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.

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.

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.

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.

Rough energy landscapes in protein folding: dimeric E. coli Trp repressor folds through three parallel channels.

PubMed

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

2001-10-05

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

Folding of 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

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.

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.

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

Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates

PubMed Central

Bhat, Shreyas; Hasenhuetl, Peter S.; Kasture, Ameya; El-Kasaby, Ali; Baumann, Michael H.; Blough, Bruce E.; Sucic, Sonja; Sandtner, Walter; Freissmuth, Michael

2017-01-01

Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na+ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to μm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-601PG602-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants. PMID:28842491

Defining the Nature of Thermal Intermediate in 3 State Folding Proteins: Apoflavodoxin, a Study Case

PubMed Central

García-Fandiño, Rebeca; Bernadó, Pau; Ayuso-Tejedor, Sara; Sancho, Javier; Orozco, Modesto

2012-01-01

The early stages of the thermal unfolding of apoflavodoxin have been determined by using atomistic multi microsecond-scale molecular dynamics (MD) simulations complemented with a variety of experimental techniques. Results strongly suggest that the intermediate is reached very early in the thermal unfolding process and that it has the properties of an “activated” form of the native state, where thermal fluctuations in the loops break loop-loop contacts. The unrestrained loops gain then kinetic energy corrupting short secondary structure elements without corrupting the core of the protein. The MD-derived ensembles agree with experimental observables and draw a picture of the intermediate state inconsistent with a well-defined structure and characteristic of a typical partially disordered protein. Our results allow us to speculate that proteins with a well packed core connected by long loops might behave as partially disordered proteins under native conditions, or alternatively behave as three state folders. Small details in the sequence, easily tunable by evolution, can yield to one or the other type of proteins. PMID:22927805

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.

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.

Mutational Analysis of the Stability of the H2A and H2B Histone Monomers

PubMed Central

Stump, Matthew R.; Gloss, Lisa M.

2008-01-01

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol−1, respectively; at 10 μM, the sum of the stability of the monomers is ~60% of the stability of the native dimer. The helical content, stability and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly-collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive inter-monomer contacts, is structured in H2B but only partially folded in H2A. PMID:18976667

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

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.

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.

Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule

PubMed Central

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

2014-01-01

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

Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule.

PubMed

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

2014-10-14

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

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

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.

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.

Folding of the four-helix bundle FF domain from a compact on-pathway intermediate state is governed predominantly by water motion.

PubMed

Sekhar, Ashok; Vallurupalli, Pramodh; Kay, Lewis E

2012-11-20

Friction plays a critical role in protein folding. Frictional forces originating from random solvent and protein fluctuations both retard motion along the folding pathway and activate protein molecules to cross free energy barriers. Studies of friction thus may provide insights into the driving forces underlying protein conformational dynamics. However, the molecular origin of friction in protein folding remains poorly understood because, with the exception of the native conformer, there generally is little detailed structural information on the other states participating in the folding process. Here, we study the folding of the four-helix bundle FF domain that proceeds via a transiently formed, sparsely populated compact on-pathway folding intermediate whose structure was elucidated previously. Because the intermediate is stabilized by both native and nonnative interactions, friction in the folding transition between intermediate and folded states is expected to arise from intrachain reorganization in the protein. However, the viscosity dependencies of rates of folding from or unfolding to the intermediate, as established by relaxation dispersion NMR spectroscopy, clearly indicate that contributions from internal friction are small relative to those from solvent, so solvent frictional forces drive the folding process. Our results emphasize the importance of solvent dynamics in mediating the interconversion between protein configurations, even those that are highly compact, and in equilibrium folding/unfolding fluctuations in general.

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.

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

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

An overlapping region between the two terminal folding units of the outer surface protein A (OspA) controls its folding behavior.

PubMed

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

2018-04-27

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

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.

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.

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

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

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments.

PubMed

Mizukami, Takuya; Abe, Yukiko; Maki, Kosuke

2015-01-01

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

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.

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

Folding thermodynamics of pseudoknotted chain conformations

PubMed Central

Kopeikin, Zoia; Chen, Shi-Jie

2008-01-01

We develop a statistical mechanical framework for the folding thermodynamics of pseudoknotted structures. As applications of the theory, we investigate the folding stability and the free energy landscapes for both the thermal and the mechanical unfolding of pseudoknotted chains. For the mechanical unfolding process, we predict the force-extension curves, from which we can obtain the information about structural transitions in the unfolding process. In general, a pseudoknotted structure unfolds through multiple structural transitions. The interplay between the helix stems and the loops plays an important role in the folding stability of pseudoknots. For instance, variations in loop sizes can lead to the destabilization of some intermediate states and change the (equilibrium) folding pathways (e.g., two helix stems unfold either cooperatively or sequentially). In both thermal and mechanical unfolding, depending on the nucleotide sequence, misfolded intermediate states can emerge in the folding process. In addition, thermal and mechanical unfoldings often have different (equilibrium) pathways. For example, for certain sequences, the misfolded intermediates, which generally have longer tails, can fold, unfold, and refold again in the pulling process, which means that these intermediates can switch between two different average end-end extensions. PMID:16674261

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

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

A small molecule chemical chaperone optimizes its unfolded state contraction and denaturant like properties

NASA Astrophysics Data System (ADS)

Sharma, Sunny; Sarkar, Suparna; Paul, Simanta Sarani; Roy, Syamal; Chattopadhyay, Krishnananda

2013-12-01

Protein aggregation is believed to occur through the formation of misfolded conformations. It is expected that, in order to minimize aggregation, an effective small molecule chaperone would destabilize these intermediates. To study the mechanism of a chemical chaperone, we have designed a series of mutant proteins in which a tryptophan residue experiences different local environments and solvent exposures. We show that these mutants correspond to a series of conformationally altered proteins with varying degree of misfolding stress and aggregation propensities. Using arginine as a model small molecule, we show that a combination of unfolded state contraction and denaturant like properties results in selective targeting and destabilization of the partially folded proteins. In comparison, the effect of arginine towards the folded like control mutant, which is not aggregation prone, is significantly less. Other small molecules, lacking either of the above two properties, do not offer any specificity towards the misfolded proteins.

Refolding of urea-denatured α-chymotrypsin by protein-folding liquid chromatography.

PubMed

Congyu, Ke; Wujuan, Sun; Qunzheng, Zhang; Xindu, Geng

2013-04-01

An approach for re-folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, α-chymotrypsin (α-Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different α-Chy states - urea-denatured (U state), its folded intermediates (M state) and nature state (N state) - were studied during protein folding. Based on the test by matrix-assisted laser desorption/ionization time of flight mass spectrometry and bioactivity, only one stable M state of the α-Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded α-Chy was found to be higher than that of commercial α-Chy as the urea concentration in the sample solution ranged from 1.0 to 3.0 m; the highest specific bioactivity at urea concentration was 1.0 m, indicating the possibility for re-folding some proteins that have partially or completely lost their bioactivity, as a dilute urea solution was employed for dissolving the sample. The experiment showed that the peak height of its M state increased with increasing urea concentration, and correspondingly decreased in the amount of the refolded α-Chy. When the urea concentration reached 6.0 m, the unfolded α-Chy could not be refolded at all. Copyright © 2012 John Wiley & Sons, Ltd.

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

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

PubMed Central

Mizukami, Takuya; Abe, Yukiko; Maki, Kosuke

2015-01-01

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

A sequential assignment procedure for proteins that have intermediate line widths in MAS NMR spectra: amyloid fibrils of human CA150.WW2.

PubMed

Becker, Johanna; Ferguson, Neil; Flinders, Jeremy; van Rossum, Barth-Jan; Fersht, Alan R; Oschkinat, Hartmut

2008-08-11

The second WW domain (WW2) of CA150, a human transcriptional activator, forms amyloid fibrils in vitro under physiological conditions. Based on experimental constraints from MAS NMR spectroscopy experiments, alanine scanning and electron microscopy, a structural model of CA150.WW2 amyloid fibrils was calculated earlier. Here, the assignment strategy is presented and suggested as a general approach for proteins that show intermediate line width. The (13)C,(13)C correlation experiments were recorded on fully or partially (13)C-labelled fibrils. The earlier (13)C assignment (26 residues) was extended to 34 of the 40 residues by direct (13)C-excitation experiments by using a deuterated sample that showed strongly improved line width. A 3D HNC-TEDOR (transferred-echo double-resonance) experiment with deuterated CA150.WW2 fibrils yielded 14 amide nitrogen and proton resonance assignments. The obtained chemical shifts were compared with the chemical shifts determined with the natively folded WW domain. TALOS (Torsion angle likelihood obtained from shift and sequence similarity) predictions confirmed that, under physiological conditions, the fibrillar form of CA150.WW2 adopts a significantly different beta structure than the native WW-domain fold.

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

Correlating folding and signaling in a photoreceptor by single molecule measurements and energy landscape calculations

NASA Astrophysics Data System (ADS)

Hoff, Wouter

2007-03-01

Receptor activation is a fundamental process in biological signaling. We study the structural changes during activation of photoactive yellow protein (PYP). This is triggered by photoisomerization of the p-coumaric acid (pCA) chromophore of PYP, which converts the initial pG state into the activated pB state. Mechanical unfolding of Cys-linked PYP multimers probed by atomic force microscopy (AFM) in the presence and absence of illumination reveals that the core of the protein is extended by 3 nm and destabilized by 30 percent in pB. These results establish a generally applicable single molecule approach for mapping functional conformational changes to selected regions of a protein and indicate that stimulus-induced partial protein unfolding can be employed as a signaling mechanism. Comparative measurements, Jarzynski-Hummer-Szabo analysis of the data, and steered MD simulations of two double-Cys PYP mutants reveal strong anisotropy in the unfolding mechanism along the two axes defined by the Cys residues. Unfolding along one axis exhibits a transition-state-like feature where six hydrogen bonds break simultaneously. The other axis displays an unpeaked force profile reflecting a non-cooperative transition, challenging the notion that cooperative unfolding is a universal feature in protein stability. MD simulations with a coarse-grained protein model show that the folding of pG is two-state, consistent with experimental observations. In contrast, the folding free energy surface of a coarse-grained model of pB involves an on-pathway partially unfolded intermediate that closely matches experimental data. The results reveal that interactions between the pCA and its binding pocket can switch the energy landscape for PYP from two- to three-state folding, and show how this can be exploited to trigger large functionally important protein conformational changes.

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

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

PubMed Central

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

2004-01-01

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

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.

The nucleotide-free state of heterotrimeric G proteins α-subunit adopts a highly stable conformation.

PubMed

Andhirka, Sai Krishna; Vignesh, Ravichandran; Aradhyam, Gopala Krishna

2017-08-01

Deciphering the mechanism of activation of heterotrimeric G proteins by their cognate receptors continues to be an intriguing area of research. The recently solved crystal structure of the ternary complex captured the receptor-bound α-subunit in an open conformation, without bound nucleotide has improved our understanding of the activation process. Despite these advancements, the mechanism by which the receptor causes GDP release from the α-subunit remains elusive. To elucidate the mechanism of activation, we studied guanine nucleotide-induced structural stability of the α-subunit (in response to thermal/chaotrope-mediated stress). Inherent stabilities of the inactive (GDP-bound) and active (GTP-bound) forms contribute antagonistically to the difference in conformational stability whereas the GDP-bound protein is able to switch to a stable intermediate state, GTP-bound protein loses this ability. Partial perturbation of the protein fold reveals the underlying influence of the bound nucleotide providing an insight into the mechanism of activation. An extra stable, pretransition intermediate, 'empty pocket' state (conformationally active-state like) in the unfolding pathway of GDP-bound protein mimics a gating system - the activation process having to overcome this stable intermediate state. We demonstrate that a relatively more complex conformational fold of the GDP-bound protein is at the core of the gating system. We report capturing this threshold, 'metastable empty pocket' conformation (the gate) of α-subunit of G protein and hypothesize that the receptor activates the G protein by enabling it to achieve this structure through mild structural perturbation. © 2017 Federation of European Biochemical Societies.

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

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.

Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry.

PubMed

Tran, Duc T; Banerjee, Sambuddha; Alayash, Abdu I; Crumbliss, Alvin L; Fitzgerald, Michael C

2012-02-07

Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C(2) proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin-haptoglobin (Hb-Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a K(d) value of 0.24 nM for the Hb-Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated in an experiment in which the protocol was used to detect the binding of zinc to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein's three-dimensional structure for successful application. © 2011 American Chemical Society

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

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.

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.

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.

Altering lipase activity and enantioselectivity in organic media using organo-soluble bases: Implication for rate-limiting proton transfer in acylation step.

PubMed

Chen, Chun-Chi; Chen, Teh-Liang; Tsai, Shau-Wei

2006-06-05

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl esters via a partially purified papaya lipase (PCPL) in water-saturated isooctane as the model system, the enzyme activity, and enantioselectivty is altered by adding a variety of organo-soluble bases that act as either enzyme activators (i.e., TEA, MP, TOA, DPA, PY, and DMA) or enzyme inhibitors (i.e., PDP, DMAP, and PP). Triethylamine (TEA) is selected as the best enzyme activator as 2.24-fold increase of the initial rate for the (S)-ester is obtained when adding 120 mM of the base. By using an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is the rate-limiting step, or more sensitive than that for the formation of tetrahedral intermediates when the enzyme activators of different pKa are added. However, no correlation for the proton transfers in the acylation step is found when adding the bases acting as enzyme deactivators. Copyright 2006 Wiley Periodicals, Inc.

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

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.

Single-molecule studies of the Im7 folding landscape.

PubMed

Pugh, Sara D; Gell, Christopher; Smith, D Alastair; Radford, Sheena E; Brockwell, David J

2010-04-23

Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I(eqm)) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I(eqm) variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na(2)SO(4) in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding. (c) 2010 Elsevier Ltd. All rights reserved.

Single-Molecule Studies of the Im7 Folding Landscape

PubMed Central

Pugh, Sara D.; Gell, Christopher; Smith, D. Alastair; Radford, Sheena E.; Brockwell, David J.

2010-01-01

Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted Ieqm) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the Ieqm variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na2SO4 in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding. PMID:20211187

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

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

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.

Elucidating quantitative stability/flexibility relationships within thioredoxin and its fragments using a distance constraint model.

PubMed

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

2006-05-05

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

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.

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.

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.

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

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 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.

Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

PubMed Central

Verespy III, Stephen; Mehta, Akul Y.; Afosah, Daniel; Al-Horani, Rami A.; Desai, Umesh R.

2016-01-01

Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (<500 nM) and high selectivity for thrombin (>150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80–100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding. PMID:27053426

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.

Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).

PubMed

Ciaccio, Natalie A; Reynolds, T Steele; Middaugh, C Russell; Laurence, Jennifer S

2012-11-05

Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation.

Enhanced picture of protein-folding intermediates using organic solvents in H/D exchange and quench-flow experiments

PubMed Central

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

2005-01-01

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

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

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.

Conditional deletion of WT1 in the septum transversum mesenchyme causes congenital diaphragmatic hernia in mice

PubMed Central

Carmona, Rita; Cañete, Ana; Cano, Elena; Ariza, Laura; Rojas, Anabel; Muñoz-Chápuli, Ramon

2016-01-01

Congenital diaphragmatic hernia (CDH) is a severe birth defect. Wt1-null mouse embryos develop CDH but the mechanisms regulated by WT1 are unknown. We have generated a murine model with conditional deletion of WT1 in the lateral plate mesoderm, using the G2 enhancer of the Gata4 gene as a driver. 80% of G2-Gata4Cre;Wt1fl/fl embryos developed typical Bochdalek-type CDH. We show that the posthepatic mesenchymal plate coelomic epithelium gives rise to a mesenchyme that populates the pleuroperitoneal folds isolating the pleural cavities before the migration of the somitic myoblasts. This process fails when Wt1 is deleted from this area. Mutant embryos show Raldh2 downregulation in the lateral mesoderm, but not in the intermediate mesoderm. The mutant phenotype was partially rescued by retinoic acid treatment of the pregnant females. Replacement of intermediate by lateral mesoderm recapitulates the evolutionary origin of the diaphragm in mammals. CDH might thus be viewed as an evolutionary atavism. DOI: http://dx.doi.org/10.7554/eLife.16009.001 PMID:27642710

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.

Probing the Non-Native H Helix Translocation in Apomyoglobin Folding Intermediates

PubMed Central

2015-01-01

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

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

Design and structure of an equilibrium protein folding intermediate: a hint into dynamical regions of proteins.

PubMed

Ayuso-Tejedor, Sara; Angarica, Vladimir Espinosa; Bueno, Marta; Campos, Luis A; Abián, Olga; Bernadó, Pau; Sancho, Javier; Jiménez, M Angeles

2010-07-23

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity. Copyright (c) 2010 Elsevier Ltd. 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

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

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

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.

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.

Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

PubMed Central

Wiech, Milena; Olszewski, Maciej B.; Tracz-Gaszewska, Zuzanna; Wawrzynow, Bartosz; Zylicz, Maciej; Zylicz, Alicja

2012-01-01

Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes. PMID:23251530

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

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

PubMed Central

Lee, Byoung-Chul; Hoff, Wouter D.

2008-01-01

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

Insights into the folding pathway of the Engrailed Homeodomain protein using replica exchange molecular dynamics simulations.

PubMed

Koulgi, Shruti; Sonavane, Uddhavesh; Joshi, Rajendra

2010-11-01

Protein folding studies were carried out by performing microsecond time scale simulations on the ultrafast/fast folding protein Engrailed Homeodomain (EnHD) from Drosophila melanogaster. It is a three-helix bundle protein consisting of 54 residues (PDB ID: 1ENH). The positions of the helices are 8-20 (Helix I), 26-36 (Helix II) and 40-53 (Helix III). The second and third helices together form a Helix-Turn-Helix (HTH) motif which belongs to the family of DNA binding proteins. The molecular dynamics (MD) simulations were performed using replica exchange molecular dynamics (REMD). REMD is a method that involves simulating a protein at different temperatures and performing exchanges at regular time intervals. These exchanges were accepted or rejected based on the Metropolis criterion. REMD was performed using the AMBER FF03 force field with the generalised Born solvation model for the temperature range 286-373 K involving 30 replicas. The extended conformation of the protein was used as the starting structure. A simulation of 600 ns per replica was performed resulting in an overall simulation time of 18 μs. The protein was seen to fold close to the native state with backbone root mean square deviation (RMSD) of 3.16 Å. In this low RMSD structure, the Helix I was partially formed with a backbone RMSD of 3.37 Å while HTH motif had an RMSD of 1.81 Å. Analysis suggests that EnHD folds to its native structure via an intermediate in which the HTH motif is formed. The secondary structure development occurs first followed by tertiary packing. The results were in good agreement with the experimental findings. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium[S

PubMed Central

Rudolph, Michael C.; Wellberg, Elizabeth A.; Lewis, Andrew S.; Terrell, Kristina L.; Merz, Andrea L.; Maluf, N. Karl; Serkova, Natalie J.; Anderson, Steven M.

2014-01-01

Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [13C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed. PMID:24771867

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.

Flame Spray Strain Gages with Improved Durability and Lifetimes

NASA Technical Reports Server (NTRS)

Fralick, Gustave (Technical Monitor); Gregory, Otto

2003-01-01

The focus of this APP research program was to improve the bond coats used in the fabrication of flame sprayed instrumentation. Typically. a bond coat is applied to a superalloy surface prior to the application of a thin dielectric coating onto which instrumentation is placed. After affixing the instrumentation, a much thicker ceramic topcoat is typically applied to protect the instrumentation from harsh environments. The fatigue life of NiCoCrAlY coated superalloys was extended beyond current state-of-the-art by relatively simple and cost effective means. Heat treatment in reduced oxygen partial pressures at 1750 to 1800 F effectively doubled the fatigue life of NiCoCrAlY coated substrates relative to as-sprayed substrates and when used in conjunction with platinum diffusion barriers yielded a four fold increase in the fatigue life of NiCoCrAlY coated substrates. Further improvements in the fatigue life of thermally sprayed coatings were made by employing intermediate coatings, which minimized thermal expansion differences between the bond coat and top coat. Combinatorial chemistry experiments yielded an optimum composition for an intermediate TCE matching coating that showed considerable promise in extending the fatigue life of thermal spray instrumentation. The intermediate coating had two functions: to reduce the surface roughness of the peaks and valleys associated with the as-sprayed NiCoCrAlY bond coat, and to produce a thin layer of a mixture of Al2O3 and NiCoCrAlY that exhibited an intermediate TCE. The optimal composition of the intermediate coating consisted of 60 wt% Al2O3 and 40 wt% NiCoCrAlY, as determined by energy dispersive analysis of x-rays (EDS). Intermediate coatings having this composition were prepared by physical vapor deposition and the resulting coating systems are being evaluated in our test facility.

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.

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.

Solubilization and partial purification of constituents of acyl-CoA elongase from Lunaria annua.

PubMed

Fehling, E; Lessire, R; Cassagne, C; Mukherjee, K D

1992-06-05

All the constituent enzymes of acyl-CoA elongase, i.e., beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase, have been solubilized from a 15,000 x g particulate fraction from developing seeds of honesty (Lunaria annua) using Triton X-100. All these activities were retained upon subsequent precipitation of the solubilized protein with polyethylene glycol and resuspension of the precipitate followed by ion exchange chromatography of the resulting protein on DEAE-cellulose. A 4.2-fold enrichment of the acyl-CoA elongase was thus obtained. Further chromatography of the DEAE fraction containing all the constituents of acyl-CoA elongase on Ultrogel yielded a major protein fraction exhibiting the activities of beta-ketoacyl-CoA synthase and beta-ketoacyl-CoA reductase only. Almost 30-fold purification of the beta-ketoacyl-CoA synthase was thus achieved. The beta-ketoacyl-CoA synthase was inhibited only at high concentrations of cerulenin, but at very low concentrations of iodoacetamide. Inhibition could be reduced by preincubation with thioesters, indicating that an enzyme thioester intermediate is involved in the condensation reaction of the acyl-CoA elongation.

