and financial deepening, as well as direction for policy makers targeting microfinance programs to alleviate poverty. Topics include treatment of financial structure general equilibrium models: testing for as-if-complete markets or other financial underpinnings; examining dual-sector models with both a perfectly intermediated sector and a sector in financial autarky, as well as a second generation of these models that embeds information problems and other obstacles to trade; designing surveys to capture measures of income, investment/savings, and flow of funds; and aggregating individuals and households to the level of network, village, or national economy. The review concludes with new directions that overcome conceptual and computational limitations. PMID:21037939

Genome-scale estimate of the metabolic turnover of E. Coli from the energy balance analysis

NASA Astrophysics Data System (ADS)

De Martino, D.

2016-02-01

In this article the notion of metabolic turnover is revisited in the light of recent results of out-of-equilibrium thermodynamics. By means of Monte Carlo methods we perform an exact sampling of the enzymatic fluxes in a genome scale metabolic network of E. Coli in stationary growth conditions from which we infer the metabolites turnover times. However the latter are inferred from net fluxes, and we argue that this approximation is not valid for enzymes working nearby thermodynamic equilibrium. We recalculate turnover times from total fluxes by performing an energy balance analysis of the network and recurring to the fluctuation theorem. We find in many cases values one of order of magnitude lower, implying a faster picture of intermediate metabolism.

Dynamic heterogeneity in the folding/unfolding transitions of FiP35

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

Mori, Toshifumi, E-mail: mori@ims.ac.jp; Saito, Shinji, E-mail: shinji@ims.ac.jp

Molecular dynamics simulations have become an important tool in studying protein dynamics over the last few decades. Atomistic simulations on the order of micro- to milliseconds are becoming feasible and are used to study the state-of-the-art experiments in atomistic detail. Yet, analyzing the high-dimensional-long-temporal trajectory data is still a challenging task and sometimes leads to contradictory results depending on the analyses. To reveal the dynamic aspect of the trajectory, here we propose a simple approach which uses a time correlation function matrix and apply to the folding/unfolding trajectory of FiP35 WW domain [Shaw et al., Science 330, 341 (2010)]. Themore » approach successfully characterizes the slowest mode corresponding to the folding/unfolding transitions and determines the free energy barrier indicating that FiP35 is not an incipient downhill folder. The transition dynamics analysis further reveals that the folding/unfolding transition is highly heterogeneous, e.g., the transition path time varies by ∼100 fold. We identify two misfolded states and show that the dynamic heterogeneity in the folding/unfolding transitions originates from the trajectory being trapped in the misfolded and half-folded intermediate states rather than the diffusion driven by a thermal noise. The current results help reconcile the conflicting interpretations of the folding mechanism and highlight the complexity in the folding dynamics. This further motivates the need to understand the transition dynamics beyond a simple free energy picture using simulations and single-molecule experiments.« less

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase*

PubMed Central

Ikehara, Yoko; Arai, Kazuhito; Furukawa, Nayuta; Ohno, Tadashi; Miyake, Tatsuya; Fushinobu, Shinya; Nakajima, Masahiro; Miyanaga, Akimasa; Taguchi, Hayao

2014-01-01

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S0.5 value 103-fold and increased the Vmax value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172–185) and MR2 (positions 211–221), form a compact core for allosteric motion, and His179 of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased Vmax 4-fold but reduced pyruvate S0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase Vmax, but 102-reduced pyruvate S0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule. PMID:25258319

Thermodynamic analysis of the disorder-to-α-helical transition of 18.5-kDa myelin basic protein reveals an equilibrium intermediate representing the most compact conformation.

PubMed

Vassall, Kenrick A; Jenkins, Andrew D; Bamm, Vladimir V; Harauz, George

2015-05-22

The intrinsically disordered, 18.5-kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that is essential to proper myelin formation in the central nervous system. MBP acts in oligodendrocytes both to adjoin membrane leaflets to each other in forming myelin and as a hub in numerous protein-protein and protein-membrane interaction networks. Like many intrinsically disordered proteins (IDPs), MBP multifunctionality arises from its high conformational plasticity and its ability to undergo reversible disorder-to-order transitions. One such transition is the disorder-to-α-helical conformational change that is induced upon MBP-membrane binding. Here, we have investigated the disorder-to-α-helical transition of MBP-derived α-peptides and the full-length 18.5-kDa protein. This transition was induced through titration of the membrane-mimetic solvent trifluoroethanol into both protein and peptide solutions, and conformational change was monitored using circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid binding, tryptophan fluorescence quenching, and Förster (fluorescence) resonance energy transfer measurements. The data suggest that the disorder-to-α-helical transition of MBP follows a 3-state model: disordered↔intermediate↔α-helical, with each of the identified equilibrium states likely representing a conformational ensemble. The disordered state is characterized by slight compaction with little regular secondary structure, whereas the intermediate is also disordered but globally more compact. Surprisingly, the α-helical conformation is less compact than the intermediate. This study suggests that multifunctionality in MBP could arise from differences in the population of energetically distinct ensembles under different conditions and also provides an example of an IDP that undergoes cooperative global conformation change. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of 2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl Ureido Based Inhibitors of Trypanosoma brucei FolD and Testing for Antiparasitic Activity

PubMed Central

2015-01-01

The bifunctional enzyme N5,N10-methylenetetrahydrofolate dehydrogenase/cyclo hydrolase (FolD) is essential for growth in Trypanosomatidae. We sought to develop inhibitors of Trypanosoma brucei FolD (TbFolD) as potential antiparasitic agents. Compound 2 was synthesized, and the molecular structure was unequivocally assigned through X-ray crystallography of the intermediate compound 3. Compound 2 showed an IC50 of 2.2 μM, against TbFolD and displayed antiparasitic activity against T. brucei (IC50 49 μM). Using compound 2, we were able to obtain the first X-ray structure of TbFolD in the presence of NADP+ and the inhibitor, which then guided the rational design of a new series of potent TbFolD inhibitors. PMID:26322631

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 mechanism for the effects of trehalose on beta-hairpin folding revealed by molecular dynamics simulation.

PubMed

Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

2008-11-01

Recent work has shown that trehalose can facilitate and inhibit protein folding, but little is known about the molecular basis of these effects. Molecular-level insights into how the osmolyte affects protein folding are of significance for the rational design of small molecular additives for enhancing or hindering the folding of proteins. To investigate the molecular mechanisms of the facilitation and inhibition effects of trehalose on protein folding, molecular dynamics (MD) simulation of a beta-hairpin peptide (Trp-Arg-Tyr-Tyr-Glu-Ser-Ser-Leu-Glu-Pro-Glu-Pro-Asp) in different trehalose concentrations (0-0.26 mol/L) is performed using an all-atom model. It is found that at a proper trehalose concentration (0.065 mol/L), the peptide folds faster than that in water, but it cannot fold to the beta-hairpin at higher trehalose concentrations. Free energy landscape analysis indicates the presence of three intermediate states in both pure water and in 0.065 mol/L trehalose, but the potential energy barriers in the folding pathway decrease greatly in 0.065 mol/L trehalose, so the peptide folding is facilitated. Moreover, at this trehalose concentration, there is a favorable balance between the peptide backbone hydrogen bonds (H-bonds) and the peptide-trehalose H-bonds, leading to the stabilization of the folded peptide. At higher trehalose concentrations, however, trehalose molecules cluster in the peptide region and interact with the peptide via many H-bonds that prevent the peptide from folding to its native structure. The energy landscape analysis indicates that the potential energy barriers increase so greatly that the peptide cannot overcome it, getting trapped in a local free energy basin. The work reported herein has elucidated the molecular mechanism of the peptide folding in the presence of trehalose.

Picosecond to nanosecond dynamics provide a source of conformational entropy for protein folding.

PubMed

Stadler, Andreas M; Demmel, Franz; Ollivier, Jacques; Seydel, Tilo

2016-08-03

Myoglobin can be trapped in fully folded structures, partially folded molten globules, and unfolded states under stable equilibrium conditions. Here, we report an experimental study on the conformational dynamics of different folded conformational states of apo- and holomyoglobin in solution. Global protein diffusion and internal molecular motions were probed by neutron time-of-flight and neutron backscattering spectroscopy on the picosecond and nanosecond time scales. Global protein diffusion was found to depend on the α-helical content of the protein suggesting that charges on the macromolecule increase the short-time diffusion of protein. With regard to the molten globules, a gel-like phase due to protein entanglement and interactions with neighbouring macromolecules was visible due to a reduction of the global diffusion coefficients on the nanosecond time scale. Diffusion coefficients, residence and relaxation times of internal protein dynamics and root mean square displacements of localised internal motions were determined for the investigated structural states. The difference in conformational entropy ΔSconf of the protein between the unfolded and the partially or fully folded conformations was extracted from the measured root mean square displacements. Using thermodynamic parameters from the literature and the experimentally determined ΔSconf values we could identify the entropic contribution of the hydration shell ΔShydr of the different folded states. Our results point out the relevance of conformational entropy of the protein and the hydration shell for stability and folding of myoglobin.

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

Role of electrostatic interactions in binding of peptides and intrinsically disordered proteins to their folded targets. 1. NMR and MD characterization of the complex between the c-Crk N-SH3 domain and the peptide Sos.

PubMed

Xue, Yi; Yuwen, Tairan; Zhu, Fangqiang; Skrynnikov, Nikolai R

2014-10-21

Intrinsically disordered proteins (IDPs) often rely on electrostatic interactions to bind their structured targets. To obtain insight into the mechanism of formation of the electrostatic encounter complex, we investigated the binding of the peptide Sos (PPPVPPRRRR), which serves as a minimal model for an IDP, to the c-Crk N-terminal SH3 domain. Initially, we measured ¹⁵N relaxation rates at two magnetic field strengths and determined the binding shifts for the complex of Sos with wild-type SH3. We have also recorded a 3 μs molecular dynamics (MD) trajectory of this complex using the Amber ff99SB*-ILDN force field. The comparison of the experimental and simulated data shows that MD simulation consistently overestimates the strength of salt bridge interactions at the binding interface. The series of simulations using other advanced force fields also failed to produce any satisfactory results. To address this issue, we have devised an empirical correction to the Amber ff99SB*-ILDN force field whereby the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across the Arg-to-Asp and Arg-to-Glu salt bridges has been increased by 3%. Implementing this correction resulted in a good agreement between the simulations and the experiment. Adjusting the strength of salt bridge interactions removed a certain amount of strain contained in the original MD model, thus improving the binding of the hydrophobic N-terminal portion of the peptide. The arginine-rich C-terminal portion of the peptide, freed from the effect of the overstabilized salt bridges, was found to interconvert more rapidly between its multiple conformational states. The modified MD protocol has also been successfully used to simulate the entire binding process. In doing so, the peptide was initially placed high above the protein surface. It then arrived at the correct bound pose within ∼2 Å of the crystallographic coordinates. This simulation allowed us to analyze the details of the dynamic binding intermediate, i.e., the electrostatic encounter complex. However, an experimental characterization of this transient, weakly populated state remains out of reach. To overcome this problem, we designed the double mutant of c-Crk N-SH3 in which mutations Y186L and W169F abrogate tight Sos binding and shift the equilibrium toward the intermediate state resembling the electrostatic encounter complex. The results of the combined NMR and MD study of this engineered system will be reported in the next part of this paper.

State-to-state modeling of non-equilibrium air nozzle flows

NASA Astrophysics Data System (ADS)

Nagnibeda, E.; Papina, K.; Kunova, O.

2018-05-01

One-dimensional non-equilibrium air flows in nozzles are studied on the basis of the state-to-state description of vibrational-chemical kinetics. Five-component mixture N2/O2/NO/N/O is considered taking into account Zeldovich exchange reactions of NO formation, dissociation, recombination and vibrational energy transitions. The equations for vibrational and chem-ical kinetics in a flow are coupled to the conservation equations of momentum and total energy and solved numerically for different conditions in a nozzle throat. The vibrational distributions of nitrogen and oxygen molecules, number densities of species as well as the gas temperature and flow velocity along a nozzle axis are analysed using the detailed state-to-state flow description and in the frame of the simplified one-temperature thermal equilibrium kinetic model. The comparison of the results showed the influence of non-equilibrium kinetics on macroscopic nozzle flow parameters. In the state-to-state approach, non-Boltzmann vibrational dis-tributions of N2 and O2 molecules with a plateau part at intermediate levels are found. The results are found with the use of the complete and simplified schemes of reactions and the impact of exchange reactions, dissociation and recombination on variation of vibrational level populations, mixture composition, gas velocity and temperature along a nozzle axis is shown.

Self-assembled materials and supramolecular chemistry within microfluidic environments: from common thermodynamic states to non-equilibrium structures.

PubMed

Sevim, S; Sorrenti, A; Franco, C; Furukawa, S; Pané, S; deMello, A J; Puigmartí-Luis, J

2018-05-01

Self-assembly is a crucial component in the bottom-up fabrication of hierarchical supramolecular structures and advanced functional materials. Control has traditionally relied on the use of encoded building blocks bearing suitable moieties for recognition and interaction, with targeting of the thermodynamic equilibrium state. On the other hand, nature leverages the control of reaction-diffusion processes to create hierarchically organized materials with surprisingly complex biological functions. Indeed, under non-equilibrium conditions (kinetic control), the spatio-temporal command of chemical gradients and reactant mixing during self-assembly (the creation of non-uniform chemical environments for example) can strongly affect the outcome of the self-assembly process. This directly enables a precise control over material properties and functions. In this tutorial review, we show how the unique physical conditions offered by microfluidic technologies can be advantageously used to control the self-assembly of materials and of supramolecular aggregates in solution, making possible the isolation of intermediate states and unprecedented non-equilibrium structures, as well as the emergence of novel functions. Selected examples from the literature will be used to confirm that microfluidic devices are an invaluable toolbox technology for unveiling, understanding and steering self-assembly pathways to desired structures, properties and functions, as well as advanced processing tools for device fabrication and integration.

α-cluster states in 46,54Cr from double-folding potentials

NASA Astrophysics Data System (ADS)

Mohr, Peter

2017-10-01

α-cluster states in 46Cr and 54Cr are investigated in the double-folding model. This study complements a recent similar work by Souza and Miyake, Eur. Phys. J. A 53, 146 (2017), which was based on a specially shaped potential. Excitation energies, reduced widths, intercluster separations, and intra-band transition strengths are calculated and compared to experimental values for the ground state bands in 46Cr and 54Cr . The α-cluster potential is also applied to elastic scattering at low and intermediate energies. Here, as a byproduct, a larger radial extent of the neutron density in 50Ti is found.

Regulation of Carbon Flow by Nitrogen and Light in the Red Alga, Gelidium coulteri.

PubMed

Macler, B A

1986-09-01

The red alga Gelidium coulteri Harv. photosynthetically fixed [(14)C] bicarbonate at high rates under defined conditions in unialgal laboratory culture. The fixation rate and flow of photosynthate into various end products were dependent on the nitrogen status of the tissue. Plants fed luxury levels of nitrogen (approximately 340 micromolar) showed fixation rates several-fold higher than those seen for plants starved for nitrogen. The addition of NO(3) (-) or NH(4) (+) to such starved plants further inhibited fixation over at least the first several hours after addition. The majority of (14)C after incubations of 30 minutes to 8 hours was found in the compounds floridoside, agar and floridean starch. In addition, amino acids and intermediate compounds of the reductive pentose phosphate pathway, glycolytic pathway and tricarboxylic acid cycle were detected. Nitrogen affected the partitioning of labeled carbon into these compounds. Plants under luxury nitrogen conditions had higher floridoside levels and markedly lower amounts of agar and starch than found in plants limited for nitrogen. Amino acid, phycobiliprotein and chlorophyll levels were also significantly higher in nitrogen-enriched plants. Addition of NO(3) (-) to starved plants led to an increase in floridoside, tricarboxylic acid cycle intermediates and amino acids within 1 hour and inhibited carbon flow into agar and starch. Carbon fixation in the dark was only 1 to 7% of that seen in the light. Dark fixation of [(14)C]bicarbonate yielded label primarily in tricarboxylic acid cycle intermediates, amino acids and polysaccharides. Nitrogen stimulated amino acid synthesis at the expense of agar and starch. Floridoside was only a minor component in the dark. Pulse-chase experiments, designed to show carbon turnover, indicated a 2-fold increase in labeling of agar over 96 hours of chase in the light. No increases were seen in the dark. Low molecular weight pools, including floridoside, decreased 2- to 5-fold over this period under both light and dark conditions. Nitrogen status did not influence turnover. There was little or no organic carbon released into the culture medium over this period. The results are consistent with biosynthetic pathways to floridoside and agar that share the common intermediate UDP-d-galactose. It is hypothesized that synthesis of floridoside is regulated by nitrogen and light at the enzymic level.

Regulation of Carbon Flow by Nitrogen and Light in the Red Alga, Gelidium coulteri1

PubMed Central

Macler, Bruce A.