Is Congo red an amyloid-specific dye?

PubMed

Khurana, R; Uversky, V N; Nielsen, L; Fink, A L

2001-06-22

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.

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

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.

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.

[Objective study of the voice quality following partial laryngectomy].

PubMed

Remacle, M; Millet, B

1991-01-01

The high resolution frequency analyzer is used for the study of the vocal quality after partial laryngectomy. The post-operative plot after speech therapy is of good quality when respecting one vocal fold. On the contrary, the heard vocal sound does not correspond to the harmonics of the fundamental frequency but to intense noise from irregular vibrations of the residual laryngeal mucosa (ventricular folds, arytenoids). High resolution frequency analysis contributes to the follow-up of the partial laryngectomy.

Microbial mats in the Black Sea that anaerobically oxidise methane

NASA Astrophysics Data System (ADS)

Nauhaus, K.; Knittel, K.; Krüger, M.; Boetius, A.; Michaelis, W.; Widdel, F.

2003-04-01

Reef-forming microbial mats were recovered from methane seeps in anoxic waters of the northwestern Black Sea (BS) shelf. The microbial mats consist mainly of archaea (ANME-1 cluster) and sulfate-reducing bacteria (Desulfosarcina/Desulfococcus group). Laboratory incubations with homogenized subsamples of the mats revealed their ability for the anaerobic oxidation of methane (AOM). The phylogentic relationship of the sulfate reducing partner is the same as in the AOM consortia studied in sediment samples from a methane hydrate area (Hydrate Ridge (HR), Oregon, USA (1,2)). The archaeal partner however belongs to a different cluster than in the HR samples (ANME-2). Methane oxidation is coupled to sulfate reduction in a 1:1 stoichiometry. Elevated methane partial pressures (0.1 to 1.1 MPa) increased the sulfate reduction rates in the Black Sea samples only two-fold in contrast to 5-fold in HR samples. The optimal temperature for the BS samples is between 10 and 25^oC. In both samples AOM was not taking place if typical inhibitors for sulfate-reduction or methanogenesis were added, thus indicating a syntrophic relationship between the partner organisms. The intermediate that is exchanged between the methane oxidizing archaea and the sulfate-reducing bacterium is still unknown. Additions of the possible intermediates (Acetate, Formate, Hydrogen) did not result in higher sulfate reduction rates in the absence of methane. (1) Boetius, A. et al. (2000) A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature. 407: 623--626 (2) Nauhaus, K., Boetius, A., Krüger, M., Widdel, F. (2002) In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area. Environ. Microbiol. 4 (5): 296--305

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.

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.

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

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.

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

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.

Gas Exchange and Carbon Partitioning in the Leaves of Celery (Apium graveolens L.) at Various Levels of Root Zone Salinity.

PubMed Central

Everard, J. D.; Gucci, R.; Kann, S. C.; Flore, J. A.; Loescher, W. H.

1994-01-01

Both mannitol and sucrose (Suc) are primary photosynthetic products in celery (Apium graveolens L.). In other biological systems mannitol has been shown to serve as a compatible solute or osmoprotectant involved in stress tolerance. Although mannitol, like Suc, is translocated and serves as a reserve carbohydrate in celery, its role in stress tolerance has yet to be resolved. Mature celery plants exposed to low (25 mM NaCl), intermediate (100 mM NaCl), and high (300 mM NaCl) salinities displayed substantial salt tolerance. Shoot fresh weight was increased at low NaCl concentrations when compared with controls, and growth continued, although at slower rates, even after prolonged exposure to high salinities. Gas-exchange analyses showed that low NaCl levels had little or no effect on photosynthetic carbon assimilation (A), but at intermediate levels decreases in stomatal conductance limited A, and at the highest NaCl levels carboxylation capacity (as measured by analyses of the CO2 assimilation response to changing internal CO2 partial pressures) and electron transport (as indicated by fluorescence measurements) were the apparent prevailing limits to A. Increasing salinities up to 300 mM, however, increased mannitol accumulation and decreased Suc and starch pools in leaf tissues, e.g. the ratio of mannitol to Suc increased almost 10-fold. These changes were due in part to shifts in photosynthetic carbon partitioning (as measured by 14C labeling) from Suc into mannitol. Salt treatments increased the activity of mannose-6-phosphate reductase (M6PR), a key enzyme in mannitol biosynthesis, 6-fold in young leaves and 2-fold in fully expanded, mature leaves, but increases in M6PR protein were not apparent in the older leaves. Mannitol biosynthetic capacity (as measured by labeling rates) was maintained despite salt treatment, and relative partitioning into mannitol consequently increased despite decreased photosynthetic capacity. The results support a suggested role for mannitol accumulation in adaptation to and tolerance of salinity stress. PMID:12232328

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

Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

PubMed

Serebryany, Eugene; Takata, Takumi; Erickson, Erika; Schafheimer, Nathaniel; Wang, Yongting; King, Jonathan A

2016-06-01

Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The βγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization. © 2016 The Protein Society.

75 FR 9442 - Lonza, Inc., Riverside Plant, Lonza Exclusive Synthesis Section, Custom Manufacturing Division...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-02

... competitive with cGMP intermediates and Active Pharmaceutical Ingredients from the subject facility to a..., Conshohocken, Pennsylvania, who are engaged in employment related to the production of cGMP intermediates and...GMP intermediates and Active Pharmaceutical Ingredients, who became totally or partially separated...

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 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.

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.

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.

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

Derivation of intermediate to silicic magma from the basalt analyzed at the Vega 2 landing site, Venus.

PubMed

Shellnutt, J Gregory

2018-01-01

Geochemical modeling using the basalt composition analyzed at the Vega 2 landing site indicates that intermediate to silicic liquids can be generated by fractional crystallization and equilibrium partial melting. Fractional crystallization modeling using variable pressures (0.01 GPa to 0.5 GPa) and relative oxidation states (FMQ 0 and FMQ -1) of either a wet (H2O = 0.5 wt%) or dry (H2O = 0 wt%) parental magma can yield silicic (SiO2 > 60 wt%) compositions that are similar to terrestrial ferroan rhyolite. Hydrous (H2O = 0.5 wt%) partial melting can yield intermediate (trachyandesite to andesite) to silicic (trachydacite) compositions at all pressures but requires relatively high temperatures (≥ 950°C) to generate the initial melt at intermediate to low pressure whereas at high pressure (0.5 GPa) the first melts will be generated at much lower temperatures (< 800°C). Anhydrous partial melt modeling yielded mafic (basaltic andesite) and alkaline compositions (trachybasalt) but the temperature required to produce the first liquid is very high (≥ 1130°C). Consequently, anhydrous partial melting is an unlikely process to generate derivative liquids. The modeling results indicate that, under certain conditions, the Vega 2 composition can generate silicic liquids that produce granitic and rhyolitic rocks. The implication is that silicic igneous rocks may form a small but important component of the northeast Aphrodite Terra.

Derivation of intermediate to silicic magma from the basalt analyzed at the Vega 2 landing site, Venus

PubMed Central

2018-01-01

Geochemical modeling using the basalt composition analyzed at the Vega 2 landing site indicates that intermediate to silicic liquids can be generated by fractional crystallization and equilibrium partial melting. Fractional crystallization modeling using variable pressures (0.01 GPa to 0.5 GPa) and relative oxidation states (FMQ 0 and FMQ -1) of either a wet (H2O = 0.5 wt%) or dry (H2O = 0 wt%) parental magma can yield silicic (SiO2 > 60 wt%) compositions that are similar to terrestrial ferroan rhyolite. Hydrous (H2O = 0.5 wt%) partial melting can yield intermediate (trachyandesite to andesite) to silicic (trachydacite) compositions at all pressures but requires relatively high temperatures (≥ 950°C) to generate the initial melt at intermediate to low pressure whereas at high pressure (0.5 GPa) the first melts will be generated at much lower temperatures (< 800°C). Anhydrous partial melt modeling yielded mafic (basaltic andesite) and alkaline compositions (trachybasalt) but the temperature required to produce the first liquid is very high (≥ 1130°C). Consequently, anhydrous partial melting is an unlikely process to generate derivative liquids. The modeling results indicate that, under certain conditions, the Vega 2 composition can generate silicic liquids that produce granitic and rhyolitic rocks. The implication is that silicic igneous rocks may form a small but important component of the northeast Aphrodite Terra. PMID:29584745

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

Heterogeneity of Equilibrium Molten Globule State of Cytochrome c Induced by Weak Salt Denaturants under Physiological Condition

PubMed Central

Rahaman, Hamidur; Alam Khan, Md. Khurshid; Hassan, Md. Imtaiyaz; Islam, Asimul; Moosavi-Movahedi, Ali Akbar; Ahmad, Faizan

2015-01-01

While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO4) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε 400) and CD ([θ]409), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO4 and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO4 and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N. PMID:25849212

Heterogeneity of equilibrium molten globule state of cytochrome c induced by weak salt denaturants under physiological condition.

PubMed

Rahaman, Hamidur; Alam Khan, Md Khurshid; Hassan, Md Imtaiyaz; Islam, Asimul; Moosavi-Movahedi, Ali Akbar; Ahmad, Faizan

2015-01-01

While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO4) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε400) and CD ([θ]409), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO4 and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO4 and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.

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.

Thermodynamic properties of an extremely rapid protein folding reaction.

PubMed

Schindler, T; Schmid, F X

1996-12-24

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

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.

Bilateral Vocal Fold Paralysis After Surgery Immediately in Adult Patient With Chiari Malformation.

PubMed

Chen, Yan; Yue, Jianhong; Yuan, Weixiu

2016-06-01

The authors report the case of a 50-year-old woman with a bilateral vocal fold paralysis after foramen magnum decompression and resection of partial cerebellar tonsil for Chiari malformation. The possible mechanisms of postoperative bilateral vocal fold paralysis are discussed.

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

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.

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.

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 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

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.

Concentration-dependent antagonistic persuasion of SDS and naphthalene derivatives on the fibrillation of stem bromelain.

PubMed

Qadeer, Atiyatul; Ahmad, Ejaz; Zaman, Masihuz; Khan, Mohd Wasif; Khan, Javed Masood; Rabbani, Gulam; Tarique, Khaja Faisal; Sharma, Gaurav; Gourinath, Samudrala; Nadeem, Sajid; Badr, Gamal; Khan, Rizwan Hasan

2013-12-01

Sodium dodecyl sulfate, a biological membrane mimetic, can be used to study the conversion of globular proteins into amyloid fibrils in vitro. Using multiple approaches, the effect of SDS was examined on stem bromelain (SB), a widely recognized therapeutic protein. SB is known to exist as a partially folded intermediate at pH 2.0, situation also encountered in the gastrointestinal tract (its site of absorption). In the presence of sub-micellar SDS concentration (500-1000 μM), this intermediate was found to exhibit great propensity to form large-sized β-sheeted aggregates with fibrillar morphology, the hall marks of amyloid structure. We also observed inhibition of fibrillation by two naphthalene-based compounds, ANS and bis-ANS. While bis-ANS significantly inhibited fibril formation at 50 μM, ANS did so at relatively higher concentration (400 μM). Alcohols, but not salts, were found to weaken the inhibitory action of these compounds suggesting the possible involvement of hydrophobic interactions in their binding to protein. Besides, isothermal titration calorimetry and molecular docking studies suggested that inhibition of fibrillation by these naphthalene derivatives is mediated not just through hydrophobic forces, but also by disruption of π-π interactions between the aromatic residues together with the inter-polypeptide chain repulsion among negatively charged ANS/bis-ANS bound SB. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Alpha-synuclein: relating metals to structure, function and inhibition.

PubMed

McDowall, J S; Brown, D R

2016-04-01

Alpha-synuclein has long been studied due to its involvement in the progression of Parkinson's disease (PD), a common neurodegenerative disorder, although a consensus on the exact function of this protein is elusive. This protein shows remarkable structural plasticity and this property is important for both correct cellular function and pathological progression of PD. Formation of intracellular oligomeric species within the substantia nigra correlates with disease progression and it has been proposed that formation of a partially folded intermediate is key to the initiation of the fibrillisation process. Many factors can influence changes in the structure of alpha-synuclein such as disease mutations and interaction with metals and neurotransmitters. High concentrations of both dopamine and metals are present in the substantia nigra making this an ideal location for both the structural alteration of alpha-synuclein and the production of toxic oxygen species. The recent proposal that alpha-synuclein is a ferrireductase is important as it can possibly catalyse the formation of such reactive species and as a result exacerbate neurodegeneration.

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates.

PubMed

Guo, Xiaoge; Jinks-Robertson, Sue

2013-12-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral introduction of UV damage can potentially compromise genetic interpretations. Copyright © 2013 Elsevier B.V. All rights reserved.

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates

PubMed Central

Guo, Xiaoge; Jinks-Robertson, Sue

2013-01-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the cillateral introduction of UV damage can potentially compromise genetic interpretations. PMID:24120148

Partial to complete wetting transitions in immiscible ternary blends with PLA: the influence of interfacial confinement.

PubMed

Zolali, Ali M; Favis, Basil D

2017-04-12

In this study it is shown that the three different intermediate phases in melt blended ternary PLA/PHBV/PBS, PLA/PBAT/PE and PLA/PE/PBAT systems all demonstrate partial wetting, but have very different wetting behaviors as a function of composition and annealing. The interfacial tension of the various components, their spreading coefficients and the contact angles of the confined partially wet droplets at the interface are examined in detail. A wetting transition from partially wet droplets to a complete layer at the interface is observed for both PHBV and PBAT by increasing the concentration and also by annealing. In contrast, in PLA/PE/PBAT, the partially wet droplets of PE at the interface of PLA/PBAT coalesce and grow in size, but remain partially wet even at a high PE concentration of 20% and after 30 min of quiescent annealing. The dewetting speed of the intermediate phase is found to be the principal factor controlling these wetting transitions. This work shows the significant potential for controlled wetting and structuring in ternary polymer systems.

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.

A similarity measure for partially folded proteins: application to unfolded and native-like conformational fluctuations

NASA Astrophysics Data System (ADS)

Larios, Edgar; Yang, Wei Y.; Schulten, K.; Gruebele, M.

2004-12-01

Computing the root-mean-square deviation (RMSD) of a partially folded protein structure from the folded state requires the two structures to be translationally and rotationally aligned. We examine the constraint matrix L that preserves orthogonality of the rotation matrix during minimization of the RMSD. L is proportional to the sensitivity of the RMSD to the rotational alignment matrix. Its trace yields an isotropic reaction coordinate, while its off-diagonal matrix elements are related to the moment of inertia derivative tensor that encodes anisotropic information about the structure. We use L to compare λ-repressor fragment 6-85 (λ 6-85) to several partially folded structures obtained from molecular dynamics simulation (MD), and find that L as a reaction coordinate indeed encodes some information about protein topology. We also apply C α RMSD, L and tryptophan sidechain mobility as criteria for native state structural fluctuations of several λ 6-85 mutants. The mutants' denaturation curves and fluorescence quenching are measured experimentally for comparison. The results are in accord with a recent proposal that structural fluctuations near the chromophore can induce increased native state fluorescence or hyperfluorescence during unfolding of proteins.

Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity

PubMed Central

Sastry, M S R; Zhou, Weibin; Baneyx, François

2009-01-01

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging. PMID:19517531

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.

Mutants Resistant to anti-Microtubule Herbicides Map to a Locus on the Uni Linkage Group in Chlamydomonas Reinhardtii

PubMed Central

James, S. W.; Ranum, LPW.; Silflow, C. D.; Lefebvre, P. A.

1988-01-01

We have used genetic analysis to study the mode of action of two anti-microtubule herbicides, amiprophos-methyl (APM) and oryzalin (ORY). Over 200 resistant mutants were selected by growth on APM- or ORY-containing plates. The 21 independently isolated mutants examined in this study are 3- to 8-fold resistant to APM and are strongly cross-resistant to ORY and butamiphos, a close analog of APM. Two Mendelian genes, apm1 and apm2, are defined by linkage and complementation analysis. There are 20 alleles of apm1 and one temperature-sensitive lethal (33°) allele of apm2. Mapping by two-factor crosses places apm1 6.5 cM centromere proximal to uni1 and within 4 cM of pf7 on the uni linkage group, a genetically circular linkage group comprising genes which affect flagellar assembly or function; apm2 maps near the centromere of linkage group VIII. Allele-specific synthetic lethality is observed in crosses between apm2 and alleles of apm1. Also, self crosses of apm2 are zygotic lethal, whereas crosses of nine apm1 alleles inter se result in normal germination and tetrad viability. The mutants are recessive to their wild-type alleles but doubly heterozygous diploids (apm1 +/+ apm2) made with apm2 and any of 15 apm1 alleles display partial intergenic noncomplementation, expressed as intermediate resistance. Diploids homozygous for mutant alleles of apm1 are 4-6-fold resistant to APM and ORY; diploids homozygous for apm2 are ts(-) and 2-fold resistant to the herbicides. Doubly heterozygous diploids complement the ts(-) phenotype of apm2, but they are typically 1.5-2-fold resistant to APM and ORY. From the results described we suggest that the gene products of apm1 and apm2 may interact directly or function in the same structure or process. PMID:8608924

Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A at low pH.

PubMed

Khan, F; Khan, R H; Muzammil, S

2000-09-29

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K(3)FeCN(6), Na(2)SO(4), KClO(4) and KCl) on the acid-induced state of alpha-chymotrypsinogen A, a predominantly beta-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K(3)FeCN(6)>Na(2)SO(4)>KClO(4)>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.

Mechanism of IAPP amyloid fibril formation involves an intermediate with a transient β-sheet

PubMed Central

Buchanan, Lauren E.; Dunkelberger, Emily B.; Tran, Huong Q.; Cheng, Pin-Nan; Chiu, Chi-Cheng; Cao, Ping; Raleigh, Daniel P.; de Pablo, Juan J.; Nowick, James S.; Zanni, Martin T.

2013-01-01

Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques. PMID:24218609

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

15. DETAIL VIEW OF MAIN LOCK MITER GATE IN PARTIALLY ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

15. DETAIL VIEW OF MAIN LOCK MITER GATE IN PARTIALLY OPENED POSITION, LOOKING NORTHWEST (UPSTREAM). COMPRESSOR BUILDING VISIBLE TO REAR ON INTERMEDIATE WALL - Upper Mississippi River 9-Foot Channel Project, Lock & Dam 27, Granite City, Madison County, IL

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

Contemporary Cambodian: The Social Institutions.

ERIC Educational Resources Information Center

Kheang, Lim Hack; And Others

"Contemporary Cambodian" consists of seven parts: the "Grammatical Sketch," the "Introduction," four intermediate modules, and a Cambodian-English glossary containing all the words in the series. After completing the "Introduction," a student can study the intermediate modules in any order, since each serves as a partial review of the others. This…

Comparison of using different bridge prosthetic designs for partial defect restoration through mathematical modeling.

PubMed

Styranivska, Oksana; Kliuchkovska, Nataliia; Mykyyevych, Nataliya

2017-01-01

To analyze the stress-strain states of bone and abutment teeth during the use of different prosthetic designs of fixed partial dentures with the use of relevant mathematical modeling principles. The use of Comsol Multiphysics 3.5 (Comsol AB, Sweden) software during the mathematical modeling of stress-strain states provided numerical data for analytical interpretation in three different clinical scenarios with fixed dentures and different abutment teeth and demountable prosthetic denture with the saddle-shaped intermediate part. Microsoft Excel Software (Microsoft Office 2017) helped to evaluate absolute mistakes of stress and strain parameters of each abutment tooth during three modeled scenarios and normal condition and to summarize data into the forms of tables. In comparison with the fixed prosthetic denture supported by the canine, first premolar, and third molar, stresses at the same abutment teeth with the use of demountable denture with the saddle-shaped intermediate part decreased: at the mesial abutment tooth by 2.8 times, at distal crown by 6.1 times, and at the intermediate part by 11.1 times, respectively, the deformation level decreased by 3.1, 1.9, and 1.4 times at each area. The methods of mathematical modeling proved that complications during the use of fixed partial dentures based on the overload effect of the abutment teeth and caused by the deformation process inside the intermediate section of prosthetic construction.

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.

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.

Fan-fold shielded electrical leads

DOEpatents

Rohatgi, Rajeev R.; Cowan, Thomas E.

1996-01-01

Fan-folded electrical leads made from copper cladded Kapton, for example, with the copper cladding on one side serving as a ground plane and the copper cladding on the other side being etched to form the leads. The Kapton is fan folded with the leads located at the bottom of the fan-folds. Electrical connections are made by partially opening the folds of the fan and soldering, for example, the connections directly to the ground plane and/or the lead. The fan folded arrangement produces a number of advantages, such as electrically shielding the leads from the environment, is totally non-magnetic, and has a very low thermal conductivity, while being easy to fabricate.

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.

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.

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

Exploring the chemical kinetics of partially oxidized intermediates by combining experiments, theory, and kinetic modeling.