1986-01-01

The red alga Gelidium coulteri Harv. photosynthetically fixed [14C] bicarbonate at high rates under defined conditions in unialgal laboratory culture. The fixation rate and flow of photosynthate into various end products were dependent on the nitrogen status of the tissue. Plants fed luxury levels of nitrogen (approximately 340 micromolar) showed fixation rates several-fold higher than those seen for plants starved for nitrogen. The addition of NO3− or NH4+ to such starved plants further inhibited fixation over at least the first several hours after addition. The majority of 14C after incubations of 30 minutes to 8 hours was found in the compounds floridoside, agar and floridean starch. In addition, amino acids and intermediate compounds of the reductive pentose phosphate pathway, glycolytic pathway and tricarboxylic acid cycle were detected. Nitrogen affected the partitioning of labeled carbon into these compounds. Plants under luxury nitrogen conditions had higher floridoside levels and markedly lower amounts of agar and starch than found in plants limited for nitrogen. Amino acid, phycobiliprotein and chlorophyll levels were also significantly higher in nitrogen-enriched plants. Addition of NO3− to starved plants led to an increase in floridoside, tricarboxylic acid cycle intermediates and amino acids within 1 hour and inhibited carbon flow into agar and starch. Carbon fixation in the dark was only 1 to 7% of that seen in the light. Dark fixation of [14C]bicarbonate yielded label primarily in tricarboxylic acid cycle intermediates, amino acids and polysaccharides. Nitrogen stimulated amino acid synthesis at the expense of agar and starch. Floridoside was only a minor component in the dark. Pulse-chase experiments, designed to show carbon turnover, indicated a 2-fold increase in labeling of agar over 96 hours of chase in the light. No increases were seen in the dark. Low molecular weight pools, including floridoside, decreased 2- to 5-fold over this period under both light and dark conditions. Nitrogen status did not influence turnover. There was little or no organic carbon released into the culture medium over this period. The results are consistent with biosynthetic pathways to floridoside and agar that share the common intermediate UDP-d-galactose. It is hypothesized that synthesis of floridoside is regulated by nitrogen and light at the enzymic level. PMID:16664980

{Phi}{sup 4} kinks: Statistical mechanics

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

Habib, S.

1995-12-31

Some recent investigations of the thermal equilibrium properties of kinks in a 1+1-dimensional, classical {phi}{sup 4} field theory are reviewed. The distribution function, kink density, correlation function, and certain thermodynamic quantities were studied both theoretically and via large scale simulations. A simple double Gaussian variational approach within the transfer operator formalism was shown to give good results in the intermediate temperature range where the dilute gas theory is known to fail.

Computation of inverse magnetic cascades

NASA Technical Reports Server (NTRS)

Montgomery, D.

1981-01-01

Inverse cascades of magnetic quantities for turbulent incompressible magnetohydrodynamics are reviewed, for two and three dimensions. The theory is extended to the Strauss equations, a description intermediate between two and three dimensions appropriate to Tokamak magnetofluids. Consideration of the absolute equilibrium Gibbs ensemble for the system leads to a prediction of an inverse cascade of magnetic helicity, which may manifest itself as a major disruption. An agenda for computational investigation of this conjecture is proposed.

Enhancing the biocatalytic manufacture of the key intermediate of atorvastatin by focused directed evolution of halohydrin dehalogenase.

PubMed

Luo, Yu; Chen, Yangzi; Ma, Hongmin; Tian, ZhenHua; Zhang, Yeqi; Zhang, Jian

2017-02-06

Halohydrin dehalogenases (HHDHs) are biocatalytically interesting enzymes due to their ability to form C-C, C-N, C-O, and C-S bonds. One of most important application of HHDH was the protein engineering of HheC (halohydrin dehalogenase from Agrobacterium radiobacter AD1) for the industrial manufacturing of ethyl (R)-4-cyano-3-hydroxybutanoate (HN), a key chiral synthon of a cholesterol-lowering drug of atorvastatin. During our development of an alternative, more efficient and economic route for chemo-enzymatic preparation of the intermediate of atorvastatin, we found that the HheC2360 previously reported for HN manufacture, had insufficient activity for the cyanolysis production of tert-butyl (3 R,5 S)-6-cyano-3,5-dihydroxyhexanoate (A7). Herein, we present the focused directed evolution of HheC2360 with higher activity and enhanced biocatalytic performance using active site mutagenesis. Through docking of the product, A7, into the crystal structure of HheC2360, 6 residues was selected for combined active sites testing (CASTing). After library screening, the variant V84G/W86F was identified to have a 15- fold increase in activity. Time course analysis of the cyanolysis reaction catalyzed by this variant, showed 2- fold increase in space time productivity compared with HheC2360. These results demonstrate the applicability of the variant V84G/W86F as a biocatalyst for the efficient and practical production of atorvastatin intermediate.

Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme.

PubMed

Lukoyanov, Dmitriy; Khadka, Nimesh; Yang, Zhi-Yong; Dean, Dennis R; Seefeldt, Lance C; Hoffman, Brian M

2016-08-24

We proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70(Val→Ile) MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN═NH) reduction level bound to the catalytic FeMo-co. EPR/ENDOR/photophysical measurements on wild type (WT) MoFe protein now establish this mechanism. They show that a state freeze-trapped during N2 reduction by WT MoFe is the same Janus intermediate, thereby establishing the α-70(Val→Ile) intermediate as a reliable guide to mechanism. Monitoring the Janus state in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby solving longstanding puzzles. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium. Relative populations of E4(2N2H)/E4(4H) freeze-trapped during WT turnover furthermore show that the reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is ∼ thermoneutral (ΔreG(0) ∼ -2 kcal/mol), whereas, by itself, hydrogenation of N2(g) is highly endergonic. These findings demonstrate that (i) re/oa accounts for the historical Key Constraints on mechanism, (ii) that Janus is central to N2 reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus, emerges a picture of the central mechanistic steps by which nitrogenase carries out one of the most challenging chemical transformations in biology.

Alloying effects on structural and thermal behavior of Ti{sub 1-x}Zr{sub x}C: A first principles study

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

Chauhan, Mamta, E-mail: mamta-physics@yahoo.co.in; Gupta, Dinesh C., E-mail: sosfizix@gmail.com

2016-05-06

The formation energy, equilibrium lattice parameter, bulk modulus, Debye temperature and heat capacity at constant volume have been calculated for TiC, ZrC, and their intermediate alloys (Ti{sub 1-x}Zr{sub x}C, x = 0,0.25.0.5,0.75,1) using first principles approach. The calculated values of lattice parameter and bulk modulus agree well with the available experimental and earlier theoretical reports. The variation of lattice parameter and bulk modulus with the change in concentration of Zr atom in Ti{sub 1-x}Zr{sub x}C has also been reported. The heat capacities of TiC, ZrC, and their intermediate alloys have been calculated by considering both vibrational and electronic contributions.

Asymmetric dark matter models in SO(10)

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

Nagata, Natsumi; Olive, Keith A.; Zheng, Jiaming, E-mail: natsumi@hep-th.phys.s.u-tokyo.ac.jp, E-mail: olive@physics.umn.edu, E-mail: zheng@physics.umn.edu

2017-02-01

We systematically study the possibilities for asymmetric dark matter in the context of non-supersymmetric SO(10) models of grand unification. Dark matter stability in SO(10) is guaranteed by a remnant Z{sub 2} symmetry which is preserved when the intermediate scale gauge subgroup of SO(10) is broken by a (\\bf 126) dimensional representation. The asymmetry in the dark matter states is directly generated through the out-of-equilibrium decay of particles around the intermediate scale, or transferred from the baryon/lepton asymmetry generated in the Standard Model sector by leptogenesis. We systematically classify possible asymmetric dark matter candidates in terms of their quantum numbers, andmore » derive the conditions for each case that the observed dark matter density is (mostly) explained by the asymmetry of dark matter particles.« less

The humidity dependence of ozone deposition onto a variety of building surfaces

NASA Astrophysics Data System (ADS)

Grøntoft, Terje; Henriksen, Jan F.; Seip, Hans M.

Measurements of the dry deposition velocity of O 3 to material samples of calcareous stone, concrete and wood at varying humidity of the air, were performed in a deposition chamber. Equilibrium surface deposition velocities were found for various humidity values by fitting a model to the time-dependent deposition data. A deposition velocity-humidity model was derived giving three separate rate constants for the surface deposition velocities, i.e. on the dry surface, on the first mono-layer of adsorbed water and on additional surface water. The variation in the dry air equilibrium surface deposition velocities among the samples correlated with variations in effective areas, with larger effective areas giving higher measured deposition velocities. A minimum for the equilibrium surface deposition velocity was generally measured at an intermediate humidity close to the humidity found to correspond to one mono-layer of water molecules on the surfaces. At low air humidity the equilibrium surface deposition velocity of O 3 was found to decrease as more adsorbed water prevented direct contact of the O 3 molecules with the surface. This was partly compensated by an increase as more adsorbed water became available for reaction with O 3. At high air humidity the equilibrium surface deposition velocity was found to increase as the mass of water on the surface increased. The deposition velocity on bulk de-ionised water at RH=90% was an order of magnitude lower than on the sample surfaces.

In silico prediction of medium effects on esterification equilibrium using the COSMO-RS method.