PubMed

Hoyermann, Karlheinz; Mauß, Fabian; Olzmann, Matthias; Welz, Oliver; Zeuch, Thomas

2017-07-19

Partially oxidized intermediates play a central role in combustion and atmospheric chemistry. In this perspective, we focus on the chemical kinetics of alkoxy radicals, peroxy radicals, and Criegee intermediates, which are key species in both combustion and atmospheric environments. These reactive intermediates feature a broad spectrum of chemical diversity. Their reactivity is central to our understanding of how volatile organic compounds are degraded in the atmosphere and converted into secondary organic aerosol. Moreover, they sensitively determine ignition timing in internal combustion engines. The intention of this perspective article is to provide the reader with information about the general mechanisms of reactions initiated by addition of atomic and molecular oxygen to alkyl radicals and ozone to alkenes. We will focus on critical branching points in the subsequent reaction mechanisms and discuss them from a consistent point of view. As a first example of our integrated approach, we will show how experiment, theory, and kinetic modeling have been successfully combined in the first infrared detection of Criegee intermediates during the gas phase ozonolysis. As a second example, we will examine the ignition timing of n-heptane/air mixtures at low and intermediate temperatures. Here, we present a reduced, fuel size independent kinetic model of the complex chemistry initiated by peroxy radicals that has been successfully applied to simulate standard n-heptane combustion experiments.

Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

PubMed Central

Haq, Imran; Irving, James A.; Faull, Sarah V.; Dickens, Jennifer A.; Ordóñez, Adriana; Belorgey, Didier; Gooptu, Bibek; Lomas, David A.

2013-01-01

The common severe Z mutation (E342K) of α1-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular ‘snapshots’, the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α1-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P14P12 and P4, but not P10P8 or P2P1′, resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P14P12 region self-inserts early during activation, while the interaction between P6P4 and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P6P4, but not P14P12, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol−1. PMID:23659468

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

Ligand-Induced Stabilization of a Duplex-like Architecture Is Crucial for the Switching Mechanism of the SAM-III Riboswitch.

PubMed

Suresh, Gorle; Srinivasan, Harini; Nanda, Shivani; Priyakumar, U Deva

2016-06-21

Riboswitches are structured RNA motifs that control gene expression by sensing the concentrations of specific metabolites and make up a promising new class of antibiotic targets. S-Adenosylmethionine (SAM)-III riboswitch, mainly found in lactic acid bacteria, is involved in regulating methionine and SAM biosynthetic pathways. SAM-III riboswitch regulates the gene expression by switching the translation process on and off with respect to the absence and presence of the SAM ligand, respectively. In this study, an attempt is made to understand the key conformational transitions involved in ligand binding using atomistic molecular dynamics (MD) simulations performed in an explicit solvent environment. G26 is found to recognize the SAM ligand by forming hydrogen bonds, whereas the absence of the ligand leads to opening of the binding pocket. Consistent with experimental results, the absence of the SAM ligand weakens the base pairing interactions between the nucleobases that are part of the Shine-Dalgarno (SD) and anti-Shine-Dalgarno (aSD) sequences, which in turn facilitates recognition of the SD sequence by ribosomes. Detailed analysis reveals that a duplex-like structure formed by nucleotides from different parts of the RNA and the adenine base of the ligand is crucial for the stability of the completely folded state in the presence of the ligand. Previous experimental studies have shown that the SAM-III riboswitch exists in equilibrium between the unfolded and partially folded states in the absence of the ligand, which completely folds upon binding of the ligand. Comparison of the results presented here to the available experimental data indicates the structures obtained using the MD simulations resemble the partially folded state. Thus, this study provides a detailed understanding of the fully and partially folded structures of the SAM-III riboswitch in the presence and absence of the ligand, respectively. This study hypothesizes a dual role for the SAM ligand, which facilitates conformational switching between partially and fully folded states by forming a stable duplex-like structure and strengthening the interactions between SD and aSD nucleotides.

Reimbursed drugs in patients with sleep-disordered breathing: A static-charge-sensitive bed study.

PubMed

Anttalainen, Ulla; Polo, Olli; Vahlberg, Tero; Saaresranta, Tarja

2010-01-01

Co-morbidities in men and women with sleep-disordered breathing (SDB) were compared retrospectively to an age-standardized, general Finnish population. The prevalence of diseases was based on the reimbursement refunds of medications. Two hundred thirty-three age- and BMI-matched male-female pairs and 368 consecutive women identified from our sleep recording database were included. Data on medication were gathered from the National Agency for Medicines and Social Insurance Institution database. Men with SDB had three-fold prevalence of reimbursed medication for diabetes and two-fold prevalence of reimbursed medication for chronic arrhythmia. Women with SDB had three-fold prevalence of reimbursed medication for thyroid insufficiency, and postmenopausal women had two-fold prevalence of reimbursed medication for psychosis. BMI and age did not explain prevalence of reimbursed medications for chronic arrhythmia or psychosis. In both genders with SDB, prevalence of reimbursed medications compared to the general population was two-fold for hypertension and seven-fold for asthma and/or chronic obstructive pulmonary disease (COPD). Partial upper airway obstruction was associated with three-fold prevalence of reimbursed medication for asthma and/or COPD in both genders and 60% reduced prevalence of reimbursed medication for hypertension in females matched for age and BMI. Co-morbidity profile differed between genders. Our results emphasize the importance of diagnosis and treatment of co-morbidities and partial upper airway obstruction. Copyright 2009 Elsevier B.V. All rights reserved.

Interaction of metronidazole with DNA repair mutants of Escherichia coli.

PubMed

Yeung, T C; Beaulieu, B B; McLafferty, M A; Goldman, P

1984-01-01

It has been proposed that one of metronidazole's partially reduced intermediates interacts either with DNA to exert a bactericidal effect or with water to form acetamide. To test this hypothesis we have examined the effect of metronidazole on several mutants of Escherichia coli that are defective in DNA repair. UV-susceptible RecA- and UvrB- point mutants have an increased susceptibility to metronidazole as manifested by both a decreased minimal inhibitory concentration and a greater bactericidal response to metronidazole in resting cultures. By these criteria, however, we find that UvrB- deletion mutants, which lack the ability to reduce nitrate and chlorate, are no more susceptible to metronidazole than is the wild type. We find, however, that these deletion mutants also lack the ability to reduce metronidazole and thus possibly to form its reactive species. When metronidazole's bactericidal effect is expressed in terms of the concurrent accumulation of acetamide derived from metronidazole, then all RecA- and UvrB- mutants are killed more efficiently than their wild types. The data are consistent, therefore, with metronidazole's lethal effect being mediated by a partially reduced intermediate on the metabolic pathway between metronidazole and acetamide. Defects in other aspects of the DNA repair system do not confer this increased susceptibility to the proposed intermediate. A Tag- mutant, for example, which is defective in 3-methyl-adenine-DNA glycosylase, does not have this increased susceptibility to the presumed precursor of acetamide. Thus, these results provide further support for the hypothesis that the bactericidal effect of metronidazole is mediated by a partially reduced intermediate in the metabolic conversion of metronidazole to acetamide and suggest that this intermediate interacts with DNA to produce a lesion similar to that caused by UV light.

Interaction of metronidazole with DNA repair mutants of Escherichia coli.

PubMed Central

Yeung, T C; Beaulieu, B B; McLafferty, M A; Goldman, P

1984-01-01

It has been proposed that one of metronidazole's partially reduced intermediates interacts either with DNA to exert a bactericidal effect or with water to form acetamide. To test this hypothesis we have examined the effect of metronidazole on several mutants of Escherichia coli that are defective in DNA repair. UV-susceptible RecA- and UvrB- point mutants have an increased susceptibility to metronidazole as manifested by both a decreased minimal inhibitory concentration and a greater bactericidal response to metronidazole in resting cultures. By these criteria, however, we find that UvrB- deletion mutants, which lack the ability to reduce nitrate and chlorate, are no more susceptible to metronidazole than is the wild type. We find, however, that these deletion mutants also lack the ability to reduce metronidazole and thus possibly to form its reactive species. When metronidazole's bactericidal effect is expressed in terms of the concurrent accumulation of acetamide derived from metronidazole, then all RecA- and UvrB- mutants are killed more efficiently than their wild types. The data are consistent, therefore, with metronidazole's lethal effect being mediated by a partially reduced intermediate on the metabolic pathway between metronidazole and acetamide. Defects in other aspects of the DNA repair system do not confer this increased susceptibility to the proposed intermediate. A Tag- mutant, for example, which is defective in 3-methyl-adenine-DNA glycosylase, does not have this increased susceptibility to the presumed precursor of acetamide. Thus, these results provide further support for the hypothesis that the bactericidal effect of metronidazole is mediated by a partially reduced intermediate in the metabolic conversion of metronidazole to acetamide and suggest that this intermediate interacts with DNA to produce a lesion similar to that caused by UV light. PMID:6367636

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

Basis for ligand discrimination between ON and OFF state riboswitch conformations: The case of the SAM-I riboswitch

PubMed Central

Boyapati, Vamsi Krishna; Huang, Wei; Spedale, Jessica; Aboul-ela, Fareed

2012-01-01

Riboswitches are RNA elements that bind to effector ligands and control gene expression. Most consist of two domains. S-Adenosyl Methionine (SAM) binds the aptamer domain of the SAM-I riboswitch and induces conformational changes in the expression domain to form an intrinsic terminator (transcription OFF state). Without SAM the riboswitch forms the transcription ON state, allowing read-through transcription. The mechanistic link between the SAM/aptamer recognition event and subsequent secondary structure rearrangement by the riboswitch is unclear. We probed for those structural features of the Bacillus subtilis yitJ SAM-I riboswitch responsible for discrimination between the ON and OFF states by SAM. We designed SAM-I riboswitch RNA segments forming “hybrid” structures of the ON and OFF states. The choice of segment constrains the formation of a partial P1 helix, characteristic of the OFF state, together with a partial antiterminator (AT) helix, characteristic of the ON state. For most choices of P1 vs. AT helix lengths, SAM binds with micromolar affinity according to equilibrium dialysis. Mutational analysis and in-line probing confirm that the mode of SAM binding by hybrid structures is similar to that of the aptamer. Altogether, binding measurements and in-line probing are consistent with the hypothesis that when SAM is present, stacking interactions with the AT helix stabilize a partially formed P1 helix in the hybrids. Molecular modeling indicates that continuous stacking between the P1 and the AT helices is plausible with SAM bound. Our findings raise the possibility that conformational intermediates may play a role in ligand-induced aptamer folding. PMID:22543867

Basis for ligand discrimination between ON and OFF state riboswitch conformations: the case of the SAM-I riboswitch.

PubMed

Boyapati, Vamsi Krishna; Huang, Wei; Spedale, Jessica; Aboul-Ela, Fareed

2012-06-01

Riboswitches are RNA elements that bind to effector ligands and control gene expression. Most consist of two domains. S-Adenosyl Methionine (SAM) binds the aptamer domain of the SAM-I riboswitch and induces conformational changes in the expression domain to form an intrinsic terminator (transcription OFF state). Without SAM the riboswitch forms the transcription ON state, allowing read-through transcription. The mechanistic link between the SAM/aptamer recognition event and subsequent secondary structure rearrangement by the riboswitch is unclear. We probed for those structural features of the Bacillus subtilis yitJ SAM-I riboswitch responsible for discrimination between the ON and OFF states by SAM. We designed SAM-I riboswitch RNA segments forming "hybrid" structures of the ON and OFF states. The choice of segment constrains the formation of a partial P1 helix, characteristic of the OFF state, together with a partial antiterminator (AT) helix, characteristic of the ON state. For most choices of P1 vs. AT helix lengths, SAM binds with micromolar affinity according to equilibrium dialysis. Mutational analysis and in-line probing confirm that the mode of SAM binding by hybrid structures is similar to that of the aptamer. Altogether, binding measurements and in-line probing are consistent with the hypothesis that when SAM is present, stacking interactions with the AT helix stabilize a partially formed P1 helix in the hybrids. Molecular modeling indicates that continuous stacking between the P1 and the AT helices is plausible with SAM bound. Our findings raise the possibility that conformational intermediates may play a role in ligand-induced aptamer folding.

Molecular cytogenetic characterization and stem rust resistance of five wheat-thinopyrum ponticum partial amphiploids

USDA-ARS?s Scientific Manuscript database

Partial amphiploids created by crossing common wheat (Triticum aestivum L.) and Thinopyrum ponticum (Podp.), Barkworth & D. R. Dewey may be resistant to major wheat diseases and are an important intermediate material in wheat breeding. In this study, we examined chromosome composition of five Xiaoy...

Characteristics of a leucine aminoacyl transfer RNA synthetase from Tritrichomonas augusta.

PubMed

Horner, J; Champney, W S; Samuels, R

1991-04-01

This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37 degrees C for 20 min, with protein at 180 micrograms ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.

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.

Method for fabricating fan-fold shielded electrical leads

DOEpatents

Rohatgi, R.R.; Cowan, T.E.

1994-12-27

Fan-folded electrical leads made from copper cladded Kapton, for example, with the copper cladding on one side serving as a ground plane and the copper cladding on the other side being etched to form the leads. The Kapton is fan folded with the leads located at the bottom of the fan-folds. Electrical connections are made by partially opening the folds of the fan and soldering, for example, the connections directly to the ground plane and/or the lead. The fan folded arrangement produces a number of advantages, such as electrically shielding the leads from the environment, is totally non-magnetic, and has a very low thermal conductivity, while being easy to fabricate. 3 figures.

Method for fabricating fan-fold shielded electrical leads

DOEpatents

Rohatgi, Rajeev R.; Cowan, Thomas E.

1994-01-01

Fan-folded electrical leads made from copper cladded Kapton, for example, with the copper cladding on one side serving as a ground plane and the copper cladding on the other side being etched to form the leads. The Kapton is fan folded with the leads located at the bottom of the fan-folds. Electrical connections are made by partially opening the folds of the fan and soldering, for example, the connections directly to the ground plane and/or the lead. The fan folded arrangement produces a number of advantages, such as electrically shielding the leads from the environment, is totally non-magnetic, and has a very low thermal conductivity, while being easy to fabricate.

Fan-fold shielded electrical leads

DOEpatents

Rohatgi, R.R.; Cowan, T.E.

1996-06-11

Disclosed are fan-folded electrical leads made from copper cladded Kapton, for example, with the copper cladding on one side serving as a ground plane and the copper cladding on the other side being etched to form the leads. The Kapton is fan folded with the leads located at the bottom of the fan-folds. Electrical connections are made by partially opening the folds of the fan and soldering, for example, the connections directly to the ground plane and/or the lead. The fan folded arrangement produces a number of advantages, such as electrically shielding the leads from the environment, is totally non-magnetic, and has a very low thermal conductivity, while being easy to fabricate. 3 figs.

Large scale wind tunnel investigation of a folding tilt rotor

NASA Technical Reports Server (NTRS)

1972-01-01

A twenty-five foot diameter folding tilt rotor was tested in a large scale wind tunnel to determine its aerodynamic characteristics in unfolded, partially folded, and fully folded configurations. During the tests, the rotor completed over forty start/stop sequences. After completing the sequences in a stepwise manner, smooth start/stop transitions were made in approximately two seconds. Wind tunnel speeds up through seventy-five knots were used, at which point the rotor mast angle was increased to four degrees, corresponding to a maneuver condition of one and one-half g.

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

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.

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

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.

Prediction of properties and elemental composition of biomass pyrolysis oils by NMR and partial least squares analysis

USDA-ARS?s Scientific Manuscript database

Several partial least squares (PLS) models were created correlating various properties and chemical composition measurements with the 1H and 13C NMR spectra of 73 different of pyrolysis bio-oil samples from various biomass sources (crude and intermediate products), finished oils and small molecule s...

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.

How the folding rates of two- and multistate proteins depend on the amino acid properties.

PubMed

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

2014-10-01

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

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.

Single-molecule Protein Unfolding in Solid State Nanopores

PubMed Central

Talaga, David S.; Li, Jiali

2009-01-01

We use single silicon nitride nanopores to study folded, partially folded and unfolded single proteins by measuring their excluded volumes. The DNA-calibrated translocation signals of β-lactoglobulin and histidine-containing phosphocarrier protein match quantitatively with that predicted by a simple sum of the partial volumes of the amino acids in the polypeptide segment inside the pore when translocation stalls due to the primary charge sequence. Our analysis suggests that the majority of the protein molecules were linear or looped during translocation and that the electrical forces present under physiologically relevant potentials can unfold proteins. Our results show that the nanopore translocation signals are sensitive enough to distinguish the folding state of a protein and distinguish between proteins based on the excluded volume of a local segment of the polypeptide chain that transiently stalls in the nanopore due to the primary sequence of charges. PMID:19530678

A molecular mechanism of chaperone-client recognition

PubMed Central

He, Lichun; Sharpe, Timothy; Mazur, Adam; Hiller, Sebastian

2016-01-01

Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature. PMID:28138538

Proceedings of the International Conference on Cyclic Nucleotides, Calcium and Protein Phosphorylation (6th) Held in Bethesda, Maryland on September 2-7, 1986. Advances in Second Messenger and Phosphorprotein Research. Volume 21A. Abstracts Volume

DTIC Science & Technology

1988-11-16

oxoireamorine. Oxotremorine inhibited Ach release for a shorter duration. The ACh release-inhibition induced by 50 PH Aech-M was only acutely blocked...and the partial agonist oxotresorine in the presence of 59K of Lit. Carbacbol showed a 10-fold lover ECS0-value (3.7liM) and oxotremorine a 3-fold...however, only a decrease in the maximal response was seen. In both tissues, the maximal, response of the partial agonist oxotremorine was suppressed

Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of high-fructose syrup.

PubMed

Singh, Ram Sarup; Dhaliwal, Rajesh; Puri, Munish

2007-05-01

An extracellular exoinulinase (2,1-beta-D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable (100%) for 3 h at the optimum temperature of 50 degrees C. Mn2+ and Ca2+ produced a 2.4-fold and 1.2-fold enhancement in enzyme activity, whereas Hg2+ and Ag2+ completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6 mg/ml and 41.3 mg/ml, respectively.

Folding cooperativity in a three-stranded beta-sheet model.

PubMed

Roe, Daniel R; Hornak, Viktor; Simmerling, Carlos

2005-09-16

The thermodynamic behavior of a previously designed three-stranded beta-sheet was studied via several microseconds of standard and replica exchange molecular dynamics simulations. The system is shown to populate at least four thermodynamic minima, including two partially folded states in which only a single hairpin is formed. Simulated melting curves show different profiles for the C and N-terminal hairpins, consistent with differences in secondary structure content in published NMR and CD/FTIR measurements, which probed different regions of the chain. Individual beta-hairpins that comprise the three-stranded beta-sheet are observed to form cooperatively. Partial folding cooperativity between the component hairpins is observed, and good agreement between calculated and experimental values quantifying this cooperativity is obtained when similar analysis techniques are used. However, the structural detail in the ensemble of conformations sampled in the simulations permits a more direct analysis of this cooperativity than has been performed on the basis of experimental data. The results indicate the actual folding cooperativity perpendicular to strand direction is significantly larger than the lower bound obtained previously.

Folding cooperativity in a 3-stranded β-sheet model

PubMed Central

Roe, Daniel R.; Hornak, Viktor

2015-01-01

Summary The thermodynamic behavior of a previously designed three-stranded β-sheet was studied via several µs of standard and replica exchange molecular dynamics simulations. The system is shown to populate at least four thermodynamic minima, including 2 partially folded states in which only a single hairpin is formed. Simulated melting curves show different profiles for the C and N-terminal hairpins, consistent with differences in secondary structure content in published NMR and CD/FTIR measurements, which probed different regions of the chain. Individual β-hairpins that comprise the 3-stranded β-sheet are observed to form cooperatively. Partial folding cooperativity between the component hairpins is observed, and good agreement between calculated and experimental values quantifying this cooperativity is obtained when similar analysis techniques are used. However, the structural detail in the ensemble of conformations sampled in the simulations permits a more direct analysis of this cooperatively than has been performed based on experimental data. The results indicate the actual folding cooperativity perpendicular to strand direction is significantly larger than the lower bound obtained previously. PMID:16095612

The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules

PubMed Central

Antoniou, Antony N.; Ford, Stuart; Alphey, Magnus; Osborne, Andrew; Elliott, Tim; Powis, Simon J.

2002-01-01

The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone-assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide-loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N-terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation. PMID:12032078

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.

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

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

EPA Science Inventory

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

The intracellular region of ClC-3 chloride channel is in a partially folded state and a monomer.

PubMed

Li, Shu Jie; Kawazaki, Masanobu; Ogasahara, Kyoko; Nakagawa, Atsushi

2006-05-01

In contrast to bacterial ClC chloride channels, all eukaryotic ClC chloride channels have a conserved long intracellular region that makes up of the carboxyl terminus of the protein and is necessary for channel functions as a channel gate. Little is known, however, about the molecular structure of the intracellular region of ClC chloride channels so far. Here, for the first time, we have expressed and purified the intracellular region of the rat ClC-3 chloride channel (C-ClC-3) as a water-soluble protein under physiological conditions, and investigated its structural characteristics and assembly behavior by means of circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), size exclusion chromatography and analytical ultracentrifugation. The far-UV CD spectra of C-ClC-3 in the native state and in the presence of urea clearly show that the protein has a significantly folded secondary structure consisting of alpha-helices and beta-sheets, while the near-UV CD spectra and DSC experiments indicate the protein is deficient in well-defined tertiary packing. Its Stokes radius is larger than its expected size as a folded globular protein, as determined on size exclusion chromatography. Furthermore, the DisEMBL program, a useful computational tool for the prediction of disordered/unstructured regions within a protein sequence, predicts that the protein is in a partially folded state. Based on these results, we conclude that C-ClC-3 is partially folded. On the other hand, both size exclusion chromatography and sedimentation equilibrium analysis show that C-ClC-3 exists as a monomer in solution, not a dimer like the whole ClC-3 molecule.

Chaperonin-based biolayer interferometry to assess the kinetic stability of metastable, aggregation-prone proteins

PubMed Central

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

2017-01-01

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

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

Experimental study on heat transfer enhancement of laminar ferrofluid flow in horizontal tube partially filled porous media under fixed parallel magnet bars

NASA Astrophysics Data System (ADS)

Sheikhnejad, Yahya; Hosseini, Reza; Saffar Avval, Majid

2017-02-01

In this study, steady state laminar ferroconvection through circular horizontal tube partially filled with porous media under constant heat flux is experimentally investigated. Transverse magnetic fields were applied on ferrofluid flow by two fixed parallel magnet bar positioned on a certain distance from beginning of the test section. The results show promising notable enhancement in heat transfer as a consequence of partially filled porous media and magnetic field, up to 2.2 and 1.4 fold enhancement were observed in heat transfer coefficient respectively. It was found that presence of both porous media and magnetic field simultaneously can highly improve heat transfer up to 2.4 fold. Porous media of course plays a major role in this configuration. Virtually, application of Magnetic field and porous media also insert higher pressure loss along the pipe which again porous media contribution is higher that magnetic field.

Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants.

PubMed

Coulter-Mackie, M B; Lian, Q

2008-07-01

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.

Mechanical development of folded chert beds in Monterey Formation, California

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

Crowther, D.; Snyder, W.S.

1988-03-01

Small-scale folds in the upper siliceous facies of the Miocene Monterey Formation, at Lions Head, California (Santa Maria basin) are of tectonic origin. Folding is well developed in the chert-dominated zones and dies out rapidly in the adjacent siliceous mudstones. A tectonic origin is evidenced by the dominantly brittle deformation of the competent chert layers. Mechanically, the folds formed through a complex interrelationship between fracture and flexural slip. Opal-CT and quartz-chert layers display brittle fractures and rotated fracture blocks that responded to shortening. Thrusting of the chert layers is common in folds where fold propagation was impeded. Dilation breccia andmore » void space occur in the hinges and reflect room problems during development of these disharmonic folds. Subsequent diagenesis has partially healed the fractures and slip surfaces, creating the erroneous appearance that ductile deformation was an important factor in the formation of the folds.« less

PA03.13. Effect of triphaladi rasayana along with yoga therapy on low grade non hodgkins lymphoma and resistant intermediate and high grade non hodgkins lymphoma

PubMed Central

Soumya, MS Surya; Sarasa, TP

2013-01-01

Purpose: 1. To find out the effect of Thriphaladi Rasayana along with Yoga Therapy on low grade Non Hodgkins Lymphoma and resistant intermediate and high grade NonHodgkins Lymphoma. 2. To apply a less costly, less morbid, well accepted method of treatment on NHL. 3.To find a simple method to increase the immunity. 4.To try a drug which is easy to prepare? Method: Purposive sampling technique was used for the study. Sample of 30 patients age range 25 75 years with histologicaly proven NonHodgkins lymphoma, attending the M.O.I.O.P of the regional cancer centre during a period of 18 months. Groups1) Low grade NonHodgkins Lymphoma 2) Resistant intermediate &High grade NonHodgkins lymphoma (failed chemotherapy) were taken. Procedure : 2 groups were given Triphaladhi Rasayana (15 grams of powder with ghee and honey) twice dailymorning& at bed time with milk as anupana for period of 1month along with selected yoga asanas and niyama? Result: Symptoms included were fever, night sweats, weight loss, lymph nodes enlargement, splenomegaly, and hepatomegaly. In low grade symptom relief was noted in almost all cases. Lymph node changes notedLow grade5 2% (complete remission), 38% (partial remission), 10% (no change), intermediate35% (CR), 52% (PR) & 13% (NC), High grade67% (CR), 33%(PR). Hepatomegaly changes :ve in low grade92.86%, intermediate 90.9% & high grade100%. Splenomegaly changes :ve in low grade92.86%, intermediate72.72% & high grade80% Over all remission status of 30 patientscomplete remission30%, partial remission 30% & no change30%? Conclusion: Thriphaladirasayana along with Yoga therapy is very effective in Low grade NonHodgkins lymphoma and resistant intermediate and high grade Non hodgkins Lymphoma?

Dynamics of partially folded and unfolded proteins investigated with quasielastic neutron spectroscopy

NASA Astrophysics Data System (ADS)

Stadler, Andreas M.

2018-05-01

Molecular dynamics in proteins animate and play a vital role for biologically relevant processes of these biomacromolecules. Quasielastic incoherent neutron scattering (QENS) is a well-suited experimental method to study protein dynamics from the picosecond to several nanoseconds and in the Ångström length-scale. In QENS experiments of protein solutions hydrogens act as reporters for the motions of methyl groups or amino acids to which they are bound. Neutron Spin-Echo spectroscopy (NSE) offers the highest energy resolution in the field of neutron spectroscopy and allows the study of slow collective motions in proteins up to several hundred nanoseconds and in the nanometer length-scale. In the following manuscript I will review recent studies that stress the relevance of molecular dynamics for protein folding and for conformational transitions of intrinsically disordered proteins (IDPs). During the folding collapse the protein is exploring its accessible conformational space via molecular motions. A large flexibility of partially folded and unfolded proteins, therefore, is mandatory for rapid protein folding. IDPs are a special case as they are largely unstructured under physiological conditions. A large flexibility is a characteristic property of IDPs as it allows, for example, the interaction with various binding partners or the rapid response to different conditions.

Partially entangled states bridge in quantum teleportation

NASA Astrophysics Data System (ADS)

Cai, Xiao-Fei; Yu, Xu-Tao; Shi, Li-Hui; Zhang, Zai-Chen

2014-10-01

The traditional method for information transfer in a quantum communication system using partially entangled state resource is quantum distillation or direct teleportation. In order to reduce the waiting time cost in hop-by-hop transmission and execute independently in each node, we propose a quantum bridging method with partially entangled states to teleport quantum states from source node to destination node. We also prove that the designed specific quantum bridging circuit is feasible for partially entangled states teleportation across multiple intermediate nodes. Compared to two traditional ways, our partially entanglement quantum bridging method uses simpler logic gates, has better security, and can be used in less quantum resource situation.

Do Partial Home Smoking Bans Signal Progress toward a Smoke-Free Home?

ERIC Educational Resources Information Center

Kegler, Michelle C.; Haardörfer, Regine; Bundy, Lucja T.; Escoffery, Cam; Berg, Carla J.; Fernandez, Maria; Williams, Rebecca; Hovell, Mel

2016-01-01

Understanding who establishes partial home smoking bans, what these bans cover, and whether they are an intermediate step in going smoke-free would help to inform smoke-free home interventions. Participants were recruited from United Way of Greater Atlanta's 2-1-1 contact center. Data were collected at baseline, 3 and 6 months via telephone…

AGGREGATION PATHWAYS OF THE AMYLOID β(1–42) PEPTIDE DEPEND ON ITS COLLOIDAL STABILITY AND ORDERED β-SHEET STACKING

PubMed Central

Jiang, Dianlu; Rauda, Iris; Han, Shubo; Chen, Shu; Zhou, Feimeng

2012-01-01

Amyloid β (Aβ) fibrils are present as a major component in senile plaques, the hallmark of Alzheimer’s disease (AD). Diffuse plaques (non-fibrous, loosely packed Aβ aggregates) containing amorphous Aβ aggregates are also formed in brain. This work examines the influence of Cu2+ complexation by Aβ on the aggregation process in the context of charge and structural variations. Changes in the surface charges of Aβ molecules due to Cu2+ binding, measured with a zeta potential measurement device, were correlated with the aggregate morphologies examined by atomic force microscopy. As a result of the charge variation, the “colloid-like” stability of the aggregation intermediates, which is essential to the fibrillation process, is affected. Consequently Cu2+ enhances the amorphous aggregate formation. By monitoring variations in the secondary structures with circular dichroism spectroscopy, a direct transformation from the unstructured conformation to the β-sheet structure was observed for all types of aggregates observed (oligomers, fibrils, and/or amorphous aggregates). Compared to the Aβ aggregation pathway in the absence of Cu2+ and taking other factors affecting Aβ aggregation (i.e., pH and temperature) into account, our investigation indicates that formations of amorphous and fibrous aggregates diverge from the same β-sheet-containing partially folded intermediate. This study suggests that the hydrophilic domain of Aβ also plays a role in the Aβ aggregation process. A kinetic model was proposed to account for the effects of the Cu2+ binding on these two aggregation pathways in terms of charge and structural variations. PMID:22870885

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.

Anti-inflammatory activity of polyphenolics from açai (Euterpe oleracea Martius) in intestinal myofibroblasts CCD-18Co cells.

PubMed

Dias, Manoela Maciel dos Santos; Martino, Hércia Stampini Duarte; Noratto, Giuliana; Roque-Andrade, Andrea; Stringheta, Paulo César; Talcott, Stephen; Ramos, Afonso Mota; Mertens-Talcott, Susanne U

2015-10-01

The demand for tropical fruits high in polyphenolics including açai (Euterpe oleracea Mart.) has been increasing based on ascribed health benefits and antioxidant properties. This study evaluated the anti-inflammatory activities of açai polyphenolics in human colon myofibroblastic CCD-18Co cells to investigate the suppression of reactive oxygen species (ROS), and mRNA and protein expression of inflammatory proteins. Non-cytotoxic concentrations of açai extract, 1-5 mg gallic acid equivalent L(-1), were selected. The generation of ROS was induced by lipopolysaccharide (LPS) and açai extract partially reversed this effect to 0.53-fold of the LPS-control. Açai extract (5 mg GAE L(-1)) down-regulated LPS-induced mRNA-expression of tumor necrosis factor alpha, TNF-α (to 0.42-fold), cyclooxygenase 2, COX-2 (to 0.61-fold), toll-like receptor-4, TLR-4 (to 0.52-fold), TNF receptor-associated factor 6, TRAF-6 (to 0.64-fold), nuclear factor kappa-B, NF-κB (to 0.76-fold), vascular cell adhesion molecule 1, VCAM-1 (to 0.71-fold) and intercellular adhesion molecule 1, ICAM-1 (to 0.68-fold). The protein levels of COX-2, TLR-4, p-NF-κB and ICAM-1 were induced by LPS and the açai extract partially reversed this effect in a dose-dependent manner. These results suggest the anti-inflammatory effect of açai polyphenolic extract in intestinal cells are at least in part mediated through the inhibition of ROS and the expression of TLR-4 and NF-κB. Results indicate the potential for açai polyphenolics in the prevention of intestinal inflammation.

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

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.

Synchronization versus decoherence of neutrino oscillations at intermediate densities

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

Raffelt, Georg G.; Tamborra, Irene

2010-12-15

We study collective oscillations of a two-flavor neutrino system with arbitrary but fixed density. In the vacuum limit, modes with different energies quickly dephase (kinematical decoherence), whereas in the limit of infinite density they lock to each other (synchronization). For intermediate densities, we find different classes of solutions. There is always a phase transition in the sense of partial synchronization occurring only above a density threshold. For small mixing angles, partial or complete decoherence can be induced by a parametric resonance, introducing a new time scale to the problem, the final outcome depending on the spectrum and mixing angle. Wemore » derive an analytic relation that allows us to calculate the late-time degree of coherence based on the spectrum alone.« less

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.

Caffeine administration alters the behaviour and development of Galleria mellonella larvae.

PubMed

Maguire, Ronan; Kunc, Martin; Hyrsl, Pavel; Kavanagh, Kevin

2017-11-01

The effect of feeding caffeine on the behaviour and neural proteome of Galleria mellonella larvae was assessed. Caffeine was administered to larvae by force feeding and the metabolites theobromine and theophylline were subsequently detected by RP-HPLC analysis. Administration of caffeine to larvae resulted in reduced movement and a reduction in the formation of pupae. The production of the muscle relaxant theophylline may contribute to the reduction in larval movement. Analysis of the changes in proteome of the brain and surrounding tissues of caffeine fed larvae revealed an increase in the abundance of immune related proteins such as immune-related Hdd1 (6.28 fold increase) and hemolin (1.68 fold increase), ATPase associated proteins such as H+ transporting ATP synthase O subunit isoform 1 (1.87 fold increase) and H+ transporting ATP synthase delta subunit (1.53 fold increase) and proteins indicative of brain trauma such as troponin T transcript variant B, partial (1.55 fold increase). Proteins involved in development and protein degradation such as SUMO-activating enzyme subunit 1 (3.08 fold decrease) and chitin deacetylase, partial (3.67 fold decrease) were decreased in abundance. The results presented here indicate that caffeine is metabolised in a similar way in G. mellonella larvae to that in mammals and results in a variety of behavioural and developmental alterations. Utilisation of insects for studying the effects of caffeine and other neuroactive compounds may offer new insights into their mode of action and reduce the need to use mammals for this type of analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Fluorinated elastomeric materials

DOEpatents

Lagow, Richard J.; Dumitru, Earl T.

1986-11-04

This invention relates to a method of making perfluorinated elastomeric materials, and to materials made by such methods. In the full synthetic scheme, a partially fluorinated polymeric compound, with moieties to prevent crystallization, is created. It is then crosslinked to a desired degree, then perfluorinated. Various intermediate materials, such as partially fluorinated crosslinked polymers, have useful properties, and are or may become commercially available. One embodiment of this invention therefore relates to perfluorination of a selected partially fluorinated, crosslinked material, which is one step of the full synthetic scheme.

Fluorinated elastomeric materials

DOEpatents

Lagow, Richard J.; Dumitru, Earl T.

1990-02-13

This invention relates to a method of making perfluorinated elastomeric materials, and to materials made by such methods. In the full synthetic scheme, a partially fluorinated polymeric compound, with moieties to prevent crystallization, is created. It is then crosslinked to a desired degree, then perfluorinated. Various intermediate materials, such as partially fluorinated crosslinked polymers, have useful properties, and are or may become commercially available. One embodiment of this invention therefore relates to perfluorination of a selected partially fluorinated, crosslinked material, which is one step of the full synthetic scheme.

Differences in the Pathways of Proteins Unfolding Induced by Urea and Guanidine Hydrochloride: Molten Globule State and Aggregates

PubMed Central

Povarova, Olga I.; Kuznetsova, Irina M.; Turoverov, Konstantin K.

2010-01-01

It was shown that at low concentrations guanidine hydrochloride (GdnHCl) can cause aggregation of proteins in partially folded state and that fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) binds with these aggregates rather than with hydrophobic clusters on the surface of protein in molten globule state. That is why the increase in ANS fluorescence intensity is often recorded in the pathway of protein denaturation by GdnHCl, but not by urea. So what was previously believed to be the molten globule state in the pathway of protein denaturation by GdnHCl, in reality, for some proteins represents the aggregates of partially folded molecules. PMID:21152408

The octahaem MccA is a haem c-copper sulfite reductase.

PubMed

Hermann, Bianca; Kern, Melanie; La Pietra, Luigi; Simon, Jörg; Einsle, Oliver

2015-04-30

The six-electron reduction of sulfite to sulfide is the pivot point of the biogeochemical cycle of the element sulfur. The octahaem cytochrome c MccA (also known as SirA) catalyses this reaction for dissimilatory sulfite utilization by various bacteria. It is distinct from known sulfite reductases because it has a substantially higher catalytic activity and a relatively low reactivity towards nitrite. The mechanistic reasons for the increased efficiency of MccA remain to be elucidated. Here we show that anoxically purified MccA exhibited a 2- to 5.5-fold higher specific sulfite reductase activity than the enzyme isolated under oxic conditions. We determined the three-dimensional structure of MccA to 2.2 Å resolution by single-wavelength anomalous dispersion. We find a homotrimer with an unprecedented fold and haem arrangement, as well as a haem bound to a CX15CH motif. The heterobimetallic active-site haem 2 has a Cu(I) ion juxtaposed to a haem c at a Fe-Cu distance of 4.4 Å. While the combination of metals is reminiscent of respiratory haem-copper oxidases, the oxidation-labile Cu(I) centre of MccA did not seem to undergo a redox transition during catalysis. Intact MccA tightly bound SO2 at haem 2, a dehydration product of the substrate sulfite that was partially turned over due to photoreduction by X-ray irradiation, yielding the reaction intermediate SO. Our data show the biometal copper in a new context and function and provide a chemical rationale for the comparatively high catalytic activity of MccA.

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

A computational model of cerebral cortex folding.

PubMed

Nie, Jingxin; Guo, Lei; Li, Gang; Faraco, Carlos; Stephen Miller, L; Liu, Tianming

2010-05-21

The geometric complexity and variability of the human cerebral cortex have long intrigued the scientific community. As a result, quantitative description of cortical folding patterns and the understanding of underlying folding mechanisms have emerged as important research goals. This paper presents a computational 3D geometric model of cerebral cortex folding initialized by MRI data of a human fetal brain and deformed under the governance of a partial differential equation modeling cortical growth. By applying different simulation parameters, our model is able to generate folding convolutions and shape dynamics of the cerebral cortex. The simulations of this 3D geometric model provide computational experimental support to the following hypotheses: (1) Mechanical constraints of the skull regulate the cortical folding process. (2) The cortical folding pattern is dependent on the global cell growth rate of the whole cortex. (3) The cortical folding pattern is dependent on relative rates of cell growth in different cortical areas. (4) The cortical folding pattern is dependent on the initial geometry of the cortex. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

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).

Simulations of flow induced structural transition of the β-switch region of glycoprotein Ibα.

PubMed

Han, Mengzhi; Xu, Ji; Ren, Ying; Li, Jinghai

2016-02-01

Binding of glycoprotein Ibα to von Willebrand factor induces platelet adhesion to injured vessel walls and initiates a multistep hemostatic process. It has been hypothesized that the flow condition could induce a loop to β-sheet conformational change in the β-switch region of glycoprotein Ibα, which regulates it binding to the von Willebrand factor and facilitates the blood clot formation and wound healing. In this work, direct molecular dynamics (MD), flow MD and metadynamics, were employed to investigate the mechanisms of this flow induced conformational transition process. Specifically, the free energy landscape of the whole transition process was drawn by metadynamics with the path collective variable approach. The results reveal that without flow, the free energy landscape has two main basins, including a random loop basin stabilized by large conformational entropy and a partially folded β-sheet basin. The free energy barrier separating these two basins is relatively high and the β-switch could not fold from loop to β-sheet state spontaneously. The fully β-sheet conformations located in high free energy regions, which are also unstable and gradually unfold into partially folded β-sheet state with flow. Relatively weak flow could trigger some folding of the β-switch but could not fold it into fully β-sheet state. Under strong flow conditions, the β-switch could readily overcome the high free energy barrier and fold into fully β-sheet state. Copyright © 2015 Elsevier B.V. All rights reserved.

Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II

NASA Astrophysics Data System (ADS)

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

2018-01-01

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

Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II.

PubMed

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

2017-12-21

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

A dianionic phosphorane intermediate and transition states in an associative A(N)+D(N) mechanism for the ribonucleaseA hydrolysis reaction.

PubMed

Elsässer, Brigitta; Valiev, Marat; Weare, John H

2009-03-25

The RNaseA enzyme efficiently cleaves phosphodiester bonds in the RNA backbone. Phosphoryl transfer plays a central role in many biochemical reactions, and this is one of the most studied enzymes. However, there remains considerable controversy about the reaction mechanism. Most of this debate centers around the roles of the conserved residues, structures of the transition state or states, the possibility of a stable intermediate, and the charge and structure of this intermediate. In this communication we report calculations of the mechanism of the hydrolysis step in this reaction using a comprehensive QM/MM theoretical approach that includes a high level calculation of the interactions in the QM region, free energy estimates along an NEB optimized reaction path, and the inclusion of the interaction of the protein surroundings and solvent. Contrary to prior calculations we find a stable pentacoordinated dianionic phosphorane intermediate in the reaction path supporting an A(N)+D(N) reaction mechanism. In the transition state in the path from the reactant to the intermediate state (with barrier of 3.96 kcal/mol and intermediate stability of 2.21 kcal/mol) a proton from the attacking water is partially transferred to the His119 residue and the PO bond only partially formed from the remaining nucleophilic OH(-) species (bond order (BO) 0.11). In passing from the intermediate to the product state (barrier 13.22 kcal/mol) the PO bond on the cyclic phosphorane intermediate is nearly broken (BO 0.28) and the transfer of the proton from the Lys41 is almost complete (Lys41-H BO 0.87). In the product state a proton has been transferred from Lys41 to the O2' position of the sugar. The role of Lys41 as the catalytic acid is a result of the relative positioning of the Lys41 and His12 in the catalytic site. This configuration is supported by calculations and docking studies.

Variation in chromosome constitution of the Xiaoyan series partial amphiploids and its relations to stripe rust and stem rust resistance

USDA-ARS?s Scientific Manuscript database

In the tertiary gene pool of wheat, tall wheatgrass Thinopyrum ponticum (2n = 10x = 70) is an excellent source of resistance genes against numerous wheat diseases. The creation of wheat-Th. ponticum partial amphiploids is an intermediate step for transferring the useful genes from Th. ponticum to w...

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.

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.

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

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.

Molecular and morphological characterization of the cercariae of Lecithodendrium linstowi (Dollfus, 1931), a trematode of bats, and incrimination of the first intermediate snail host, Radix balthica.

PubMed

Enabulele, Egie E; Lawton, Scott P; Walker, Anthony J; Kirk, Ruth S

2018-03-01

Lecithodendrium linstowi is one of the most prevalent and abundant trematodes of bats, but the larval stages and intermediate hosts have not been identified. We present the first molecular and morphological characterization of the cercariae of L. linstowi based on a phylogenetic analysis of partial fragments of LSU and ITS2 rDNA. The first intermediate host was incriminated as Radix balthica by DNA barcoding using cox1 and ITS2 sequences, although the snail morphologically resembled Radix peregra, emphasizing the requirement for molecular identification of lymnaeids as important intermediate hosts of medical and veterinary impact. The application of molecular data in this study has enabled linkage of life cycle stages and accurate incrimination of the first intermediate host.

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

Mechanism of salt-induced activity enhancement of a marine-derived laccase, Lac15.