PubMed

Fermeglia, Maurizio; Braiuca, Paolo; Gardossi, Lucia; Pricl, Sabrina; Halling, Peter J

2006-01-01

This paper presents a new approach for predicting solvent effects on esterification reactions of industrial importance in the field of biocatalysis. The COSMO-RS method has been used to calculate the activity coefficients of the chemical species involved in various reactions, carried out in different solvents. For comparison we also used the traditional UNIFAC method. Three lipase-catalyzed esterifications were considered: (1) 1-dodecanoic acid with menthol in n-hexane, n-heptane, cyclohexane, 2,2,4-trimethylpentane, toluene, acetonitrile, and 2-methyl-2-butanol; (2) 1-dodecanoic acid and 1-dodecanol in n-hexane, n-heptane, cyclohexane, 2,2,4-trimethylpentane, and toluene; and (3) glycerol and n-octanoic acid in acetonitrile, benzene, and toluene and in the neat reaction mixture (without any solvent). Predicted activities were used to calculate the thermodynamic equilibrium ratio. This should be independent of medium, and the variation in COSMO-RS values is at most 9-fold (corresponding to a DeltaG degrees of about 5.5 kJ/mol, which would still be a very useful prediction) and often only 2-fold (corresponding to less than 2 kJ/mol or 0.5 kcal/mol, therefore comparable with experimental error). UNIFAC is weaker, especially when important roles are played by conformational freedom, intramolecular interactions, strong polar effects, and charge distribution of molecules in the mixture. The relative percent deviations from the mean of equilibrium constants in different solvents range between 17 and 49 for COSMO-RS versus 32 to 65 for UNIFAC. The COSMO-RS method opens up new perspectives for the development of theoretical models for solvent selection with general applicability.

Van’t Hoff global analyses of variable temperature isothermal titration calorimetry data

PubMed Central

Freiburger, Lee A.; Auclair, Karine; Mittermaier, Anthony K.

2016-01-01

Isothermal titration calorimetry (ITC) can provide detailed information on the thermodynamics of biomolecular interactions in the form of equilibrium constants, KA, and enthalpy changes, ΔHA. A powerful application of this technique involves analyzing the temperature dependences of ITC-derived KA and ΔHA values to gain insight into thermodynamic linkage between binding and additional equilibria, such as protein folding. We recently developed a general method for global analysis of variable temperature ITC data that significantly improves the accuracy of extracted thermodynamic parameters and requires no prior knowledge of the coupled equilibria. Here we report detailed validation of this method using Monte Carlo simulations and an application to study coupled folding and binding in an aminoglycoside acetyltransferase enzyme. PMID:28018008

Conservation of protein structure over four billion years.

PubMed

Ingles-Prieto, Alvaro; Ibarra-Molero, Beatriz; Delgado-Delgado, Asuncion; Perez-Jimenez, Raul; Fernandez, Julio M; Gaucher, Eric A; Sanchez-Ruiz, Jose M; Gavira, Jose A

2013-09-03

Little is known about the evolution of protein structures and the degree of protein structure conservation over planetary time scales. Here, we report the X-ray crystal structures of seven laboratory resurrections of Precambrian thioredoxins dating up to approximately four billion years ago. Despite considerable sequence differences compared with extant enzymes, the ancestral proteins display the canonical thioredoxin fold, whereas only small structural changes have occurred over four billion years. This remarkable degree of structure conservation since a time near the last common ancestor of life supports a punctuated-equilibrium model of structure evolution in which the generation of new folds occurs over comparatively short periods and is followed by long periods of structural stasis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Laws controlling crystallization and melting in bulk polymers

NASA Astrophysics Data System (ADS)

Strobl, Gert

2007-03-01

When the fundamentals of the structure of semi-crystalline polymers - layer-like crystallites with fold surfaces being embedded in an amorphous matrix - were revealed in the Fifties, considerations about the mechanism of formation started immediately. In the Sixties and Seventies, they became a major field of research and a focus of interest. In the years which followed the approach put forward by Hoffman, Lauritzen and their co-workers [1] gained superiority. The picture envisaged by the treatment - a crystalline lamella with an ordered fold surface and smooth lateral faces, growing layer by layer with a secondary nucleation as rate determining step - is easy to grasp and yields simple relationships. Supercooling below the equilibrium melting point Tf^∞ is the control parameter determining both the thickness dc and the lateral growth rate of the crystallites G. Experiments carried out during the last decade provided new insights and are now completely changing the understanding. They showed in particular - that dc is inversely proportional to the distance to a temperature Tc^∞ distinctly above Tf^∞- that the activation energy determining G diverges at a temperature Tzero clearly below Tf^∞.Further simple relationships concern- recrystallization processes: dc is again inversely proportional to the distance to Tc^∞- the extension of ordered regions within the lamellar crystallites: it is proportional to dc. We interpret the observations as indication that the pathway followed in the growth of polymer crystallites includes an intermediate phase of mesomorphic character. A thin layer with mesomorphic inner structure forms between the lateral crystal face and the melt, stabilized by epitaxial forces. The first step in the growth process is an attachment of chain sequences from the melt onto the growth face of the mesomorphic layer. The high mobility of the chains in the layer allows a spontaneous thickening, up to a critical thickness, where the layer solidifies under formation of block-like crystallites. The last step is a perfectioning of the crystallites, leading to a further stabilization. We constructed a thermodynamic scheme dealing with the transitions between melt, mesomorphic layers and lamellar crystallites, assuming for the latter ones that they exist both in an initial native and a final stabilized form. Tc^∞ and Tzero are identified with the temperatures Tmc and Tam of the (hidden) transitions mesomorphic -> crystalline and amorphous-> mesomorphic, respectively. Application of the scheme in a quantitative evaluation of small angle X-ray scattering and calorimetric results yields the equilibrium transition temperatures between the various phases, latent heats of transition and surface free energies [2]. [1] J.D Hoffman, G.T Davis, and J.I. Lauritzen. In Treatise on Solid State Chemistry Vol.3, N.B.Hannay Ed., page 497. Plenum, 1976. [2] G. Strobl.Eur.Phys.J.E, 18:295, 2005.

Chemical exchange in biomacromolecules: Past, present, and future

PubMed Central

Palmer, Arthur G.

2014-01-01

The perspective reviews quantitative investigations of chemical exchange phenomena in proteins and other biological macromolecules using NMR spectroscopy, particularly relaxation dispersion methods. The emphasis is on techniques and applications that quantify the populations, interconversion kinetics, and structural features of sparsely populated conformational states in equilibrium with a highly populated ground state. Applications to folding, mol ecular recognition, catalysis, and allostery by proteins and nucleic acids are highlighted. PMID:24656076

Wang-Landau density of states based study of the folding-unfolding transition in the mini-protein Trp-cage (TC5b)

NASA Astrophysics Data System (ADS)

Singh, Priya; Sarkar, Subir K.; Bandyopadhyay, Pradipta

2014-07-01

We present the results of a high-statistics equilibrium study of the folding/unfolding transition for the 20-residue mini-protein Trp-cage (TC5b) in water. The ECEPP/3 force field is used and the interaction with water is treated by a solvent-accessible surface area method. A Wang-Landau type simulation is used to calculate the density of states and the conditional probabilities for the various values of the radius of gyration and the number of native contacts at fixed values of energy—along with a systematic check on their convergence. All thermodynamic quantities of interest are calculated from this information. The folding-unfolding transition corresponds to a peak in the temperature dependence of the computed specific heat. This is corroborated further by the structural signatures of folding in the distributions for radius of gyration and the number of native contacts as a function of temperature. The potentials of mean force are also calculated for these variables, both separately and jointly. A local free energy minimum, in addition to the global minimum, is found in a temperature range substantially below the folding temperature. The free energy at this second minimum is approximately 5 kBT higher than the value at the global minimum.

Expression and function of transforming growth factor-β isoforms and cognate receptors in the rat urinary bladder following cyclophosphamide-induced cystitis

PubMed Central

Gonzalez, Eric J.; Girard, Beatrice M.

2013-01-01

Numerous proinflammatory cytokines have been implicated in the reorganization of lower urinary tract function following cyclophosphamide (CYP)-induced cystitis. The present study investigated the functional profile of three pleiotropic transforming growth factor-β (TGF-β) isoforms and receptor (TβR) variants in the normal and inflamed (CYP-induced cystitis) rat urinary bladder. Our findings indicate that TGF-β (1, 2, and 3) and TβR (1, 2, and 3) transcript and protein expression were regulated to varying degrees in the urothelium or detrusor smooth muscle following intermediate (48 h; 150 mg/kg ip) or chronic (75 mg/kg ip; once every 3 days for 10 days), but not acute (4 h; 150 mg/kg ip), CYP-induced cystitis. Conscious, open-outlet cystometry was performed to determine whether aberrant TGF-β signaling contributes to urinary bladder dysfunction following intermediate (48 h) CYP-induced cystitis. TβR-1 inhibition with SB505124 (5 μM) significantly (p ≤ 0.001) decreased voiding frequency and increased bladder capacity (2.5-fold), void volume (2.6-fold), and intercontraction intervals (2.5-fold) in CYP-treated (48 h) rats. Taken together, these results provide evidence for 1) the involvement of TGF-β in lower urinary tract neuroplasticity following urinary bladder inflammation, 2) a functional role of TGF-β signaling in the afferent limb of the micturition reflex, and 3) urinary bladder TβR-1 as a viable target to reduce voiding frequency with cystitis. PMID:23926183

Combination of cephalosporins with vancomycin or teicoplanin enhances antibacterial effect of glycopeptides against heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and VISA.