PubMed

Li, Jie; Xie, Yanan; Wang, Rui; Fang, Zemin; Fang, Wei; Zhang, Xuecheng; Xiao, Yazhong

2018-04-01

Laccase (benzenediol: oxygen oxidoreductases, EC1.10.3.2) is a multi-copper oxidase capable of oxidizing a variety of phenolic and other aromatic organic compounds. The catalytic power of laccase makes it an attractive candidate for potential applications in many areas of industry including biodegradation of organic pollutants and synthesis of novel drugs. Most laccases are vulnerable to high salt and have limited applications. However, some laccases are not only tolerant to but also activated by certain concentrations of salt and thus have great application potential. The mechanisms of salt-induced activity enhancement of laccases are unclear as yet. In this study, we used dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, intrinsic fluorescence emission, circular dichroism, ultraviolet-visible light absorption, and an enzymatic assay to investigate the potential correlation between the structure and activity of the marine-derived laccase, Lac15, whose activity is promoted by low concentrations of NaCl. The results showed that low concentrations of NaCl exert little influence on the protein structure, which was partially folded in the absence of the salt; moreover, the partially folded rather than the fully folded state seemed to be favorable for enzyme activity, and this partially folded state was distinctive from the so-called 'molten globule' occasionally observed in active enzymes. More data indicated that salt might promote laccase activity through mechanisms involving perturbation of specific local sites rather than a change in global structure. Potential binding sites for chloride ions and their roles in enzyme activity promotion are proposed.

Minimizing donor-site morbidity following bilateral pedicled TRAM breast reconstruction with the double mesh fold over technique.

PubMed

Bharti, Gaurav; Groves, Leslie; Sanger, Claire; Thompson, James; David, Lisa; Marks, Malcolm

2013-05-01

Transverse rectus abdominus muscle flaps (TRAM) can result in significant abdominal wall donor-site morbidity. We present our experience with bilateral pedicle TRAM breast reconstruction using a double-layered polypropylene mesh fold over technique to repair the rectus fascia. A retrospective study was performed that included patients with bilateral pedicle TRAM breast reconstruction and abdominal reconstruction using a double-layered polypropylene mesh fold over technique. Thirty-five patients met the study criteria with a mean age of 49 years old and mean follow-up of 7.4 years. There were no instances of abdominal hernia and only 2 cases (5.7%) of abdominal bulge. Other abdominal complications included partial umbilical necrosis (14.3%), seroma (11.4%), partial wound dehiscence (8.6%), abdominal weakness (5.7%), abdominal laxity (2.9%), and hematoma (2.9%). The TRAM flap is a reliable option for bilateral autologous breast reconstruction. Using the double mesh repair of the abdominal wall can reduce instances of an abdominal bulge and hernia.

Phase behavior and reactive transport of partial melt in heterogeneous mantle model

NASA Astrophysics Data System (ADS)

Jordan, J.; Hesse, M. A.

2013-12-01

The reactive transport of partial melt is the key process that leads to the chemical and physical differentiation of terrestrial planets and smaller celestial bodies. The essential role of the lithological heterogeneities during partial melting of the mantle is increasingly recognized. How far can enriched melts propagate while interacting with the ambient mantle? Can the melt flow emanating from a fertile heterogeneity be localized through a reactive infiltration feedback in a model without exogenous factors or contrived initial conditions? A full understanding of the role of heterogeneities requires reactive melt transport models that account for the phase behavior of major elements. Previous work on reactive transport in the mantle focuses on trace element partitioning; we present the first nonlinear chromatographic analysis of reactive melt transport in systems with binary solid solution. Our analysis shows that reactive melt transport in systems with binary solid solution leads to the formation of two separate reaction fronts: a slow melting/freezing front along which enthalpy change is dominant and a fast dissolution/precipitation front along which compositional changes are dominated by an ion-exchange process over enthalpy change. An intermediate state forms between these two fronts with a bulk-rock composition and enthalpy that are not necessarily bounded by the bulk-rock composition and enthalpy of either the enriched heterogeneity or the depleted ambient mantle. The formation of this intermediate state makes it difficult to anticipate the porosity changes and hence the stability of reaction fronts. Therefore, we develop a graphical representation for the solution that allows identification of the intermediate state by inspection, for all possible bulk-rock compositions and enthalpies of the heterogeneity and the ambient mantle. We apply the analysis to the partial melting of an enriched heterogeneity. This leads to the formation of moving precipitation front that followes a stationary melting front which creates low porosity intermediate states. Therefore, localization of the melt flow is not observed because the precipitation front is stable and the melting front is always stationary under these conditions. This analysis illustrates the counterintuitive behavior that can arise when the phase behavior is taken into account and is a first step to understanding reactive melt transport and the reactive constraints on channelization in partial melts. ¬¬

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.

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.

Development and Validation of a Novel Robotic Procedure Specific Simulation Platform: Partial Nephrectomy.

PubMed

Hung, Andrew J; Shah, Swar H; Dalag, Leonard; Shin, Daniel; Gill, Inderbir S

2015-08-01

We developed a novel procedure specific simulation platform for robotic partial nephrectomy. In this study we prospectively evaluate its face, content, construct and concurrent validity. This hybrid platform features augmented reality and virtual reality. Augmented reality involves 3-dimensional robotic partial nephrectomy surgical videos overlaid with virtual instruments to teach surgical anatomy, technical skills and operative steps. Advanced technical skills are assessed with an embedded full virtual reality renorrhaphy task. Participants were classified as novice (no surgical training, 15), intermediate (less than 100 robotic cases, 13) or expert (100 or more robotic cases, 14) and prospectively assessed. Cohort performance was compared with the Kruskal-Wallis test (construct validity). Post-study questionnaire was used to assess the realism of simulation (face validity) and usefulness for training (content validity). Concurrent validity evaluated correlation between virtual reality renorrhaphy task and a live porcine robotic partial nephrectomy performance (Spearman's analysis). Experts rated the augmented reality content as realistic (median 8/10) and helpful for resident/fellow training (8.0-8.2/10). Experts rated the platform highly for teaching anatomy (9/10) and operative steps (8.5/10) but moderately for technical skills (7.5/10). Experts and intermediates outperformed novices (construct validity) in efficiency (p=0.0002) and accuracy (p=0.002). For virtual reality renorrhaphy, experts outperformed intermediates on GEARS metrics (p=0.002). Virtual reality renorrhaphy and in vivo porcine robotic partial nephrectomy performance correlated significantly (r=0.8, p <0.0001) (concurrent validity). This augmented reality simulation platform displayed face, content and construct validity. Performance in the procedure specific virtual reality task correlated highly with a porcine model (concurrent validity). Future efforts will integrate procedure specific virtual reality tasks and their global assessment. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Mechanistic Insights into the Catalytic Oxidation of Carboxylic Acids on Au/TiO 2: Partial Oxidation of Propionic and Butyric Acid to Gold Ketenylidene through Unsaturated Acids

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

McEntee, Monica; Tang, Wenjie; Neurock, Matthew

Here, the partial oxidation of model C 2–C 4 (acetic, propionic, and butyric) carboxylic acids on Au/TiO 2 catalysts consisting of Au particles ~3 nm in size was investigated using transmission infrared spectroscopy and density functional theory. All three acids readily undergo oxidative dehydrogenation on Au/TiO 2. Propionic and butyric acid dehydrogenate at the C2–C3 positions, whereas acetic acid dehydrogenates at the C1–C2 position. The resulting acrylate and crotonate intermediates are subsequently oxidized to form β-keto acids that decarboxylate. All three acids form a gold ketenylidene intermediate, Au 2C=C=O, along the way to their full oxidation to form CO 2.more » Infrared measurements of Au 2C=C=O formation as a function of time provides a surface spectroscopic probe of the kinetics for the activation and oxidative dehydrogenation of the alkyl groups in the carboxylate intermediates that form.« less

Mechanistic Insights into the Catalytic Oxidation of Carboxylic Acids on Au/TiO 2: Partial Oxidation of Propionic and Butyric Acid to Gold Ketenylidene through Unsaturated Acids

DOE PAGES

McEntee, Monica; Tang, Wenjie; Neurock, Matthew; ...

2014-12-12

Here, the partial oxidation of model C 2–C 4 (acetic, propionic, and butyric) carboxylic acids on Au/TiO 2 catalysts consisting of Au particles ~3 nm in size was investigated using transmission infrared spectroscopy and density functional theory. All three acids readily undergo oxidative dehydrogenation on Au/TiO 2. Propionic and butyric acid dehydrogenate at the C2–C3 positions, whereas acetic acid dehydrogenates at the C1–C2 position. The resulting acrylate and crotonate intermediates are subsequently oxidized to form β-keto acids that decarboxylate. All three acids form a gold ketenylidene intermediate, Au 2C=C=O, along the way to their full oxidation to form CO 2.more » Infrared measurements of Au 2C=C=O formation as a function of time provides a surface spectroscopic probe of the kinetics for the activation and oxidative dehydrogenation of the alkyl groups in the carboxylate intermediates that form.« less

Pressure-induced structural transition of mature HIV-1 protease from a combined NMR/MD simulation approach.

PubMed

Roche, Julien; Louis, John M; Bax, Ad; Best, Robert B

2015-12-01

We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer, using residual dipolar coupling (RDC) measurements in a weakly oriented solution. (1)DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared with 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

PREFACE Protein folding: lessons learned and new frontiers Protein folding: lessons learned and new frontiers

NASA Astrophysics Data System (ADS)

Pappu, Rohit V.; Nussinov, Ruth

2009-03-01

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

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.

The forgotten cause of stridor in the emergency department.

PubMed

Ng, Tian-Tee

2017-01-01

Paradoxical Vocal Fold Movement Disorder is where the larynx exhibits paradoxical vocal cords closure during respiration, creating partial airway obstruction. Causes of vocal fold movement disorder are multifactorial, and patients describe tightness of throat, difficulty getting air in, have stridor, and do not respond to inhalers. We propose using transnasal laryngoscopy examination, which will show narrowing of vocal cords on inspiration, and The Pittsburgh Vocal Cord Dysfunction Index with a cutoff score of ≥4 to distinguish vocal fold movement disorder from asthma and other causes of stridor. Management of paradoxical vocal fold movement disorder involves a combination of pharmacological, psychological, psychiatric, and speech training. Paradoxical vocal fold movement disorder is a very treatable cause of stridor, so long as it is identified and other organic causes are excluded.

Molecular dynamics studies of protein folding and aggregation

NASA Astrophysics Data System (ADS)

Ding, Feng

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

Kinetic analysis of human CYP24A1 metabolism of vitamin D via the C24-oxidation pathway.

PubMed

Tieu, Elaine W; Tang, Edith K Y; Tuckey, Robert C

2014-07-01

CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via the C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its metabolism of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] and the intermediates of the C24-oxidation pathway in a phospholipid-vesicle reconstituted system. The C24-oxidation pathway intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3, were enzymatically produced from 1,25(OH)2 D3 using rat CYP24A1. Both 1,25(OH)2 D3 and 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2 D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate·mol phospholipid(-1) ) that determines their rate of metabolism. CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol·mol phospholipid(-1) , with 24-oxo-1,23,25-trihydroxyvitamin D3 [24-oxo-1,23,25(OH)3 D3] displaying the lowest and 1,24,25-trihydroxyvitamin D3 [1,24,25(OH)3 D3] displaying the highest. The kcat values varied by up to 3.8-fold, with 1,24,25(OH)3 D3 displaying the highest kcat (34 min(-1) ) and 24-oxo-1,23,25(OH)3 D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3 D3 occurs with the highest catalytic efficiency (kcat /Km ) and produces 1-hydroxy-23-oxo-24,25,26,27-tetranorvitamin D3 and not 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the enzyme, which manifests as an accumulation of intermediates, indicating that they dissociate after each catalytic step. © 2014 FEBS.

Selection of stably folded proteins by phage-display with proteolysis.

PubMed

Bai, Yawen; Feng, Hanqiao

2004-05-01

To facilitate the process of protein design and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteolysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. Therefore, in principle it can be applied to any protein. Since its first demonstration in 1998, the method has been used to create hyperthermophilic proteins, to evolve novel folded domains from a library generated by combinatorial shuffling of polypeptide segments and to convert a partially unfolded structure to a fully folded protein.

Design and Study of Efflux Function of EGFP Fused MexAB-OprM Membrane Transporter in Pseudomonas aeruginosa Using Fluorescence Spectroscopy

PubMed Central

Ding, Feng; Lee, Kerry J.; Vahedi-Faridi, Ardeschir; Yoneyama, Hiroshi; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

2014-01-01

Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in ΔMexB (MexB deletion) strain of Pseudomonas aeruginosa to create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined their expression in live cells by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy. Efflux function of the new strain was studied by measuring its accumulation kinetics of ethidium bromide (EtBr, a pump substrate) using fluorescence spectroscopy, which was compared with the cells (WT, ΔMexM, ΔABM, and nalB1) with various expression levels of MexAB-OprM. The new strain shows 6-fold lower accumulation rates of EtBr (15 μM) than ΔABM, 4-fold lower than ΔMexB, but only 1.1-fold higher than WT. As EtBr concentration increases to 40 μM, the new strain has nearly the same accumulation rate of EtBr as ΔMexB, but 1.4-fold higher than WT. We observed the nearly same level of inhibitory effect of CCCP (carbonyl cyanide-m-chlorophenylhydrazone) on the efflux of EtBr by the new strain and WT. Antibiotic susceptibility study shows that the minimum inhibitory concentrations (MICs) of aztreonam (AZT) and chloramphenicol (CP) for the new strain are 6-fold or 3-fold lower than WT, respectively, and 2-fold higher than those of ΔMexB. Taken together, the results suggest that the fusion protein partially retains the efflux function of MexAB-OprM. Modeled structure of the fusion protein shows that the position and orientation of the N-terminal fused EGFP domain may either partially block the translocation pore or restrict the movement of the individual pump domains, which leads to partially restrict efflux activity. PMID:24781334

An improved method to detect correct protein folds using partial clustering.

PubMed

Zhou, Jianjun; Wishart, David S

2013-01-16

Structure-based clustering is commonly used to identify correct protein folds among candidate folds (also called decoys) generated by protein structure prediction programs. However, traditional clustering methods exhibit a poor runtime performance on large decoy sets. We hypothesized that a more efficient "partial" clustering approach in combination with an improved scoring scheme could significantly improve both the speed and performance of existing candidate selection methods. We propose a new scheme that performs rapid but incomplete clustering on protein decoys. Our method detects structurally similar decoys (measured using either C(α) RMSD or GDT-TS score) and extracts representatives from them without assigning every decoy to a cluster. We integrated our new clustering strategy with several different scoring functions to assess both the performance and speed in identifying correct or near-correct folds. Experimental results on 35 Rosetta decoy sets and 40 I-TASSER decoy sets show that our method can improve the correct fold detection rate as assessed by two different quality criteria. This improvement is significantly better than two recently published clustering methods, Durandal and Calibur-lite. Speed and efficiency testing shows that our method can handle much larger decoy sets and is up to 22 times faster than Durandal and Calibur-lite. The new method, named HS-Forest, avoids the computationally expensive task of clustering every decoy, yet still allows superior correct-fold selection. Its improved speed, efficiency and decoy-selection performance should enable structure prediction researchers to work with larger decoy sets and significantly improve their ab initio structure prediction performance.

Effect of temperature on the conformation of natively unfolded protein 4E-BP1 in aqueous and mixed solutions containing trifluoroethanol and hexafluoroisopropanol.

PubMed

Hackl, Ellen V

2015-02-01

Natively unfolded (intrinsically disordered) proteins have attracted growing attention due to their high abundance in nature, involvement in various signalling and regulatory pathways and direct association with many diseases. In the present work the combined effect of temperature and alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), on the natively unfolded 4E-BP1 protein was studied to elucidate the balance between temperature-induced folding and unfolding in intrinsically disordered proteins. It was shown that elevated temperatures induce reversible partial folding of 4E-BP1 both in buffer and in the mixed solutions containing denaturants. In the mixed solutions containing TFE (HFIP) 4E-BP1 adopts a partially folded helical conformation. As the temperature increases, the initial temperature-induced protein folding is replaced by irreversible unfolding/melting only after a certain level of the protein helicity has been reached. Onset unfolding temperature decreases with TFE (HFIP) concentration in solution. It was shown that an increase in the temperature induces two divergent processes in a natively unfolded protein--hydrophobicity-driven folding and unfolding. Balance between these two processes determines thermal behaviour of a protein. The correlation between heat-induced protein unfolding and the amount of helical content in a protein is revealed. Heat-induced secondary structure formation can be a valuable test to characterise minor changes in the conformations of natively unfolded proteins as a result of site-directed mutagenesis. Mutants with an increased propensity to fold into a structured form reveal different temperature behaviour.

Characterization of a transient +2 sulfur oxidation state intermediate from the oxidation of aqueous sulfide

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

Vairavmurthy, M.A.; Zhou, Weiqing

1995-04-01

The oxidation H{sub 2}S to sulfate involves a net transfer of eight electrons and occurs through the formation of several partially oxidized intermediates with oxidation states ranging from {minus}1 to +5. Known intermediates include elemental sulfur (oxidation state 0), polysulfides (outer sulfur: {minus}1, inner sulfur: 0), sulfite (+4) and thiosulfate (outer sulfur: {minus}1, inner sulfur: +5). A noticeable gap in this series of intermediates is that of a +2 sulfur oxidation state oxoacid/oxoanion species, which was never detected experimentally. Here, we present evidence of the transient existence of +2 oxidation state intermediate in the Ni(II)-catalyzed oxidation of aqueous sulfide. X-raymore » absorption near-edge structure (XANES) spectroscopy and Fourier-transform-infrared (FT-IR) spectroscopy were used to characterize this species; they suggest that it has a sulfoxylate ion (SO{sub 2}{sup 2{minus}}) structure.« less

Merits of using mechanical treatments to stimulate cone production of slash and longleaf pine

Treesearch

James P. Barnett

1993-01-01

four mechanical treatments (untreated, partial girdling in the spring, partial girdling in summer, and banding in spring) stimulated cone production of pole-sized slash and longleaf pines. A 2- to 3-fold increase in slash pine seed production was limited to the first crop originating after the treatments were applied. However, the treatments killed half the longleaf...

The Coupled Photochemistry of Ammonia and Acetylene: Applications to the Atmospheric Chemistry on Jupiter

NASA Astrophysics Data System (ADS)

Keane, Thomas Christopher

1995-01-01

The existence of hydrogen cyanide (HCN) in the highly reducing atmosphere of Jupiter was a surprising discovery (Tokunaga et al., 1981). Previous studies that tested the theoretical proposal of Kaye and Strobel (1983a) that the HCN observed on Jupiter is the result of NH _3 photolysis in the presence of C _2H_2 established that acetonitrile (CH_3CN) and acetaldazine (CH _3CH=NN=CHCH_3) are important intermediates in HCN formation (Ferris and Ishikawa, 1988). In this study the rates of formation of these compounds, and of other recently detected intermediates, have been determined in static photolysis experiments at 296 K and at temperatures which are closer to those found in the Jovian atmosphere. Experiments were also performed, using a photochemical flow reactor, that allowed for a better approximation of the mixing ratios of reactant gases (8 times 10^{ -4} for NH_3 and 1 times 10^{-5} for C_2H_2) and the process of advection in the Jovian atmosphere. An overall reaction pathway for HCN formation is proposed. Major intermediates and products found in these laboratory simulations that have not yet been observed on Jupiter are acetonitrile (CH_3CN), acetaldazine (CH_3CH=NN=CHCH _3), acetaldehyde hydrazone (CH_3 CH=NNH_2), N-ethylethylideneimine (CH_3CH=NC_2H _5), ethylamine (C_2H _5NH_2) and methylamine (CH _3NH_2). HCN is formed by the photolysis of NH_3/C _2H_2 mixtures (40:5 torr) at 296 K and at low temperature (208 K, 195 K and 180 K) with the highest quantum efficiency for HCN formation observed at 180 K. In static experiments using a high partial pressure of H_2 the quantum yield for HCN formation decreased three-fold relative to the 296 K photolyses when no H_2 was used. An additional ten-fold decrease in the quantum yield for HCN formation occurred when using the flow system. The quantum yields for acetaldazine and acetaldehyde hydrazone formation were found to vary inversely to that for HCN formation. For those static experiments which best simulate Jovian reaction conditions (H_2: NH_3 : C_2H_2 = 600: 7.5: 5 torr, 180 K) the following products and their quantum yields for formation were obtained: C_2H_4 (0.129), CH_3 CH=NN=CHCH_3 (0.079), CH _3CH=NNH_2 (0.049), C_2H_5NH_2 (0.038), CH_3NH_2 (0.003), CH_3CN (0.002), HCN (0.001) and CH_3CH=NC _2H_5 (0.001).

Biomechanics of fundamental frequency regulation: Constitutive modeling of the vocal fold lamina propria.

PubMed

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

2009-12-01

Accurate characterization of biomechanical characteristics of the vocal fold is critical for understanding the regulation of vocal fundamental frequency (F(0)), which depends on the active control of the intrinsic laryngeal muscles as well as the passive biomechanical response of the vocal fold lamina propria. Specifically, the tissue stress-strain response and viscoelastic properties under cyclic tensile deformation are relevant, when the vocal folds are subjected to length and tension changes due to posturing. This paper describes a constitutive modeling approach quantifying the relationship between vocal fold stress and strain (or stretch), and establishes predictions of F(0) with the string model of phonation based on the constitutive parameters. Results indicated that transient and time-dependent changes in F(0), including global declinations in declarative sentences, as well as local F(0) overshoots and undershoots, can be partially attributed to the time-dependent viscoplastic response of the vocal fold cover.

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 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.

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

Enhanced Scattering of Diffuse Ions on Front of the Earth's Quasi-Parallel Bow Shock: a Case Study

NASA Astrophysics Data System (ADS)

Kis, A.; Matsukiyo, S.; Otsuka, F.; Hada, T.; Lemperger, I.; Dandouras, I. S.; Barta, V.; Facsko, G. I.

2017-12-01

In the analysis we present a case study of three energetic upstream ion events at the Earth's quasi-parallel bow shock based on multi-spacecraft data recorded by Cluster. The CIS-HIA instrument onboard Cluster provides partial energetic ion densities in 4 energy channels between 10 and 32 keV.The difference of the partial ion densities recorded by the individual spacecraft at various distances from the bow shock surface makes possible the determination of the spatial gradient of energetic ions.Using the gradient values we determined the spatial profile of the energetic ion partial densities as a function of distance from the bow shock and we calculated the e-folding distance and the diffusion coefficient for each event and each ion energy range. Results show that in two cases the scattering of diffuse ions takes place in a normal way, as "by the book", and the e-folding distance and diffusion coefficient values are comparable with previous results. On the other hand, in the third case the e-folding distance and the diffusion coefficient values are significantly lower, which suggests that in this case the scattering process -and therefore the diffusive shock acceleration (DSA) mechanism also- is much more efficient. Our analysis provides an explanation for this "enhanced" scattering process recorded in the third case.

A long serendipitous XMM-Newton observation of the intermediate polar XY Ari1

NASA Astrophysics Data System (ADS)

Zengin Ćamurdan, D.; Balman, Ş.; Burwitz, V.

2018-07-01

XY Ari is one of the few known eclipsing intermediate polars. We present results from a detailed analysis of an unpublished archival observation using XMM-Newton EPIC pn and MOS data in a quiescent state of XY Ari. The X-ray orbital modulation and spin-pulse variations were investigated for energy-dependent modulations in different energy bands. The broad orbital modulation observed with various observations was confirmed with XMM-Newton in the hard X-ray (>1.6 keV). The EPIC light curves folded at the spin phases show a double-peak profile, as expected from two-pole accretion. The pulse profile is found to be energy-dependent. Hardness-ratio variations and energy modulation depth during spin modulation can be explained by photoelectric absorption. The simultaneously fitted EPIC spectra with a multi-temperature plasma emission model (CEVMKL) model yield a maximum plasma temperature of 28^{+3.1}_{-2.9} keV with an iron abundance Fe/Fe_{⊙}=0.37^{+0.06}_{-0.05}. We find two intrinsic partial covering absorption columns of 6.2^{+1.0}_{-0.9} × 10^{22} and 105.3^{+35.4}_{-30.4} × 10^{22} cm^{-2} with covering fractions of 0.53^{+0.05}_{-0.04}, 0.41^{+0.14}_{-0.13}, respectively. In addition, a Gaussian emission line at 6.43^{+0.01}_{-0.02} keV with an equivalent width of 51^{+12}_{-10} eV is required to account for fluorescent emission from neutral iron. The X-ray luminosity of the source is 4.2 × 1032 erg s-1 in the 0.2-10.0 keV energy band.

Alternative modes of client binding enable functional plasticity of Hsp70

NASA Astrophysics Data System (ADS)

Mashaghi, Alireza; Bezrukavnikov, Sergey; Minde, David P.; Wentink, Anne S.; Kityk, Roman; Zachmann-Brand, Beate; Mayer, Matthias P.; Kramer, Günter; Bukau, Bernd; Tans, Sander J.

2016-11-01

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

Prolonged fasting increases purine recycling in post-weaned northern elephant seals.

PubMed

Soñanez-Organis, José Guadalupe; Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Aguilar, Andres; Crocker, Daniel E; Ortiz, Rudy M

2012-05-01

Northern elephant seals are naturally adapted to prolonged periods (1-2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, food deprivation stimulates ATP degradation and decreases ATP synthesis, resulting in the accumulation of purines (ATP degradation byproducts). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism, which also promotes oxidant production. The contributions of HGPRT to purine recycling during prolonged food deprivation in marine mammals are not well defined. In the present study we cloned and characterized the complete and partial cDNA sequences that encode for HGPRT and xanthine oxidoreductase (XOR) in northern elephant seals. We also measured XO protein expression and circulating activity, along with xanthine and hypoxanthine plasma content in fasting northern elephant seal pups. Blood, adipose and muscle tissue samples were collected from animals after 1, 3, 5 and 7 weeks of their natural post-weaning fast. The complete HGPRT and partial XOR cDNA sequences are 771 and 345 bp long and encode proteins of 218 and 115 amino acids, respectively, with conserved domains important for their function and regulation. XOR mRNA and XO protein expression increased 3-fold and 1.7-fold with fasting, respectively, whereas HGPRT mRNA (4-fold) and protein (2-fold) expression increased after 7 weeks in adipose tissue and muscle. Plasma xanthine (3-fold) and hypoxanthine (2.5-fold) levels, and XO (1.7- to 20-fold) and HGPRT (1.5- to 1.7-fold) activities increased during the last 2 weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to ameliorating oxidant production and enhancing the supply of ATP, both of which would be beneficial during prolonged food deprivation and appear to be adaptive in this species.

Prolonged fasting increases purine recycling in post-weaned northern elephant seals

PubMed Central

Soñanez-Organis, José Guadalupe; Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Aguilar, Andres; Crocker, Daniel E.; Ortiz, Rudy M.

2012-01-01

SUMMARY Northern elephant seals are naturally adapted to prolonged periods (1–2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, food deprivation stimulates ATP degradation and decreases ATP synthesis, resulting in the accumulation of purines (ATP degradation byproducts). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism, which also promotes oxidant production. The contributions of HGPRT to purine recycling during prolonged food deprivation in marine mammals are not well defined. In the present study we cloned and characterized the complete and partial cDNA sequences that encode for HGPRT and xanthine oxidoreductase (XOR) in northern elephant seals. We also measured XO protein expression and circulating activity, along with xanthine and hypoxanthine plasma content in fasting northern elephant seal pups. Blood, adipose and muscle tissue samples were collected from animals after 1, 3, 5 and 7 weeks of their natural post-weaning fast. The complete HGPRT and partial XOR cDNA sequences are 771 and 345 bp long and encode proteins of 218 and 115 amino acids, respectively, with conserved domains important for their function and regulation. XOR mRNA and XO protein expression increased 3-fold and 1.7-fold with fasting, respectively, whereas HGPRT mRNA (4-fold) and protein (2-fold) expression increased after 7 weeks in adipose tissue and muscle. Plasma xanthine (3-fold) and hypoxanthine (2.5-fold) levels, and XO (1.7- to 20-fold) and HGPRT (1.5- to 1.7-fold) activities increased during the last 2 weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to ameliorating oxidant production and enhancing the supply of ATP, both of which would be beneficial during prolonged food deprivation and appear to be adaptive in this species. PMID:22496280

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.

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

Asphyxia by Drowning Induces Massive Bleeding Due To Hyperfibrinolytic Disseminated Intravascular Coagulation.

PubMed

Schwameis, Michael; Schober, Andreas; Schörgenhofer, Christian; Sperr, Wolfgang Reinhard; Schöchl, Herbert; Janata-Schwatczek, Karin; Kürkciyan, Erol Istepan; Sterz, Fritz; Jilma, Bernd

2015-11-01

To date, no study has systematically investigated the impact of drowning-induced asphyxia on hemostasis. Our objective was to test the hypothesis that asphyxia induces bleeding by hyperfibrinolytic disseminated intravascular coagulation. Observational study. A 2,100-bed tertiary care facility in Vienna, Austria, Europe. All cases of drowning-induced asphyxia (n=49) were compared with other patients with cardiopulmonary resuscitation (n=116) and to patients with acute promyelocytic leukemia (n=83). Six drowning victims were investigated prospectively. To study the mechanism, a forearm-ischemia model was used in 20 volunteers to investigate whether hypoxia releases tissue plasminogen activator. None. Eighty percent of patients with drowning-induced asphyxia developed overt disseminated intravascular coagulation within 24 hours. When compared with nondrowning cardiac arrest patients, drowning patients had a 13 times higher prevalence of overt disseminated intravascular coagulation at admission (55% vs 4%; p<0.001). Despite comparable disseminated intravascular coagulation scores, acute promyelocytic leukemia patients had higher fibrinogen but lower d-dimer levels and platelet counts than drowning patients (p<0.001). Drowning victims had a three-fold longer activated partial thromboplastin time (124 s; p<0.001) than both nondrowning cardiac arrest and acute promyelocytic leukemia patients. Hyperfibrinolysis was reflected by up to 1,000-fold increased d-dimer levels, greater than 5-fold elevated plasmin antiplasmin levels, and a complete absence of thrombelastometric clotting patterns, which was reversed by antifibrinolytics and heparinase. Thirty minutes of forearm-ischemia increased tissue plasminogen activator 31-fold (p<0.001). The vast majority of drowning patients develops overt hyperfibrinolytic disseminated intravascular coagulation, partly caused by hypoxia induced tissue plasminogen activator release. Antifibrinolytics and heparinase partially reverse the abnormal clotting patterns. Severe activated partial thromboplastin time prolongation may be a marker of combined hyperfibrinolytic afibrinogenemia and autoheparinization in drowning-related asphyxia.

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".

Michelin tire baby syndrome--a case report and literature review.

PubMed

Farooqi, Ghazala A; Mulla, Shafeek A; Ahmad, Mohammad

2010-09-01

Michelin tire syndrome is described in a two month old infant of Filipino-Saudi parents. The infant had generalized excessive folding of skin and facial dysmorphism. The skin biopsy showed excessive adipose tissue in reticular dermis, papillary dermis and around adnexa. Spontaneous partial improvement in skin folding was noted on follow up. To the best of our knowledge, this is the first case ever reported locally of Michelin tire.

Unfolding Kinetics of β-Lactoglobulin Induced by Surfactant and Denaturant: A Stopped-Flow/Fluorescence Study

PubMed Central

Viseu, Maria Isabel; Melo, Eduardo P.; Carvalho, Teresa Isabel; Correia, Raquel F.; Costa, Sílvia M. B.

2007-01-01

The β→α transition of β-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine β-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the α-helical content increases, leading to the so-called α-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I1) with mostly native secondary structure but loose tertiary structure appears between the native (β) and α-states; in GnHCl, another intermediate (I2) appears between states β and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species. PMID:17693475

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.

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.

Tracking of BMI, fatness and cardiorespiratory fitness from adolescence to middle adulthood: the Zagreb Growth and Development Longitudinal Study.

PubMed

Sorić, Maroje; Jembrek Gostović, Mirjana; Gostović, Mladen; Hočevar, Marija; Mišigoj-Duraković, Marjeta

2014-01-01

Effective intervention strategies aiming to improve cardiorespiratory fitness and to decrease body fatness are needed. However, long-term stability of these traits is not well understood. To assess long-term tracking of cardiorespiratory fitness and body fatness from late adolescence to middle adulthood. The sample consisted of 50 participants (31 boys) from the Zagreb Growth and Development Longitudinal Study who were followed up in adulthood (median age = 43). Fatness was evaluated through BMI and skin-folds, while cardiorespiratory fitness was assessed using a cardiopulmonary exercise test. Inter-age partial correlation coefficients were calculated to evaluate tracking. Body mass index and skin-folds showed moderate tracking from age 15 years to middle adulthood (partial r = 0.55, p < 0.001 and partial r = 0.52, p < 0.001, respectively), while tracking of subcutaneous fat distribution was somewhat lower (partial r = 0.38, p < 0.01). At the same time, the observed tracking of peak oxygen uptake was low-to-moderate (partial r = 0.30, p = 0.03), while ventilatory aerobic and anaerobic thresholds did not show significant tracking. The results of this study indicate that preventive efforts aiming to increase cardiorespiratory fitness should include all adolescents, irrespective of their cardiorespiratory fitness status. Conversely, strategies aiming at obesity prevention should focus on high-risk groups of adolescents.

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

Aggregation of γ-crystallins associated with human cataracts via domain swapping at the C-terminal β-strands

PubMed Central

Das, Payel; King, Jonathan A.; Zhou, Ruhong

2011-01-01

The prevalent eye disease age-onset cataract is associated with aggregation of human γD-crystallins, one of the longest-lived proteins. Identification of the γ-crystallin precursors to aggregates is crucial for developing strategies to prevent and reverse cataract. Our microseconds of atomistic molecular dynamics simulations uncover the molecular structure of the experimentally detected aggregation-prone folding intermediate species of monomeric native γD-crystallin with a largely folded C-terminal domain and a mostly unfolded N-terminal domain. About 30 residues including a, b, and c strands from the Greek Key motif 4 of the C-terminal domain experience strong solvent exposure of hydrophobic residues as well as partial unstructuring upon N-terminal domain unfolding. Those strands comprise the domain–domain interface crucial for unusually high stability of γD-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors, which reveals domain-swapped dimeric structures. In the simulated dimeric structures, the N-terminal domain of one monomer is frequently found in contact with residues 135–164 encompassing the a, b, and c strands of the Greek Key motif 4 of the second molecule. The present results suggest that γD-crystallin may polymerize through successive domain swapping of those three C-terminal β-strands leading to age-onset cataract, as an evolutionary cost of its very high stability. Alanine substitutions of the hydrophobic residues in those aggregation-prone β-strands, such as L145 and M147, hinder domain swapping as a pathway toward dimerization. These findings thus provide critical molecular insights onto the initial stages of age-onset cataract, which is important for understanding protein aggregation diseases. PMID:21670251

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.

Improved production of an enzyme that hydrolyses raw yam starch by Penicillium sp. S-22 using fed-batch fermentation.

PubMed

Sun, Hai-Yan; Ge, Xiang-Yang; Zhang, Wei-Guo

2006-11-01

A newly isolated strain, Penicillium sp. S-22, was used to produce an enzyme that hydrolyses raw yam starch [raw yam starch digesting enzyme (RYSDE)]. The enzyme activity and overall enzyme productivity were respectively 16 U/ml and 0.19 U/ml h in the batch culture. The enzyme activity increased to 85 U/ml by feeding of partially hydrolyzed raw yam starch. When a mixture containing partially hydrolyzed raw yam starch and peptone was fed by a pH-stat strategy, the enzyme activity reached 366 U/ml, 23-fold of that obtained in the batch culture, and the overall productivity reached 3.4 U/ml h, which was 18-fold of that in the batch culture.

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

Ameliorating effects of fluorocarbon emulsion on sickle red blood cell-induced obstruction in an ex vivo vasculature.

PubMed

Kaul, D K; Liu, X; Nagel, R L

2001-11-15

In sickle cell (SS) vaso-occlusion, the culminating event is blockage of blood vessels by sickled red blood cells (SS RBCs). As shown in animal models, SS RBC-induced vaso-occlusion is often partial, allowing for a residual flow, hence oxygen delivery to partially occluded vessels could reduce vaso-occlusion. The efficacy of an oxygenated perflubron-based fluorocarbon emulsion (PFE) was tested for its anti-vaso-occlusive effects in the ex vivo mesocecum vasculature of the rat. Microvascular obstruction was induced by the infusion of deoxygenated SS RBCs into ex vivo preparations with or without pretreatment with platelet-activating factor (PAF). PAF induced enhanced SS RBC-endothelium interactions, leading to greater vaso-occlusion. Microvascular blockage resulted in increased peripheral resistance units (PRU). Deoxygenated SS RBCs caused a persistent 1.5-fold PRU increase in untreated preparations and approximately a 2-fold PRU increase in PAF-treated preparations. The greater PRU in PAF-treated preparations was caused by widespread adhesion and postcapillary blockage. Oxygenated PFE, but not deoxygenated PFE, resulted in PRU decreases to baseline values in both groups of experiments (with or without PAF). The PRU decrease caused by oxygenated PFE infusion was caused by unsickling of SS RBCs in partially occluded vessels, with no antiadhesive effect on already adherent SS RBCs as assessed by intravital microscopy. PFE had no effect on vascular tone. The efficacy of PFE appears to result from its greater capacity to dissolve oxygen (10-fold higher than plasma). The dislodgement of trapped SS RBCs and an increase in wall shear rates will help reverse the partial obstruction. Thus, oxygenated PFE is capable of reducing SS RBC-induced vaso-occlusion, and further development of this approach is advisable.

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

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

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.

Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification.

PubMed

Dey, Tapati Bhanja; Banerjee, Rintu

2014-01-01

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T(660 nm) = 97.0%) of juice was attained after 2 h of incubation at 50 °C in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.

Partial wave analysis for folded differential cross sections

NASA Astrophysics Data System (ADS)

Machacek, J. R.; McEachran, R. P.

2018-03-01

The value of modified effective range theory (MERT) and the connection between differential cross sections and phase shifts in low-energy electron scattering has long been recognized. Recent experimental techniques involving magnetically confined beams have introduced the concept of folded differential cross sections (FDCS) where the forward (θ ≤ π/2) and backward scattered (θ ≥ π/2) projectiles are unresolved, that is the value measured at the angle θ is the sum of the signal for particles scattered into the angles θ and π - θ. We have developed an alternative approach to MERT in order to analyse low-energy folded differential cross sections for positrons and electrons. This results in a simplified expression for the FDCS when it is expressed in terms of partial waves and thereby enables one to extract the first few phase shifts from a fit to an experimental FDCS at low energies. Thus, this method predicts forward and backward angle scattering (0 to π) using only experimental FDCS data and can be used to determine the total elastic cross section solely from experimental results at low-energy, which are limited in angular range.

Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification

PubMed Central

Dey, Tapati Bhanja; Banerjee, Rintu

2014-01-01

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T660nm = 97.0%) of juice was attained after 2 h of incubation at 50 °C in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property. PMID:24948919

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.

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.

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

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

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.

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

Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp

We present that challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperonemore » Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.« less

Visualizing chaperone-assisted protein folding

DOE PAGES

Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; ...

2016-05-30

We present that challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperonemore » Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.« less

Effect of plasma nitriding on the structural stability and hydrogen absorption capability of Pd-coated Nb during thermal treatment

NASA Astrophysics Data System (ADS)

Ohtsu, Naofumi; Kozuka, Taro; Shibata, Yuga; Yamane, Misao

2017-11-01

Plasma nitriding was explored for improving the thermal stability of a composite hydrogen permeable membrane comprising a Pd coating on Nb substrate. A NbN intermediate layer was formed on the Nb substrate, and the progress of interdiffusion and deterioration of hydrogen absorption behavior after a thermal treatment at 573 and 773 K, respectively, were investigated. The intermediate layer significantly suppressed the interdiffusion between the coating and the substrate. Furthermore, an increase in the NbN concentration of the intermediate layer enhanced the suppression efficiency. However, the hydrogen permeability of the intermediate layer was significantly low, and hence, an increase in NbN concentration further decreased the hydrogen permeability. We concluded that the nitride layer with a high NbN content was unsuitable as an intermediate layer owing to its low hydrogen permeability, while the partial nitride layer with a low NbN content was inefficient in suppressing the interdiffusion.

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

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

Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

PubMed Central

Anderson, J. W.; Fowden, L.

1970-01-01

1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [32P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase. PMID:5493505

Partial entrainment of gravel bars during floods

USGS Publications Warehouse

Konrad, Christopher P.; Booth, Derek B.; Burges, Stephen J.; Montgomery, David R.

2002-01-01

Spatial patterns of bed material entrainment by floods were documented at seven gravel bars using arrays of metal washers (bed tags) placed in the streambed. The observed patterns were used to test a general stochastic model that bed material entrainment is a spatially independent, random process where the probability of entrainment is uniform over a gravel bar and a function of the peak dimensionless shear stress τ0* of the flood. The fraction of tags missing from a gravel bar during a flood, or partial entrainment, had an approximately normal distribution with respect to τ0* with a mean value (50% of the tags entrained) of 0.085 and standard deviation of 0.022 (root‐mean‐square error of 0.09). Variation in partial entrainment for a given τ0* demonstrated the effects of flow conditioning on bed strength, with lower values of partial entrainment after intermediate magnitude floods (0.065 < τ0*< 0.08) than after higher magnitude floods. Although the probability of bed material entrainment was approximately uniform over a gravel bar during individual floods and independent from flood to flood, regions of preferential stability and instability emerged at some bars over the course of a wet season. Deviations from spatially uniform and independent bed material entrainment were most pronounced for reaches with varied flow and in consecutive floods with small to intermediate magnitudes.

Starch and the Control of Kernel Number in Maize at Low Water Potentials1

PubMed Central

Zinselmeier, Christopher; Jeong, Byeong-Ryong; Boyer, John S.

1999-01-01

After reproduction is initiated in plants, subsequent reproductive development is sometimes interrupted, which decreases the final number of seeds and fruits. We subjected maize (Zea mays L.) to low water potentials (ψw) that frequently cause this kind of failure. We observed metabolite pools and enzyme activities in the developing ovaries while we manipulated the sugar stream by feeding sucrose (Suc) to the stems. Low ψw imposed for 5 d around pollination allowed embryos to form, but abortion occurred and kernel number decreased markedly. The ovary contained starch that nearly disappeared during this abortion. Analyses showed that all of the intermediates in starch synthesis were depleted. However, when labeled Suc was fed to the stems, label arrived at the ovaries. Solute accumulated and caused osmotic adjustment. Suc accumulated, but other intermediates did not, showing that a partial block in starch synthesis occurred at the first step in Suc utilization. This step was mediated by invertase, which had low activity. Because of the block, Suc feeding only partially prevented starch disappearance and abortion. These results indicate that young embryos abort when the sugar stream is interrupted sufficiently to deplete starch during early ovary development, and this abortion results in a loss of mature seeds and fruits. At low ψw, maintaining the sugar stream partially prevented the abortion, but invertase regulated the synthesis of ovary starch and partially prevented full recovery. PMID:10482657

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

Fat digestion by lingual lipase: mechanism of lipolysis in the stomach and upper small intestine.