PubMed

Lai, Chih-Cheng; Chen, Chi-Chung; Chuang, Yin-Ching; Tang, Hung-Jen

2017-01-31

Eight heterogeneous vancomycin-intermediate S. aureus (h-VISA) and seven VISA clinical isolates confirmed by the population analysis profile/area under the curve ratio (PAP/AUC) were collected. We further performed the PAP/AUC, time-killing methods and MIC tests using vancomycin/teicoplanin alone or combination with susceptible breakpoint concentrations of cefazolin, cefmetazole, cefotaxime, and cefepime for these isolates. The PAP/AUC MIC curve shifted left after addition of cephalosporins with vancomycin or teicoplanin for both h-VISA and VISA isolates. With the combination of different cephalosporins with vancomycin or teicoplanin, the AUC/Mu3 AUC ratio decreased to <0.9 for the standard Mu3 isolate which are compatible with the definition of vancomycin susceptible S. aureus. These decreases ranged between 1.81-2.02 and 2.37-2.85-fold for h-VISA treated with cephalosporins and vancomycin or teicoplanin, and 2.05-4.59, and 2.93-4,89-fold for VISA treated with cephalosporins with vancomycin or teicoplanin. As measured by time-killing assays, the combinations of different cephalosporins with vancomycin concentrations at 1/2 and 1/4 MIC, exhibited a bactericidal and bacteriostatic effect in VISA. The mean fold of MIC decline for vancomycin base combinations ranged from 1.81-3.83 and 2.71-9.33 for h-VISA and VISA, respectively. Overall, this study demonstrated the enhanced antibacterial activity of vancomycin/teicoplanin after adding cephalosporins against clinical h-VISA/VISA isolates.

Multiphase Simulated Annealing Based on Boltzmann and Bose-Einstein Distribution Applied to Protein Folding Problem.

PubMed

Frausto-Solis, Juan; Liñán-García, Ernesto; Sánchez-Hernández, Juan Paulo; González-Barbosa, J Javier; González-Flores, Carlos; Castilla-Valdez, Guadalupe

2016-01-01

A new hybrid Multiphase Simulated Annealing Algorithm using Boltzmann and Bose-Einstein distributions (MPSABBE) is proposed. MPSABBE was designed for solving the Protein Folding Problem (PFP) instances. This new approach has four phases: (i) Multiquenching Phase (MQP), (ii) Boltzmann Annealing Phase (BAP), (iii) Bose-Einstein Annealing Phase (BEAP), and (iv) Dynamical Equilibrium Phase (DEP). BAP and BEAP are simulated annealing searching procedures based on Boltzmann and Bose-Einstein distributions, respectively. DEP is also a simulated annealing search procedure, which is applied at the final temperature of the fourth phase, which can be seen as a second Bose-Einstein phase. MQP is a search process that ranges from extremely high to high temperatures, applying a very fast cooling process, and is not very restrictive to accept new solutions. However, BAP and BEAP range from high to low and from low to very low temperatures, respectively. They are more restrictive for accepting new solutions. DEP uses a particular heuristic to detect the stochastic equilibrium by applying a least squares method during its execution. MPSABBE parameters are tuned with an analytical method, which considers the maximal and minimal deterioration of problem instances. MPSABBE was tested with several instances of PFP, showing that the use of both distributions is better than using only the Boltzmann distribution on the classical SA.

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

Coarse-Grained Models Reveal Functional Dynamics – II. Molecular Dynamics Simulation at the Coarse-Grained Level – Theories and Biological Applications

PubMed Central

Chng, Choon-Peng; Yang, Lee-Wei

2008-01-01

Molecular dynamics (MD) simulation has remained the most indispensable tool in studying equilibrium/non-equilibrium conformational dynamics since its advent 30 years ago. With advances in spectroscopy accompanying solved biocomplexes in growing sizes, sampling their dynamics that occur at biologically interesting spatial/temporal scales becomes computationally intractable; this motivated the use of coarse-grained (CG) approaches. CG-MD models are used to study folding and conformational transitions in reduced resolution and can employ enlarged time steps due to the absence of some of the fastest motions in the system. The Boltzmann-Inversion technique, heavily used in parameterizing these models, provides a smoothed-out effective potential on which molecular conformation evolves at a faster pace thus stretching simulations into tens of microseconds. As a result, a complete catalytic cycle of HIV-1 protease or the assembly of lipid-protein mixtures could be investigated by CG-MD to gain biological insights. In this review, we survey the theories developed in recent years, which are categorized into Folding-based and Molecular-Mechanics-based. In addition, physical bases in the selection of CG beads/time-step, the choice of effective potentials, representation of solvent, and restoration of molecular representations back to their atomic details are systematically discussed. PMID:19812774

Stability of a family of uniform vortices related to vortex configurations before merging

NASA Astrophysics Data System (ADS)

Luzzatto-Fegiz, P.; Williamson, C. H. K.

2006-11-01

Motivated by the merger of two corotating vortices, Cerretelli & Williamson (JFM 2003) discovered a family of uniform vorticity patches representing the continuation of two corotating vortices into a single ``dumbbell'' shape. This branch of solutions passes through a bifurcation from the Kirchhoff ellipses (discovered by Kamm 1987 and Saffman 1988) and ends into a cat's eye shape. By using a more accurate method for equilibrium shape calculation, we find some differences in the equilibrium shapes to those discovered by Cerretelli & Williamson, particularly near the topological change (from a two-vortex to a single vortex shape). We implement the approach of Dritschel (1985), and show that all the simply connected shapes are unstable to a three-fold perturbation, while a regime of the two-vortex shapes nearing the topological change is unstable to a two-fold antisymmetric perturbation. The stability of two patches has been source of debate in the literature. Saffman & Szeto (1980) predicted exchange of stability at an extremum in energy and angular momentum; on the other hand, Dritschel (1985) found that conditions for instability from linear analysis did not match those coming from the energy criterion. In the present work, we find precise agreement between results from linear analysis and energy criterion, in accordance with the more recent work of Kamm (1987) and Dritschel (1995).

Fluid flow plate for decreased density of fuel cell assembly

DOEpatents

Vitale, Nicholas G.

1999-01-01

A fluid flow plate includes first and second outward faces. Each of the outward faces has a flow channel thereon for carrying respective fluid. At least one of the fluids serves as reactant fluid for a fuel cell of a fuel cell assembly. One or more pockets are formed between the first and second outward faces for decreasing density of the fluid flow plate. A given flow channel can include one or more end sections and an intermediate section. An interposed member can be positioned between the outward faces at an interface between an intermediate section, of one of the outward faces, and an end section, of that outward face. The interposed member can serve to isolate the reactant fluid from the opposing outward face. The intermediate section(s) of flow channel(s) on an outward face are preferably formed as a folded expanse.

A Native to Amyloidogenic Transition Regulated by a Backbone Trigger

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

Eakin,C.; Berman, A.; Miranker, A.

2006-01-01

Many polypeptides can self-associate into linear, aggregated assemblies termed amyloid fibers. High-resolution structural insights into the mechanism of fibrillogenesis are elusive owing to the transient and mixed oligomeric nature of assembly intermediates. Here, we report the conformational changes that initiate fiber formation by beta-2-microglobulin (beta2m) in dialysis-related amyloidosis. Access of beta2m to amyloidogenic conformations is catalyzed by selective binding of divalent cations. The chemical basis of this process was determined to be backbone isomerization of a conserved proline. On the basis of this finding, we designed a beta2m variant that closely adopts this intermediate state. The variant has kinetic, thermodynamicmore » and catalytic properties consistent with its being a fibrillogenic intermediate of wild-type beta2m. Furthermore, it is stable and folded, enabling us to unambiguously determine the initiating conformational changes for amyloid assembly at atomic resolution.« less

Evidence for an intermediate in tau filament formation.

PubMed

Chirita, Carmen N; Kuret, Jeff

2004-02-17

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of full-length, unphosphorylated recombinant tau can be induced under near-physiological conditions by treatment with various agents, including anionic surfactants. Here we examine the pathway through which anionic surfactants promote tau fibrillization using a combination of electron microscopy and fluorescence spectroscopy. Protein and surfactant first interacted in solution to form micelles, which then provided negatively charged surfaces that accumulated tau aggregates. Surface aggregation of tau protein was followed by the time-dependent appearance of a thioflavin S reactive intermediate that accumulated over a period of hours. The intermediate was unstable in the absence of anionic surfaces, suggesting it was not filamentous. Fibrillization proceeded after intermediate formation with classic nucleation-dependent kinetics, consisting of lag phase followed by the exponential increase in filament lengths, followed by an equilibrium phase reached in approximately 24 h. The pathway did not require protein insertion into the micelle hydrophobic core or conformational change arising from mixed micelle formation, because anionic microspheres constructed from impermeable polystyrene were capable of qualitatively reproducing all aspects of the fibrillization reaction. It is proposed that the progression from amorphous aggregation through intermediate formation and fibrillization may underlie the activity of other inducers such as hyperphosphorylation and may be operative in vivo.

Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies

PubMed Central

Buchmueller, Karen L.; Weeks, Kevin M.

2004-01-01

Native polyacrylamide gel electrophoresis is a powerful approach for visualizing RNA folding states and folding intermediates. Tris-borate has a high-buffering capacity and is therefore widely used in electrophoresis-based investigations of RNA structure and folding. However, the effectiveness of Tris-borate as a counterion for RNA has not been systematically investigated. In a recirculated Hepes/KCl buffer, the catalytic core of the bI5 group I intron RNA undergoes a conformational collapse characterized by a bulk transition midpoint, or Mg1/2, of ∼3 mM, consistent with extensive independent biochemical experiments. In contrast, in Tris-borate, RNA collapse has a much smaller apparent Mg1/2, equal to 0.1 mM, because in this buffer the RNA undergoes a different, large amplitude, folding transition at low Mg2+ concentrations. Analysis of structural neighbors using a short-lived, RNA-tethered, photocrosslinker indicates that the global RNA structure eventually converges in the two buffer systems, as the divalent ion concentration approaches ∼1 mM Mg2+. The weak capacity of Tris-borate to stabilize RNA folding may reflect relatively unfavorable interactions between the bulky Tris-borate ion and RNA or partial coordination of RNA functional groups by borate. Under some conditions, Tris-borate is a poor counterion for RNA and its use merits careful evaluation in RNA folding studies. PMID:15601995

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

Native characterization of nucleic acid motif thermodynamics via non-covalent catalysis

PubMed Central

Wang, Chunyan; Bae, Jin H.; Zhang, David Yu

2016-01-01

DNA hybridization thermodynamics is critical for accurate design of oligonucleotides for biotechnology and nanotechnology applications, but parameters currently in use are inaccurately extrapolated based on limited quantitative understanding of thermal behaviours. Here, we present a method to measure the ΔG° of DNA motifs at temperatures and buffer conditions of interest, with significantly better accuracy (6- to 14-fold lower s.e.) than prior methods. The equilibrium constant of a reaction with thermodynamics closely approximating that of a desired motif is numerically calculated from directly observed reactant and product equilibrium concentrations; a DNA catalyst is designed to accelerate equilibration. We measured the ΔG° of terminal fluorophores, single-nucleotide dangles and multinucleotide dangles, in temperatures ranging from 10 to 45 °C. PMID:26782977

Thermodynamic Bounds on the Ultra- and Infra-affinity of Hsp70 for Its Substrates

NASA Astrophysics Data System (ADS)

Nguyen, Basile; Hartich, David; Seifert, Udo; Rios, Paolo De Los

2017-07-01

The 70 kDa Heat Shock Proteins Hsp70 have several essential functions in living systems, such as protecting cells against protein aggregation, assisting protein folding, remodeling protein complexes and driving the translocation into organelles. These functions require high affinity for non-specific amino-acid sequences that are ubiquitous in proteins. It has been recently shown that this high affinity, called ultra-affinity, depends on a process driven out of equilibrium by ATP hydrolysis. Here we establish the thermodynamic bounds for ultra-affinity, and further show that the same reaction scheme can in principle be used both to strengthen and to weaken affinities (leading in this case to infra-affinity). We show that cofactors are essential to achieve affinity beyond the equilibrium range. Finally, biological implications are discussed.

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.

Comparison of rate constants for (PO3-) transfer by the Mg(II), Cd(II), and Li(I) forms of phosphoglucomutase.

PubMed

Ray, W J; Post, C B; Puvathingal, J M

1989-01-24

Net rate constants that define the steady-state rate through a sequence of steps and the corresponding effective energy barriers for two (PO3-)-transfer steps in the phosphoglucomutase reaction were compared as a function of metal ion, M, where M = Mg2+ and Cd2+. These steps involve the reaction of either the 1-phosphate or the 6-phosphate of glucose 1,6-bisphosphate (Glc-P2) bound to the dephosphoenzyme (ED) to produce the phosphoenzyme (EP) and the free monophosphates, glucose 1-phosphate (Glc-1-P) or glucose 6-phosphate (Glc-6-P): EP.M + Glc-1-P----ED.M.Glc-P2----EP.M.Glc-6-P6. Before this comparison was made, net rate constants for the Cd2+ enzyme, obtained at high enzyme concentration via 31P NMR saturation-transfer studies [Post, C. B., Ray, W. J., Jr., & Gorenstein, D. G. (1989) Biochemistry (preceding paper in this issue)], were appropriately scaled by using the observed constants to calculate both the expected isotope-transfer rate at equilibrium and the steady-state rate under initial velocity conditions and comparing the calculated values with those measured in dilute solution. For the Mg2+ enzyme, narrow limits on possible values of the corresponding net rate constants were imposed on the basis of initial velocity rate constants for the forward and reverse directions plus values for the equilibrium distribution of central complexes, since direct measurement is not feasible. The effective energy barriers for both the Mg2+ and Cd2+ enzymes, calculated from the respective net rate constants, together with previously values for the equilibrium distribution of complexes in both enzymic systems [Ray, W. J., Jr., & Long, J. W. (1976) Biochemistry 15, 4018-4025], show that the 100-fold decrease in the kappa cat for the Cd2+ relative to the Mg2+ enzyme is caused by two factors: the increased stability of the intermediate bisphosphate complex and the decreased ability to cope with the phosphate ester involving the 1-hydroxyl group of the glucose ring. In fact, it is unlikely that the efficiency of (PO3-) transfer to the 6-hydroxyl group of bound Glc-1-P (thermodynamically favorable direction) is reduced by more than an order of magnitude in the Cd2+ enzyme. By contrast, the efficiency of the Li+ enzyme in the same (PO3-)-transfer step is less than 4 x 10(-8) that of the Mg2+ enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterisation of transition state structures for protein folding using 'high', 'medium' and 'low' {Phi}-values.

PubMed

Geierhaas, Christian D; Salvatella, Xavier; Clarke, Jane; Vendruscolo, Michele

2008-03-01

It has been suggested that Phi-values, which allow structural information about transition states (TSs) for protein folding to be obtained, are most reliably interpreted when divided into three classes (high, medium and low). High Phi-values indicate almost completely folded regions in the TS, intermediate Phi-values regions with a detectable amount of structure and low Phi-values indicate mostly unstructured regions. To explore the extent to which this classification can be used to characterise in detail the structure of TSs for protein folding, we used Phi-values divided into these classes as restraints in molecular dynamics simulations. This type of procedure is related to that used in NMR spectroscopy to define the structure of native proteins from the measurement of inter-proton distances derived from nuclear Overhauser effects. We illustrate this approach by determining the TS ensembles of five proteins and by showing that the results are similar to those obtained by using as restraints the actual numerical Phi-values measured experimentally. Our results indicate that the simultaneous consideration of a set of low-resolution Phi-values can provide sufficient information for characterising the architecture of a TS for folding of a protein.

Characterisation of transition state structures for protein folding using ‘high’, ‘medium’ and ‘low’ Φ-values

PubMed Central

Geierhaas, Christian D.; Salvatella, Xavier; Clarke, Jane; Vendruscolo, Michele

2008-01-01

It has been suggested that Φ-values, which allow structural information about transition states (TSs) for protein folding to be obtained, are most reliably interpreted when divided into three classes (high, medium and low). High Φ-values indicate almost completely folded regions in the TS, intermediate Φ-values regions with a detectable amount of structure and low Φ-values indicate mostly unstructured regions. To explore the extent to which this classification can be used to characterise in detail the structure of TSs for protein folding, we used Φ-values divided into these classes as restraints in molecular dynamics simulations. This type of procedure is related to that used in NMR spectroscopy to define the structure of native proteins from the measurement of inter-proton distances derived from nuclear Overhauser effects. We illustrate this approach by determining the TS ensembles of five proteins and by showing that the results are similar to those obtained by using as restraints the actual numerical Φ-values measured experimentally. Our results indicate that the simultaneous consideration of a set of low-resolution Φ-values can provide sufficient information for characterising the architecture of a TS for folding of a protein. PMID:18299294

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

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.

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.