PubMed

Liao, T H; Hamosh, P; Hamosh, M

1984-05-01

Ten to 30% of dietary fat is hydrolyzed in the stomach by lingual lipase, an enzyme secreted from lingual serous glands. We investigated the substrate specificity of this enzyme as well as the potential of lingual lipase to act in the upper small intestine i.e., in the presence of bile salts and lecithin. The data presented show that partially purified preparations of rat lingual lipase and the lipase in gastric aspirates of newborn infants have identical substrate specificity: medium-chain triglycerides were hydrolyzed at rates 5-8-fold higher than long-chain triglycerides; the rat and human enzymes do not hydrolyze the ester bond of lecithin or cholesteryl-ester. In contrast to pancreatic lipase, the hydrolysis of triglycerides by lingual lipase is not inhibited by lecithin. But, similar to pancreatic lipase the activity of lingual lipase is inhibited by bile salts, the extent of inhibition varying with its nature and concentration. This inactivation is not prevented by colipase but is partially averted by lipids and protein, suggesting that lingual lipase can remain active in the duodenum. The pH optimum of the enzyme (2.2-6.5 in the rat and 3.5-6.0 in human gastric aspirates) is compatible with continued activity in the upper small intestine, especially during the neonatal period, when the luminal pH is under 6.5. The marked variation in lipase activity levels in gastric aspirates of newborn infants is probably due to individual variations in enzyme amounts. The characteristics of the lipase are however identical in infants with low, intermediate or high activity levels.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Clusters of isoleucine, leucine, and valine side chains define cores of stability in high-energy states of globular proteins: Sequence determinants of structure and stability.

PubMed

Kathuria, Sagar V; Chan, Yvonne H; Nobrega, R Paul; Özen, Ayşegül; Matthews, C Robert

2016-03-01

Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates. © 2015 The Protein Society.

Partial oxidation of alkanes by dioxiranes formed in situ at low temperature.

PubMed

Yacob, Sara; Caulfield, Michael J; Barckholtz, Timothy A

2018-01-13

Partial oxidation catalysts capable of efficiently operating at low temperatures may limit the over-oxidation of alkane substrates and thereby improve selectivity. This work focuses on examining alkane oxidation using completely metal-free organocatalysts, dioxiranes. The dioxiranes employed here are synthesized by oxidation of a ketone using a terminal oxidant, such as hydrogen peroxide. Our work generates the dioxirane in situ , so that the process can be catalytic with respect to the ketone. To date, we have demonstrated selective partial oxidation of adamantane using ketone catalysts resulting in yields upwards of 60% towards 1-adamantanol with greater than 99% selectivity. Furthermore, we have demonstrated that changing the electrophilic character of the ketone R groups to contain more electron-donating ligands facilitates the dioxirane ring formation and improves overall oxidation yields. Isotopic labelling studies using H 2 18 O 2 show the preferential incorporation of an 18 O label into the parent ketone, providing evidence for a dioxirane intermediate formed in situ The isotopic labelling studies, along with solvent effect studies, suggest the formation of peracetic acid as a reactive intermediate.This article is part of a discussion meeting issue 'Providing sustainable catalytic solutions for a rapidly changing world'. © 2017 The Author(s).

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.

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

Method for producing metal oxide aerogels

DOEpatents

Tillotson, Thomas M.; Poco, John F.; Hrubesh, Lawrence W.; Thomas, Ian M.

1995-01-01

A two-step hydrolysis-condensation method was developed to form metal oxide aerogels of any density, including densities of less than 0.003g/cm.sup.3 and greater than 0.27g/cm.sup.3. High purity metal alkoxide is reacted with water, alcohol solvent, and an additive to form a partially condensed metal intermediate. All solvent and reaction-generated alcohol is removed, and the intermediate is diluted with a nonalcoholic solvent. The intermediate can be stored for future use to make aerogels of any density. The aerogels are formed by reacting the intermediate with water, nonalcoholic solvent, and a catalyst, and extracting the nonalcoholic solvent directly. The resulting monolithic aerogels are hydrophobic and stable under atmospheric conditions, and exhibit good optical transparency, high clarity, and homogeneity. The aerogels have high thermal insulation capacity, high porosity, mechanical strength and stability, and require shorter gelation times than aerogels formed by conventional methods.

Method for producing metal oxide aerogels

DOEpatents

Tillotson, T.M.; Poco, J.F.; Hrubesh, L.W.; Thomas, I.M.

1995-04-25

A two-step hydrolysis-condensation method was developed to form metal oxide aerogels of any density, including densities of less than 0.003g/cm{sup 3} and greater than 0.27g/cm{sup 3}. High purity metal alkoxide is reacted with water, alcohol solvent, and an additive to form a partially condensed metal intermediate. All solvent and reaction-generated alcohol is removed, and the intermediate is diluted with a nonalcoholic solvent. The intermediate can be stored for future use to make aerogels of any density. The aerogels are formed by reacting the intermediate with water, nonalcoholic solvent, and a catalyst, and extracting the nonalcoholic solvent directly. The resulting monolithic aerogels are hydrophobic and stable under atmospheric conditions, and exhibit good optical transparency, high clarity, and homogeneity. The aerogels have high thermal insulation capacity, high porosity, mechanical strength and stability, and require shorter gelation times than aerogels formed by conventional methods. 8 figs.

Unraveling metamaterial properties in zigzag-base folded sheets.

PubMed

Eidini, Maryam; Paulino, Glaucio H

2015-09-01

Creating complex spatial objects from a flat sheet of material using origami folding techniques has attracted attention in science and engineering. In the present work, we use the geometric properties of partially folded zigzag strips to better describe the kinematics of known zigzag/herringbone-base folded sheet metamaterials such as Miura-ori. Inspired by the kinematics of a one-degree of freedom zigzag strip, we introduce a class of cellular folded mechanical metamaterials comprising different scales of zigzag strips. This class of patterns combines origami folding techniques with kirigami. Using analytical and numerical models, we study the key mechanical properties of the folded materials. We show that our class of patterns, by expanding on the design space of Miura-ori, is appropriate for a wide range of applications from mechanical metamaterials to deployable structures at small and large scales. We further show that, depending on the geometry, these materials exhibit either negative or positive in-plane Poisson's ratios. By introducing a class of zigzag-base materials in the current study, we unify the concept of in-plane Poisson's ratio for similar materials in the literature and extend it to the class of zigzag-base folded sheet materials.

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

PubMed

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

2015-04-14

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

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.

Spatially resolving the secretome within the mycelium of the cell factory Aspergillus niger.

PubMed

Krijgsheld, Pauline; Altelaar, A F Maarten; Post, Harm; Ringrose, Jeffrey H; Müller, Wally H; Heck, Albert J R; Wösten, Han A B

2012-05-04

Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature.

Preventing α-synuclein aggregation: the role of the small heat-shock molecular chaperone proteins.

PubMed

Cox, Dezerae; Carver, John A; Ecroyd, Heath

2014-09-01

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

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

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.

The electromagnetic interchange mode in a partially ionized collisional plasma. [spread F region

NASA Technical Reports Server (NTRS)

Hudson, M. K.; Kennel, C. F.

1974-01-01

A collisional electromagnetic dispersion relation is derived from two-fluid theory for the interchange mode coupled to the Alfven, acoustic, drift and entropy modes in a partially ionized plasma. The fundamental electromagnetic nature of the interchange model is noted; coupling to the intermediate Alfven mode is strongly stabilizing for finite k sub z. Both ion viscous and ion-neutral stabilization are included, and it was found that collisions destroy the ion finite Larmor radius cutoff at short perpendicular wavelengths.

Multiple-basin energy landscapes for large-amplitude conformational motions of proteins: Structure-based molecular dynamics simulations

PubMed Central

Okazaki, Kei-ichi; Koga, Nobuyasu; Takada, Shoji; Onuchic, Jose N.; Wolynes, Peter G.

2006-01-01

Biomolecules often undergo large-amplitude motions when they bind or release other molecules. Unlike macroscopic machines, these biomolecular machines can partially disassemble (unfold) and then reassemble (fold) during such transitions. Here we put forward a minimal structure-based model, the “multiple-basin model,” that can directly be used for molecular dynamics simulation of even very large biomolecular systems so long as the endpoints of the conformational change are known. We investigate the model by simulating large-scale motions of four proteins: glutamine-binding protein, S100A6, dihydrofolate reductase, and HIV-1 protease. The mechanisms of conformational transition depend on the protein basin topologies and change with temperature near the folding transition. The conformational transition rate varies linearly with driving force over a fairly large range. This linearity appears to be a consequence of partial unfolding during the conformational transition. PMID:16877541

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

Plane flame furnace combustion tests on JPL desulfurized coal

NASA Technical Reports Server (NTRS)

Reuther, J. J.; Kim, H. T.; Lima, J. G. H.

1982-01-01

The combustion characteristics of three raw bituminous (PSOC-282 and 276) and subbituminous (PSOC-230) coals, the raw coals partially desulfurized (ca -60%) by JPL chlorinolysis, and the chlorinated coals more completely desulfurized (ca -75%) by JPL hydrodesulfurization were determined. The extent to which the combustion characteristics of the untreated coals were altered upon JPL sulfur removal was examined. Combustion conditions typical of utility boilers were simulated in the plane flame furnace. Upon decreasing the parent coal voltaile matter generically by 80% and the sulfur by 75% via the JPL desulfurization process, ignition time was delayed 70 fold, burning velocity was retarded 1.5 fold, and burnout time was prolonged 1.4 fold. Total flame residence time increased 2.3 fold. The JPL desulfurization process appears to show significant promise for producing technologically combustible and clean burning (low SO3) fuels.

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.

Baraitser and Winter syndrome with growth hormone deficiency.

PubMed

Chentli, Farida; Zellagui, Hadjer

2014-01-01

Baraitser-Winter syndrome (BWS), first reported in 1988, is apparently due to genetic abnormalities that are still not well-defined, although many gene abnormalities are already discovered and de novo missense changes in the cytoplasmic actin-encoding genes (called ACTB and ACTG1) have been recently discovered. The syndrome combines facial and cerebral malformations. Facial malformations totally or partially present in the same patient are: Iris coloboma, bilateral ptosis, hypertelorism, broad nasal bridge, and prominent epicanthic folds. The various brain malformations are probably responsible for growth and mental retardation. To the best of our knowledge, the syndrome is very rare as few cases have been reported so far. Our aim was to describe a child with a phenotype that looks like BWS with proved partial growth hormone (GH) deficiency which was not reported before. A girl aged 7-year-old of consanguineous parents was referred for short stature and mental retardation. Clinical examination showed dwarfism and a delay in her mental development. Other clinical features included: Strabismus, epicanthic folds, broad nasal bridge, and brain anomalies such as lissencephaly, bilateral hygroma, and cerebral atrophy. Hormonal assessment showed partial GH deficiency without other endocrine disorders. Our case looks exactly like BWS. However, apart from facial and cerebral abnormalities, there is a partial GH deficiency which can explain the harmonious short stature. This case seems worth to be reported as it adds GH deficiency to the very rare syndrome.

Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation

PubMed Central

Ghosh, Dhiman; Singh, Pradeep K.; Sahay, Shruti; Jha, Narendra Nath; Jacob, Reeba S.; Sen, Shamik; Kumar, Ashutosh; Riek, Roland; Maji, Samir K.

2015-01-01

Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases. PMID:25784353

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.

Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I.

PubMed Central

Zhukov, I.; Jaroszewski, L.; Bierzyński, A.

2000-01-01

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process. PMID:10716179

Solvent sensitivity of protein unfolding: dynamical study of chicken villin headpiece subdomain in water-ethanol binary mixture.

PubMed

Ghosh, Rikhia; Roy, Susmita; Bagchi, Biman

2013-12-12

We carry out a series of long atomistic molecular dynamics simulations to study the unfolding of a small protein, chicken villin headpiece (HP-36), in water-ethanol (EtOH) binary mixture. The prime objective of this work is to explore the sensitivity of protein unfolding dynamics toward increasing concentration of the cosolvent and unravel essential features of intermediates formed in search of a dynamical pathway toward unfolding. In water-ethanol binary mixtures, HP-36 is found to unfold partially, under ambient conditions, that otherwise requires temperature as high as ∼600 K to denature in pure aqueous solvent. However, an interesting course of pathway is observed to be followed in the process, guided by the formation of unique intermediates. The first step of unfolding is essentially the separation of the cluster formed by three hydrophobic (phenylalanine) residues, namely, Phe-7, Phe-11, and Phe-18, which constitute the hydrophobic core, thereby initiating melting of helix-2 of the protein. The initial steps are similar to temperature-induced unfolding as well as chemical unfolding using DMSO as cosolvent. Subsequent unfolding steps follow a unique path. As water-ethanol shows composition-dependent anomalies, so do the details of unfolding dynamics. With an increase in cosolvent concentration, different partially unfolded intermediates are found to be formed. This is reflected in a remarkable nonmonotonic composition dependence of several order parameters, including fraction of native contacts and protein-solvent interaction energy. The emergence of such partially unfolded states can be attributed to the preferential solvation of the hydrophobic residues by the ethyl groups of ethanol. We further quantify the local dynamics of unfolding by using a Marcus-type theory.

Episodic growth of a Late Cretaceous and Paleogene intrusive complex of pegmatitic leucogranite, Ruby Mountains core complex, Nevada, USA

USGS Publications Warehouse

Howard, Keith A.; Wooden, J.L.; Barnes, C.G.; Premo, W.R.; Snoke, A.W.; Lee, S.-Y.

2011-01-01

Gneissic pegmatitic leucogranite forms a dominant component (>600 km3) of the midcrustal infrastructure of the Ruby Mountains–East Humboldt Range core complex (Nevada, USA), and was assembled and modified episodically into a batholithic volume by myriad small intrusions from ca. 92 to 29 Ma. This injection complex consists of deformed sheets and other bodies emplaced syntectonically into a stratigraphic framework of marble, calc-silicate rocks, quartzite, schist, and other granitoids. Bodies of pegmatitic granite coalesce around host-rock remnants, which preserve relict or ghost stratigraphy, thrusts, and fold nappes. Intrusion inflated but did not disrupt the host-rock structure. The pegmatitic granite increases proportionally downward from structurally high positions to the bottoms of 1-km-deep canyons where it constitutes 95%–100% of the rock. Zircon and monazite dated by U-Pb (sensitive high-resolution ion microprobe, SHRIMP) for this rock type cluster diffusely at ages near 92, 82(?), 69, 38, and 29 Ma, and indicate successive or rejuvenated igneous crystallization multiple times over long periods of the Late Cretaceous and the Paleogene. Initial partial melting of unexposed pelites may have generated granite forerunners, which were remobilized several times in partial melting events. Sources for the pegmatitic granite differed isotopically from sources of similar-aged interleaved equigranular granites. Dominant Late Cretaceous and fewer Paleogene ages recorded from some pegmatitic granite samples, and Paleogene-only ages from the two structurally deepest samples, together with varying zircon trace element contents, suggest several disparate ages of final emplacement or remobilization of various small bodies. Folded sills that merge with dikes that cut the same folds suggest that there may have been in situ partial remobilization. The pegmatitic granite intrusions represent prolonged and recurrent generation, assembly, and partial melting modification of a batholithic volume even while the regional tectonic environment varied dramatically from contractile thickening to extension and mafic underplating.

Mapping the energy landscape for second-stage folding of a single membrane protein

PubMed Central

Min, Duyoung; Jefferson, Robert E; Bowie, James U; Yoon, Tae-Young

2016-01-01

Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (ΔG = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ~3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure. PMID:26479439

Predicting folding-unfolding transitions in proteins without a priori knowledge of the folded state

NASA Astrophysics Data System (ADS)

Okan, Osman; Turgut, Deniz; Garcia, Angel; Ozisik, Rahmi

2013-03-01

The common computational method of studying folding transitions in proteins is to compare simulated conformations against the folded structure, but this method obviously requires the folded structure to be known beforehand. In the current study, we show that the use of bond orientational order parameter (BOOP) Ql [Steinhardt PJ, Nelson DR, Ronchetti M, Phys. Rev. B 1983, 28, 784] is a viable alternative to the commonly adopted root mean squared distance (RMSD) measure in probing conformational transitions. Replica exchange molecular dynamics simulations of the trp-cage protein (with 20 residues) in TIP-3P water were used to compare BOOP against RMSD. The results indicate that the correspondence between BOOP and RMSD time series become stronger with increasing l. We finally show that robust linear models that incorporate different Ql can be parameterized from a given replica run and can be used to study other replica trajectories. This work is partially supported by NSF DUE-1003574.

A molten globule-like intermediate state detected in the thermal transition of cytochrome c under low salt concentration.

PubMed

Nakamura, Shigeyoshi; Baba, Takayuki; Kidokoro, Shun-Ichi

2007-04-01

To understand the stabilization mechanism of the transient intermediate state in protein folding, it is very important to understand the structure and stability of the molten globule state under a native condition, in which the native state exists stably. The thermal transitions of horse cytochrome c were thermodynamically evaluated by highly precise differential scanning calorimetry (DSC) at pH 3.8-5.0. The heat capacity functions were analyzed using double deconvolution and the nonlinear least-squares method. An intermediate (I) state is clearly confirmed in the thermal native (N)-to-denatured (D) transition of horse cytochrome c. The mole fraction of the intermediate state shows the largest value, 0.4, at nearly 70 degrees C at pH 4.1. This intermediate state was also detected by the circular dichroism (CD) method and was found to have the properties of the molten globule-like structure by three-state analysis of the CD data. The Gibbs free-energy change between N and I, DeltaG(NI), and that between N and D, DeltaG(ND), were evaluated to be 9-22 kJ mol(-1) and 41-45 kJ mol(-1), respectively at 15( ) degrees C and pH 4.1.

Ambiguities in model-independent partial-wave analysis

NASA Astrophysics Data System (ADS)

Krinner, F.; Greenwald, D.; Ryabchikov, D.; Grube, B.; Paul, S.

2018-06-01

Partial-wave analysis is an important tool for analyzing large data sets in hadronic decays of light and heavy mesons. It commonly relies on the isobar model, which assumes multihadron final states originate from successive two-body decays of well-known undisturbed intermediate states. Recently, analyses of heavy-meson decays and diffractively produced states have attempted to overcome the strong model dependences of the isobar model. These analyses have overlooked that model-independent, or freed-isobar, partial-wave analysis can introduce mathematical ambiguities in results. We show how these ambiguities arise and present general techniques for identifying their presence and for correcting for them. We demonstrate these techniques with specific examples in both heavy-meson decay and pion-proton scattering.

A quantitative approach to nucleophilic organocatalysis

PubMed Central

Lakhdar, Sami; Maji, Biplab; Ofial, Armin R

2012-01-01

Summary The key steps in most organocatalytic cyclizations are the reactions of electrophiles with nucleophiles. Their rates can be calculated by the linear free-energy relationship log k(20 °C) = s N(E + N), where electrophiles are characterized by one parameter (E) and nucleophiles are characterized by the solvent-dependent nucleophilicity (N) and sensitivity (s N) parameters. Electrophilicity parameters in the range –10 < E < –5 were determined for iminium ions derived from cinnamaldehyde and common organocatalysts, such as pyrrolidines and imidazolidinones, by studying the rates of their reactions with reference nucleophiles. Iminium activated reactions of α,β-unsaturated aldehydes can, therefore, be expected to proceed with nucleophiles of 2 < N < 14, because such nucleophiles are strong enough to react with iminium ions but weak enough not to react with their precursor aldehydes. With the N parameters of enamines derived from phenylacetaldehyde and MacMillan’s imidazolidinones one can rationalize why only strong electrophiles, such as stabilized carbenium ions (–8 < E < –2) or hexachlorocyclohexadienone (E = –6.75), are suitable electrophiles for enamine activated reactions with imidazolidinones. Several mechanistic controversies concerning iminium and enamine activated reactions could thus be settled by studying the reactivities of independently synthesized intermediates. Kinetic investigations of the reactions of N-heterocyclic carbenes (NHCs) with benzhydrylium ions showed that they have similar nucleophilicities to common organocatalysts (e.g., PPh3, DMAP, DABCO) but are much stronger (100–200 kJ mol–1) Lewis bases. While structurally analogous imidazolylidenes and imidazolidinylidenes have comparable nucleophilicities and Lewis basicities, the corresponding deoxy Breslow intermediates differ dramatically in reactivity. The thousand-fold higher nucleophilicity of 2-benzylidene-imidazoline relative to 2-benzylidene-imidazolidine is explained by the gain of aromaticity during electrophilic additions to the imidazoline derivatives. O-Methylated Breslow intermediates are a hundred-fold less nucleophilic than deoxy Breslow intermediates. PMID:23019481

Multidimensional free energy surface of unfolding of HP-36: Microscopic origin of ruggedness

NASA Astrophysics Data System (ADS)

Ghosh, Rikhia; Roy, Susmita; Bagchi, Biman

2014-10-01

The protein folding funnel paradigm suggests that folding and unfolding proceed as directed diffusion in a multidimensional free energy surface where a multitude of pathways can be traversed during the protein's sojourn from initial to final state. However, finding even a single pathway, with the detail chronicling of intermediates, is an arduous task. In this work we explore the free energy surface of unfolding pathway through umbrella sampling, for a small globular α-helical protein chicken-villin headpiece (HP-36) when the melting of secondary structures is induced by adding DMSO in aqueous solution. We find that the unfolding proceeds through the initial separation or melting of aggregated hydrophobic core that comprises of three phenylalanine residues (Phe7, Phe11, and Phe18). This separation is accompanied by simultaneous melting of the second helix. Unfolding is found to be a multistage process involving crossing of three consecutive minima and two barriers at the initial stage. At a molecular level, Phe18 is observed to reorient itself towards other hydrophobic grooves to stabilize the intermediate states. We identify the configuration of the intermediates and correlate the intermediates with those obtained in our previous works. We also give an estimate of the barriers for different transition states and observe the softening of the barriers with increasing DMSO concentration. We show that higher concentration of DMSO tunes the unfolding pathway by destabilizing the third minimum and stabilizing the second one, indicating the development of a solvent modified, less rugged pathway. The prime outcome of this work is the demonstration that mixed solvents can profoundly transform the nature of the energy landscape and induce unfolding via a modified route. A successful application of Kramer's rate equation correlating the free energy simulation results shows faster rate of unfolding with increasing DMSO concentration. This work perhaps presents the first systematic theoretical study of the effect of a chemical denaturant on the microscopic free energy surface and rates of unfolding of HP-36.