Unfolding single RNA molecules: bridging the gap between equilibrium and non-equilibrium statistical thermodynamics.

PubMed

Bustamante, Carlos

2005-11-01

During the last 15 years, scientists have developed methods that permit the direct mechanical manipulation of individual molecules. Using this approach, they have begun to investigate the effect of force and torque in chemical and biochemical reactions. These studies span from the study of the mechanical properties of macromolecules, to the characterization of molecular motors, to the mechanical unfolding of individual proteins and RNA. Here I present a review of some of our most recent results using mechanical force to unfold individual molecules of RNA. These studies make it possible to follow in real time the trajectory of each molecule as it unfolds and characterize the various intermediates of the reaction. Moreover, if the process takes place reversibly it is possible to extract both kinetic and thermodynamic information from these experiments at the same time that we characterize the forces that maintain the three-dimensional structure of the molecule in solution. These studies bring us closer to the biological unfolding processes in the cell as they simulate in vitro, the mechanical unfolding of RNAs carried out in the cell by helicases. If the unfolding process occurs irreversibly, I show here that single-molecule experiments can still provide equilibrium, thermodynamic information from non-equilibrium data by using recently discovered fluctuation theorems. Such theorems represent a bridge between equilibrium and non-equilibrium statistical mechanics. In fact, first derived in 1997, the first experimental demonstration of the validity of fluctuation theorems was obtained by unfolding mechanically a single molecule of RNA. It is perhaps a sign of the times that important physical results are these days used to extract information about biological systems and that biological systems are being used to test and confirm fundamental new laws in physics.

The effects of intraspecific competition and stabilizing selection on a polygenic trait.

PubMed Central

Bürger, Reinhard; Gimelfarb, Alexander

2004-01-01

The equilibrium properties of an additive multilocus model of a quantitative trait under frequency- and density-dependent selection are investigated. Two opposing evolutionary forces are assumed to act: (i) stabilizing selection on the trait, which favors genotypes with an intermediate phenotype, and (ii) intraspecific competition mediated by that trait, which favors genotypes whose effect on the trait deviates most from that of the prevailing genotypes. Accordingly, fitnesses of genotypes have a frequency-independent component describing stabilizing selection and a frequency- and density-dependent component modeling competition. We study how the equilibrium structure, in particular, number, degree of polymorphism, and genetic variance of stable equilibria, is affected by the strength of frequency dependence, and what role the number of loci, the amount of recombination, and the demographic parameters play. To this end, we employ a statistical and numerical approach, complemented by analytical results, and explore how the equilibrium properties averaged over a large number of genetic systems with a given number of loci and average amount of recombination depend on the ecological and demographic parameters. We identify two parameter regions with a transitory region in between, in which the equilibrium properties of genetic systems are distinctively different. These regions depend on the strength of frequency dependence relative to pure stabilizing selection and on the demographic parameters, but not on the number of loci or the amount of recombination. We further study the shape of the fitness function observed at equilibrium and the extent to which the dynamics in this model are adaptive, and we present examples of equilibrium distributions of genotypic values under strong frequency dependence. Consequences for the maintenance of genetic variation, the detection of disruptive selection, and models of sympatric speciation are discussed. PMID:15280253

Solvent Isotope-induced Equilibrium Perturbation for Isocitrate Lyase

PubMed Central

Quartararo, Christine E.; Hadi, Timin; Cahill, Sean M.; Blanchard, John S.

2014-01-01

Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacteria’s life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage of D2OV = 2.0 ± 0.1 and D2O[V/Kisocitrate] = 2.2 ± 0.3 arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate and succinate prepared in D2O, would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of NMR spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by 1H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed di-deuterated succinate, protiated glyoxylate, and mono-deuterated isocitrate, with the transient appearance and disappearance of mono-deuterated succinate. A model for the equilibrium perturbation of substrate species, and their time-dependent isotopic composition is presented. PMID:24261638

Kinetic theory for DNA melting with vibrational entropy

NASA Astrophysics Data System (ADS)

Sensale, Sebastian; Peng, Zhangli; Chang, Hsueh-Chia

2017-10-01

By treating DNA as a vibrating nonlinear lattice, an activated kinetic theory for DNA melting is developed to capture the breakage of the hydrogen bonds and subsequent softening of torsional and bending vibration modes. With a coarse-grained lattice model, we identify a key bending mode with GHz frequency that replaces the hydrogen vibration modes as the dominant out-of-phase phonon vibration at the transition state. By associating its bending modulus to a universal in-phase bending vibration modulus at equilibrium, we can hence estimate the entropic change in the out-of-phase vibration from near-equilibrium all-atom simulations. This and estimates of torsional and bending entropy changes lead to the first predictive and sequence-dependent theory with good quantitative agreement with experimental data for the activation energy of melting of short DNA molecules without intermediate hairpin structures.

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

Knapp, R.J.; Sharma, S.D.; Toth, G.

(D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sitesmore » in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.« less

Studies of the Initial Stages of Epitaxial Growth of Germanium on Silicon

NASA Astrophysics Data System (ADS)

Krishnamurthy, Mohan

The epitaxial growth of ultra-thin films (< 1nm thick) of Ge on Si(100) has been studied in -situ in an Ultra High Vacuum-Scanning Transmission Electron Microscope. Ge was deposited on clean Si(100) using molecular beam techniques to study two types of growth processes, Molecular Beam Epitaxy (MBE) and Solid Phase MBE. Ge grows in the Stranski-Krastanov growth mode, forming islands after initial layer growth. This islanding transition has been studied with high spatial resolution biassed Secondary Electron Imaging and Auger spectroscopy and imaging. Ex -situ Transmission Electron Microscopy (TEM) and Reflection High Energy Diffraction (RHEED) were also used to characterize the transition. The islanding process and its subsequent evolution was monitored with the help of island size distributions, sensitive to islands above 2nm in size. The studies indicate that Ge forms islands in equilibrium with a 3 monolayer (ML) thick intermediate layer. These islands may initially grow coherently strained (dislocation free) with radii usually below 10nm under the conditions. The strain in these islands reduces the adatom sticking coefficient and strongly influences the microstructural evolution. The intermediate layer may grow metastably under certain conditions to as much as 7 ML before collapsing to its equilibrium form. The influence of three types of adatom sinks--strained islands, dislocated islands and contaminant particles have been studied. The contaminant particles are the strongest sinks, followed by dislocated islands and strained islands. Stepped (vicinal) surfaces (1^circ and 5 ^circ toward {110 }) had no significant influence possibly due to the steps being weak adatom sinks. The coarsening of Ge islands does not follow the Ostwald ripening model at the early stages and is influenced by the supersaturation in the intermediate layer and the strain in the coherent islands. A novel mechanism has been observed, where the larger (dislocated) islands grow at the expense of the unstable intermediate layer while the distribution of smaller (strained) islands is constant. This is possibly due to the lower sticking coefficient at the strained islands.

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

Kojima, S.; Yokosawa, M.; Matsuyama, M.

To study the practical application of a tritium separation process using Self-Developing Gas Chromatography (SDGC) using a Pd-Pt alloy, intermediate scale-up experiments (22 mm ID x 2 m length column) and the development of a computational simulation method have been conducted. In addition, intermediate scale production of Pd-Pt powder has been developed for the scale-up experiments.The following results were obtained: (1) a 50-fold scale-up from 3 mm to 22 mm causes no significant impact on the SDGC process; (2) the Pd-Pt alloy powder is applicable to a large size SDGC process; and (3) the simulation enables preparation of a conceptualmore » design of a SDGC process for tritium separation.« less

Dynamic study of sub-micro sized LiFePO4 cathodes by in-situ tender X-ray absorption near edge structure

NASA Astrophysics Data System (ADS)

Wang, Dongniu; Wang, Huixin; Yang, Jinli; Zhou, Jigang; Hu, Yongfeng; Xiao, Qunfeng; Fang, Haitao; Sham, Tsun-Kong

2016-01-01

Olivine-type phosphates (LiMPO4, M = Fe, Mn, Co) are promising cathode materials for lithium-ion batteries that are generally accepted to follow first order equilibrium phase transformations. Herein, the phase transformation dynamics of sub-micro sized LiFePO4 particles with limited rate capability at a low current density of 0.14 C was investigated. An in-situ X-ray Absorption Near Edge Structure (XANES) measurement was conducted at the Fe and P K-edge for the dynamic studies upon lithiation and delithiation. Fe K-edge XANES spectra demonstrate that not only lithium-rich intermediate phase LixFePO4 (x = 0.6-0.75), but also lithium-poor intermediate phase LiyFePO4 (y = 0.1-0.25) exist during the charge and discharge, respectively. Furthermore, during charge and discharge, a fluctuation of the FePO4 and LiFePO4 fractions obtained by liner combination fitting around the imaginary phase fractions followed Faraday's law and the equilibrium first-order two-phase transformation versus reaction time is present, respectively. The charging and discharging process has a reversible phase transformation dynamics with symmetric structural evolution routes. P K-edge XANES spectra reveal an enrichment of PF6-1 anions at the surface of the electrode during charging.

Structural origins of pH and ionic strength effects on protein stability. Acid denaturation of sperm whale apomyoglobin.

PubMed

Yang, A S; Honig, B

1994-04-15

A recently developed approach to calculate the pH dependence of protein stability from three-dimensional structure information is applied to the analysis of acid denaturation of sperm whale apomyoglobin. The finite difference Poisson-Boltzmann method is used to calculate pKa values and these are used to obtain titration curves for the folded protein as well as for compact intermediates. The total electrostatic free energy change involved in apomyoglobin unfolding is then evaluated. Calculations are carried out of the unfolding free energy of the native (N) and the compact intermediate (I) of apomyoglobin relative to the unfolded state (U) over a range of pH at various ionic strengths. The contributions from key ionizable groups to the unfolding process are discussed. For the acid-induced partial unfolding of apomyoglobin near pH 5, the transition from N to I is found to be driven by three histidines that are exposed when the B, C, D and E helices unfold. Similarly, the unfolding of the compact intermediate I consisting of the A, G and H helices is driven primarily by a few carboxylic acids with low pKa values in the compact state. This picture is in contrast to the view which attributes acid denaturation to electrostatic repulsion resulting from the build up of positive charge. In fact, charge-charge interactions in myoglobin are found to be attractive at all pH values where the protein unfolds. pH-dependent changes in these interactions contribute to acid denaturation but other electrostatic effects, such as hydrogen bonding and solvation, are important as well. The effect of increasing ionic strength on unfolding is attributed to the decrease of attractive charge-charge interactions which destabilize the N state relative to I, but stabilize the I state relative to U by reducing the pKa shifts of a few critical carboxylic acids. The I state is found to be more stable than U at neutral pH thus accounting for its presence as an intermediate on the protein folding pathway. Our results have implications for the origins of compact intermediates or "molten globule" states.

Effect of light on the kinetics and equilibrium of the textile dye (Reactive Red 120) adsorption by Helianthus annuus hairy roots.

PubMed

Srikantan, Chitra; Suraishkumar, G K; Srivastava, Smita

2018-06-01

The study demonstrates for the first time that light influences the adsorption equilibrium and kinetics of a dye by root culture system. The azo dye (Reactive Red 120) adsorption by the hairy roots of H. annuus followed a pseudo first-order kinetic model and the adsorption equilibrium parameters were best estimated using Langmuir isotherm. The maximum dye adsorption capacity of the roots increased 6-fold, from 0.26 mg g -1 under complete dark conditions to 1.51 mg g -1 under 16/8 h light/dark photoperiod. Similarly, adsorption rate of the dye and removal (%) also increased in the presence of light, irrespective of the initial concentration of the dye (20-110 mg L -1 ). The degradation of the azo dye upon adsorption by the hairy roots of H. annuus was also confirmed. In addition, a strategy for simultaneous dye removal and increased alpha-tocopherol (industrially relevant) production by H. annuus hairy root cultures has been proposed and demonstrated. Copyright © 2018 Elsevier Ltd. All rights reserved.

Internal friction and nonequilibrium unfolding of polymeric globules.

PubMed

Alexander-Katz, Alfredo; Wada, Hirofumi; Netz, Roland R

2009-07-10

The stretching response of a single collapsed homopolymer is studied using Brownian dynamic simulations. The irreversibly dissipated work is found to be dominated by internal friction effects below the collapse temperature, and the internal viscosity grows exponentially with the effective cohesive strength between monomers. These results explain friction effects of globular DNA and are relevant for dissipation at intermediate stages of protein folding.

Novel Inhibitor Cystine Knot Peptides from Momordica charantia

PubMed Central

Clark, Richard J.; Tang, Jun; Zeng, Guang-Zhi; Franco, Octavio L.; Cantacessi, Cinzia; Craik, David J.; Daly, Norelle L.; Tan, Ning-Hua

2013-01-01

Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature. PMID:24116036

Can misfolded proteins be beneficial? The HAMLET case.

PubMed

Pettersson-Kastberg, Jenny; Aits, Sonja; Gustafsson, Lotta; Mossberg, Anki; Storm, Petter; Trulsson, Maria; Persson, Filip; Mok, K Hun; Svanborg, Catharina

2009-01-01

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

Microsecond simulations of the folding/unfolding thermodynamics of the Trp-cage mini protein

PubMed Central

Day, Ryan; Paschek, Dietmar; Garcia, Angel E.

2012-01-01

We study the unbiased folding/unfolding thermodynamics of the Trp-cage miniprotein using detailed molecular dynamics simulations of an all-atom model of the protein in explicit solvent, using the Amberff99SB force field. Replica-exchange molecular dynamics (REMD) simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states, and at two densities. The obtained ensembles are shown to reach equilibrium in the 1 μs per replica timescale. The total simulation time employed in the calculations exceeds 100 μs. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ΔG(P,T), over the whole region of temperature and pressures sampled in the simulations. The ΔG(P,T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (Tf = 321 K), free energy and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semi-empirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins. PMID:20408169

Voice rest after vocal fold surgery: current practice and evidence.

PubMed

Coombs, A C; Carswell, A J; Tierney, P A

2013-08-01

Voice rest is commonly recommended after vocal fold surgery, but there is a lack of evidence base and no standard protocol. The aim of this study was to establish common practice regarding voice rest following vocal fold surgery. An online survey was circulated via e-mail invitation to members of the ENT UK Expert Panel between October and November 2011. The survey revealed that 86.5 per cent of respondents agreed that 'complete voice rest' means no sound production at all, but there was variability in how 'relative voice rest' was defined. There was no dominant type of voice rest routinely recommended after surgery for laryngeal papillomatosis or intermediate pathologies. There was considerable variability in the duration of voice rest recommended, with no statistically significant, most popular response (except for malignant lesions). Surgeons with less than 10 years of experience were more likely to recommend fewer days of voice rest. There is a lack of consistency in advice given to patients after vocal fold surgery, in terms of both type and length of voice rest. This may arise from an absence of robust evidence on which to base practice.

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.

Early events in the folding of an amphipathic peptide: A multinanosecond molecular dynamics study

NASA Technical Reports Server (NTRS)

Chipot, C.; Maigret, B.; Pohorille, A.

1999-01-01

Folding of the capped LQQLLQQLLQL peptide is investigated at the water-hexane interface by molecular dynamics simulations for 161.5 ns. Initially placed in the aqueous phase as a beta-strand, the peptide rapidly adsorbs to the interface, where it adopts an amphipathic conformation. The marginal presence of nonamphipathic structures throughout the complete trajectory indicates that the corresponding conformations are strongly disfavored at the interface. It is further suggestive that folding in an interfacial environment proceeds through a pathway of successive amphipathic intermediates. The energetic and entropic penalties involved in the conformational changes along this pathway markedly increase the folding time scales of LQQLLQQLLQL, explaining why the alpha-helix, the hypothesized lowest free energy structure for a sequence with a hydrophobic periodicity of 3.6, has not been reached yet. The formation of a type I beta-turn at the end of the simulation confirms the importance of such motifs as initiation sites allowing the peptide to coalesce towards a secondary structure. Proteins 1999;36:383-399. Copyright 1999 Wiley-Liss, Inc.

Biomolecular dynamics studied with IR-spectroscopy using quantum cascade lasers combined with nanosecond perturbation techniques

NASA Astrophysics Data System (ADS)

Popp, Alexander; Scheerer, David; Heck, Benjamin; Hauser, Karin

2017-06-01

Early events of protein folding can be studied with fast perturbation techniques triggering non-equilibrium relaxation dynamics. A nanosecond laser-excited pH-jump or temperature-jump (T-jump) was applied to initiate helix folding or unfolding of poly-L-glutamic acid (PGA). PGA is a homopolypeptide with titratable carboxyl side-chains whose protonation degree determines the PGA conformation. A pH-jump was realized by the photochemical release of protons and induces PGA folding due to protonation of the side-chains. Otherwise, the helical conformation can be unfolded by a T-jump. We operated under conditions where PGA does not aggregate and temperature and pH are the regulatory properties of its conformation. The experiments were performed in such a manner that the folding/unfolding jump proceeded to the same PGA conformation. We quantified the increase/decrease in helicity induced by the pH-/T-jump and demonstrated that the T-jump results in a relatively small change in helical content in contrast to the pH-jump. This is caused by the strong pH-dependence of the PGA conformation. The conformational changes were detected by time-resolved single wavelength IR-spectroscopy using quantum cascade lasers (QCL). We could independently observe the kinetics for α-helix folding and unfolding in PGA by using different perturbation techniques and demonstrate the high sensitivity of time-resolved IR-spectroscopy to study protein folding mechanisms.