External validation of the modified Glasgow prognostic score for renal cancer

PubMed Central

Tai, Caroline G.; Johnson, Timothy V.; Abbasi, Ammara; Herrell, Lindsey; Harris, Wayne B.; Kucuk, Omer; Canter, Daniel J.; Ogan, Kenneth; Pattaras, John G.; Nieh, Peter T.; Master, Viraj A.

2014-01-01

Purpose: The modified Glasgow prognostic Score (mGPS) incorporates C-reactive protein and albumin as a clinically useful marker of tumor behavior. The ability of the mGPS to predict metastasis in localized renal cell carcinoma (RCC) remains unknown in an external validation cohort. Patients and Methods: Patients with clinically localized clear cell RCC were followed for 1 year post-operatively. Metastases were identified radiologically. Patients were categorized by mGPS score as low-risk (mGPS = 0 points), intermediate-risk (mGPS = 1 point) and high-risk (mGPS = 2 points). Univariate, Kaplan-Meier and multivariate Cox regression analyses examined Recurrence -free survival (RFS) across patient and disease characteristics. Results: Of the 129 patients in this study, 23.3% developed metastases. Of low, intermediate and high risk patients, 10.1%, 38.9% and 89.9% recurred during the study. After accounting for various patient and tumor characteristics in multivariate analysis including stage and grade, only mGPS was significantly associated with RFS. Compared with low-risk patients, intermediate- and high-risk patients experienced a 4-fold (hazard ratios [HR]: 4.035, 95% confidence interval [CI]: 1.312-12.415, P = 0.015) and 7-fold (HR: 7.012, 95% CI: 2.126-23.123 P < 0.001) risk of metastasis, respectively. Conclusions: mGPS is a robust predictor of metastasis following potentially curative nephrectomy for localized RCC. Clinicians may consider mGPS as an adjunct to identify high-risk patients for possible enrollment into clinical trials or for patient counseling PMID:24497679

Preoperative Erythrocyte Sedimentation Rate Independently Predicts Overall Survival in Localized Renal Cell Carcinoma following Radical Nephrectomy

PubMed Central

Cross, Brian W.; Johnson, Timothy V.; DeRosa, Austin B.; Ogan, Kenneth; Pattaras, John G.; Nieh, Peter T.; Kucuk, Omer; Harris, Wayne B.; Master, Viraj A.

2012-01-01

Objectives. To determine the relationship between preoperative erythrocyte sedimentation rate (ESR) and overall survival in localized renal cell carcinoma (RCC) following nephrectomy. Methods. 167 patients undergoing nephrectomy for localized RCC had ESR levels measured preoperatively. Receiver Operating Characteristics curves were used to determine Area Under the Curve and relative sensitivity and specificity of preoperative ESR in predicting overall survival. Cut-offs for low (0.0–20.0 mm/hr), intermediate (20.1–50.0 mm/hr), and high risk (>50.0 mm/hr) groups were created. Kaplan-Meier analysis was conducted to assess the univariate impact of these ESR-based groups on overall survival. Univariate and multivariate Cox regression analysis was conducted to assess the potential of these groups to predict overall survival, adjusting for other patient and tumor characteristics. Results. Overall, 55.2% were low risk, while 27.0% and 17.8% were intermediate and high risk, respectively. Median (95% CI) survival was 44.1 (42.6–45.5) months, 35.5 (32.3–38.8) months, and 32.1 (25.5–38.6) months, respectively. After controlling for other patient and tumor characteristics, intermediate and high risk groups experienced a 4.5-fold (HR: 4.509, 95% CI: 0.735–27.649) and 18.5-fold (HR: 18.531, 95% CI: 2.117–162.228) increased risk of overall mortality, respectively. Conclusion. Preoperative ESR values represent a robust predictor of overall survival following nephrectomy in localized RCC. PMID:22900160

Getting into the Jet Stream.

ERIC Educational Resources Information Center

Boggs, Elizabeth M.

1992-01-01

This paper reacts to previous symposium papers (EC 604 155-161) concerning regulations and quality assurance in Intermediate Care Facilities for the Mentally Retarded (ICF/MR). Contributions of the Home and Community Based Services program model, which is seen as a partial solution to overregulation, are highlighted. (DB)

Simple triple-state polymer actuators with controllable folding characteristics

NASA Astrophysics Data System (ADS)

Chen, Shuyang; Li, Jing; Fang, Lichen; Zhu, Zeyu; Kang, Sung Hoon

2017-03-01

Driven by the interests in self-folding, there have been studies developing artificial self-folding structures at different length scales based on various polymer actuators that can realize dual-state actuation. However, their unidirectional nature limits the applicability of the actuators for a wide range of multi-state self-folding behaviors. In addition, complex fabrication and programming procedures hinder broad applications of existing polymer actuators. Moreover, few of the existing polymer actuators are able to show the self-folding behaviors with the precise control of curvature and force. To address these issues, we report an easy-to-fabricate triple-state actuator with controllable folding behaviors based on bilayer polymer composites with different glass transition temperatures. Initially, the fabricated actuator is in the flat state, and it can sequentially self-fold to angled folding states of opposite directions as it is heated up. Based on an analytical model and measured partial recovery behaviors of polymers, we can accurately control the folding characteristics (curvature and force) for the rational design. To demonstrate an application of our triple-state actuator, we have developed a self-folding transformer robot which self-folds from a two-dimensional sheet into a three-dimensional boat-like configuration and transforms from the boat shape to a car shape with the increase in the temperature applied to the actuator. Our findings offer a simple approach to generate multiple configurations from a single system by harnessing behaviors of polymers with the rational design.

Protein folding on Biosensor tips: Folding of Maltodextrin glucosidase monitored by its interactions with GroEL

PubMed Central

Pastor, Ashutosh; Singh, Amit K.; Fisher, Mark T.; Chaudhuri, Tapan K.

2016-01-01

Protein folding has been extensively studied for past four decades by employing solution based experiments such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR and HD exchange. However, for rapid analysis of the folding process, solution based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work we demonstrate that a Bio-Layer Interferometry (BLI) chaperonin detection system can be potentially applied to identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on Bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation prone protein Maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the Bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution. PMID:27367928

Influence of high-pressure processing on the profile of polyglutamyl 5-methyltetrahydrofolate in selected vegetables.

PubMed

Wang, Chao; Riedl, Ken M; Somerville, Jeremy; Balasubramaniam, V M; Schwartz, Steven J

2011-08-24

In plants, folate occurs predominantly as 5-methyltetrahydrofolate (5MTHF) polyglutamyl forms. Differences in stability and bioavailability of food folate compared to synthetic folic acid have been attributed to the presence of the polyglutamyl chain. High-pressure processing (HPP) was tested for whether it might shorten polyglutamyl chains of 5MTHF species in fresh vegetables by enabling action of native γ-glutamylhydrolase (GGH). A validated ultrahigh-performance reversed-phase liquid chromatography-tandem mass spectrometry method using stable isotope as internal standard was applied for characterizing 5MTHF polyglutamyl profiles. HPP conditions included 300, 450, and 600 MPa at 30 °C for 0 or 5 min, and vegetables were vacuum-packed before treatment. Investigated vegetables included cauliflower (Brassica oleracea), baby carrots (Daucus carota), and carrot greens (D. carota). HPP treatment caused conversion of polyglutamyl 5MTHF species to short-chain and monoglutamyl forms. Maximal conversion of polyglutamyl folate to monoglutamyl folate occurred at the highest pressure/time combination investigated, 600 MPa/30 °C/5 min. Under this condition, cauliflower monoglutamyl folate increased nearly 4-fold, diglutamyl folate 32-fold, and triglutamyl folate 8-fold; carrot monoglutamyl increased 23-fold and diglutamyl 32-fold; and carrot greens monoglutamyl increased 2.5-fold and the diglutamyl form 19-fold. Although some folate degradation was observed at certain intermediate HPP conditions, total 5MTHF folate was largely preserved at 600 MPa/5 min. Thus, HPP of raw vegetables is a feasible strategy for enhancing vegetable monoglutamate 5MTHF.

Influence of High-Pressure Processing on the Profile of Polyglutamyl 5-Methyltetrahydrofolate in Selected Vegetables

PubMed Central

Wang, Chao; Riedl, Ken M.; Somerville, Jeremy; Balasubramaniam, V. M.; Schwartz, Steven J.

2013-01-01

In plants, folate occurs predominantly as 5-methyltetrahydrofolate (5MTHF) polyglutamyl forms. Differences in stability and bioavailability of food folate compared to synthetic folic acid have been attributed to the presence of the polyglutamyl chain. High-pressure processing (HPP) was tested for whether it might shorten polyglutamyl chains of 5MTHF species in fresh vegetables by enabling action of native γ-glutamylhydrolase (GGH). A validated ultrahigh-performance reversed-phase liquid chromatography–tandem mass spectrometry method using stable isotope as internal standard was applied for characterizing 5MTHF polyglutamyl profiles. HPP conditions included 300, 450, and 600 MPa at 30 °C for 0 or 5 min, and vegetables were vacuum-packed before treatment. Investigated vegetables included cauliflower (Brassica oleracea), baby carrots (Daucus carota), and carrot greens (D. carota). HPP treatment caused conversion of polyglutamyl 5MTHF species to short-chain and monoglutamyl forms. Maximal conversion of polyglutamyl folate to monoglutamyl folate occurred at the highest pressure/time combination investigated, 600 MPa/30 °C/5 min. Under this condition, cauliflower monoglutamyl folate increased nearly 4-fold, diglutamyl folate 32-fold, and triglutamyl folate 8-fold; carrot monoglutamyl increased 23-fold and diglutamyl 32-fold; and carrot greens monoglutamyl increased 2.5-fold and the diglutamyl form 19-fold. Although some folate degradation was observed at certain intermediate HPP conditions, total 5MTHF folate was largely preserved at 600 MPa/5 min. Thus, HPP of raw vegetables is a feasible strategy for enhancing vegetable monoglutamate 5MTHF. PMID:21770413

Folding kinetics of WW domains with the united residue force field for bridging microscopic motions and experimental measurements

PubMed Central

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

2014-01-01

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

Effects of desolvation barriers and sidechains on local-nonlocal coupling and chevron behaviors in coarse-grained models of protein folding.

PubMed

Chen, Tao; Chan, Hue Sun

2014-04-14

Local-nonlocal coupling is an organizational principle in protein folding. It envisions a cooperative energetic interplay between local conformational preferences and favorable nonlocal contacts. Previous theoretical studies by our group showed that two classes of native-centric coarse-grained models can capture the experimentally observed high degrees of protein folding cooperativity and diversity in folding rates. These models either embody an explicit local-nonlocal coupling mechanism or incorporate desolvation barriers in the models' pairwise interactions. Here a conceptual connection is made between these two paradigmatic coarse-grained interaction schemes by showing that desolvation barriers enhance local-nonlocal coupling. Furthermore, we find that a class of coarse-grained protein models with a single-site representation of sidechains also increases local-nonlocal coupling relative to mainchain models without sidechains. Enhanced local-nonlocal coupling generally leads to higher folding cooperativity and chevron plots with more linear folding arms. For the sidechain models studied, the chevron plot simulated with entirely native-centric intrachain interactions behaves very similarly to the corresponding chevron plots simulated with interactions that are partly modulated by sequence- and denaturant-dependent transfer free energies. In these essentially native-centric models, the mild chevron rollovers in the simulated folding arm are caused by occasionally populated intermediates as well as the movement of the unfolded and putative folding transition states. The strength and limitation of the models are analyzed by comparison with experiment. New formulations of sidechain models that may provide a physical account for nonnative interactions are also explored.

Folding kinetics of WW domains with the united residue force field for bridging microscopic motions and experimental measurements.

PubMed

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

2014-12-23

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

Modeling of wind gap formation and development of sedimentary basins during fold growth: application to the Zagros Fold Belt, Iran.

NASA Astrophysics Data System (ADS)

Collignon, Marine; Yamato, Philippe; Castelltort, Sébastien; Kaus, Boris

2016-04-01

Mountain building and landscape evolution are controlled by the interactions between river dynamics and tectonic forces. Such interactions have been largely studied but a quantitative evaluation of tectonic/geomorphic feedbacks remains required for understanding sediments routing within orogens and fold-and-thrust belts. Here, we employ numerical simulations to assess the conditions of uplift and river incision necessary to deflect an antecedent drainage network during the growth of one or several folds. We propose that a partitioning of the river network into internal (endorheic) and longitudinal drainage arises as a result of lithological differences within the deforming crustal sedimentary cover. We show with examples from the Zagros Fold Belt (ZFB) that drainage patterns can be linked to the incision ratio R between successive lithological layers, corresponding to the ratio between their relative erodibilities or incision coefficients. Transverse drainage networks develop for uplift rates smaller than 0.8 mm.yr-1 and -10 < R < 10. Intermediate drainage network are obtained for uplift rates up to 2 mm.yr-1 and incision ratios of 20. Parallel drainage networks and formation of sedimentary basins occur for large values of incision ratio (R >20) and uplift rates between 1 and 2 mm.yr-1. These results have implications for predicting the distribution of sediment depocenters in fold-and-thrust belts, which can be of direct economic interest for hydrocarbon exploration.

Injection Laryngoplasty Using Micronized Acellular Dermis for Vocal Fold Paralysis: Long-term Voice Outcomes.

PubMed

Hernandez, Stephen C; Sibley, Haley; Fink, Daniel S; Kunduk, Melda; Schexnaildre, Mell; Kakade, Anagha; McWhorter, Andrew J

2016-05-01

Micronized acellular dermis has been used for nearly 15 years to correct glottic insufficiency. With previous demonstration of safety and efficacy, this study aims to evaluate intermediate and long-term voice outcomes in those who underwent injection laryngoplasty for unilateral vocal fold paralysis. Technique and timing of injection were also reviewed to assess their impact on outcomes. Case series with chart review. Tertiary care center. Patients undergoing injection laryngoplasty from May 2007 to September 2012 were reviewed for possible inclusion. Pre- and postoperative Voice Handicap Index (VHI) scores, as well as senior speech-language pathologists' blinded assessment of voice, were collected for analysis. The final sample included patients who underwent injection laryngoplasty for unilateral vocal fold paralysis, 33 of whom had VHI results and 37 of whom had voice recordings. Additional data were obtained, including technique and timing of injection. Analysis was performed on those patients above with VHI and perceptual voice grades before and at least 6 months following injection. Mean VHI improved by 28.7 points at 6 to 12 months and 22.8 points at >12 months (P = .001). Mean perceptual voice grades improved by 17.6 points at 6 to 12 months and 16.3 points at >12 months (P < .001). No statistically significant difference was found with technique or time to injection. Micronized acellular dermis is a safe injectable that improved both patient-completed voice ratings and blinded reviewer voice gradings at intermediate and long-term follow-up. Further investigation may be warranted regarding technique and timing of injection. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.

Combination of cephalosporins with vancomycin or teicoplanin enhances antibacterial effect of glycopeptides against heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and VISA

PubMed Central

Lai, Chih-Cheng; Chen, Chi-Chung; Chuang, Yin-Ching; Tang, Hung-Jen

2017-01-01

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. PMID:28139739

CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

PubMed Central

Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

2014-01-01

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

Cenozoic structural evolution, thermal history, and erosion of the Ukrainian Carpathians fold-thrust belt

NASA Astrophysics Data System (ADS)

Nakapelyukh, Mykhaylo; Bubniak, Ihor; Bubniak, Andriy; Jonckheere, Raymond; Ratschbacher, Lothar

2018-01-01

The Carpathians are part of the Alpine-Carpathian-Dinaridic orogen surrounding the Pannonian basin. Their Ukrainian part constitutes an ancient subduction-accretion complex that evolved into a foreland fold-thrust belt with a shortening history that was perpendicular to the orogenic strike. Herein, we constrain the evolution of the Ukrainian part of the Carpathian fold-thrust belt by apatite fission-track dating of sedimentary and volcanic samples and cross-section balancing and restoration. The apatite fission-track ages are uniform in the inner―southwestern part of the fold-thrust belt, implying post-shortening erosion since 12-10 Ma. The ages in the leading and trailing edges record provenance, i.e., sources in the Trans-European suture zone and the Inner Carpathians, respectively, and show that these parts of the fold-thrust were not heated to more than 100 °C. Syn-orogenic strata show sediment recycling: in the interior of the fold-thrust belt―the most thickened and most deeply eroded nappes―the apatite ages were reset, eroded, and redeposited in the syn-orogenic strata closer to the fore- and hinterland; the lag times are only a few million years. Two balanced cross sections, one constructed for this study and based on field and subsurface data, reveal an architecture characterized by nappe stacks separated by high-displacement thrusts; they record 340-390 km shortening. A kinematic forward model highlights the fold-thrust belt evolution from the pre-contractional configuration over the intermediate geometries during folding and thrusting and the post-shortening, erosional-unloading configuration at 12-10 Ma to the present-day geometry. Average shortening rates between 32-20 Ma and 20-12 Ma amounted to 13 and 21 km/Ma, respectively, implying a two-phased deformation of the Ukrainian fold-thrust belt.

On the Preservation of Intergranular Coesite in UHP Eclogite at Yangkou Bay, Sulu belt of eastern China

NASA Astrophysics Data System (ADS)

Wang, L.; Wang, S.; Brown, M.

2016-12-01

In contrast to coesite that occurs as inclusions in zircon and rock-forming minerals, intergranular coesite is preserved in UHP eclogite at Yangkou in the Sulu belt. The survival of intergranular coesite is intriguing because the eclogite experienced phengite growth and partial melting during exhumation. The coesite eclogite occurs as rootless isoclinal fold noses within quartz-rich schist which contains 10-20 vol% phengite, whereas phengite is absent from coesite eclogite in the fold noses. To evaluate the factors that control preservation of intergranular coesite, four samples representative of different stages along the retrograde P-T path were selected for study. For each sample we determined the number of intergranular coesite grains per cm2 and the OH content of garnet and omphacite. As the number of coesite grains decreases, the bulk rock OH content increases from <200 ppm in phengite-free coesite eclogite to 200-260 ppm in phengite-bearing (<5 vol%) coesite eclogite and up to a maximum of 430-438 ppm in quartz eclogite ( 10 vol% phengite). However, the OH content drops to a minimum of 59 ppm in residual eclogite resulting from melt drainage. This trend implies that the volume of fluid increased sufficiently during exhumation to facilitate the growth of phengite and the transformation to quartz of intergranular coesite outside of the fold noses. The fluid is inferred to have been a supercritical fluid probably residual from prograde dehydration but also derived by dissolution of nominally anhydrous minerals. Post-metamorphic-peak deformation combined with fluid percolation along sheared fold limbs induced phengite growth during initial exhumation and then facilitated partial melting. In contrast, fold hinges in competent layers are unfavourable sites for fluid penetration. At Yangkou, the intergranular coesite is preserved in the fold noses where it was protected from both penetrative deformation and fluid ingress. Therefore, the fold noses maintained a relatively dry environment that allowed preservation of the intergranular coesite. Thus, deformation partitioning and strain localization impose local controls on fluid distribution and migration in UHP eclogite. This study informs our understanding of variations in fluid regime during exhumation of deeply subducted continental crust.

Metabolomics revealed an association of metabolite changes and defective growth in Methylobacterium extorquens AM1 overexpressing ecm during growth on methanol

DOE PAGES

Cui, Jinyu; Good, Nathan M.; Hu, Bo; ...

2016-04-26

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targetedmore » metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)-meso-diaminopimelic acid (mDAP) and Ala-mDAP-Ala, each over 45-fold higher in the ecm overexpressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. Lastly, this research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.« less

Metabolomics revealed an association of metabolite changes and defective growth in Methylobacterium extorquens AM1 overexpressing ecm during growth on methanol

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

Cui, Jinyu; Good, Nathan M.; Hu, Bo

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multi-carbon compounds. The ethylmalonyl-CoA (EMC) pathway is one of the central assimilatory pathways in M. extorquens during growth on C1 and C2 substrates. Previous studies had shown that ethylmalonyl-CoA mutase functioned as a control point during the transition from growth on succinate to growth on ethylamine. In this study we overexpressed ecm, phaA, mcmAB and found that upregulating ecm by expressing it from the strong constitutive mxaF promoter caused a 27% decrease in growth rate on methanol compared to the strain with an empty vector. Targetedmore » metabolomics demonstrated that most of the central intermediates in the ecm over-expressing strain did not change significantly compared to the control strain; However, poly-β-hydroxybutyrate (PHB) was 4.5-fold lower and 3-hydroxybutyryl-CoA was 1.6-fold higher. Moreover, glyoxylate, a toxic and highly regulated essential intermediate, was determined to be 2.6-fold higher when ecm was overexpressed. These results demonstrated that overexpressing ecm can manipulate carbon flux through the EMC pathway and divert it from the carbon and energy storage product PHB, leading to an accumulation of glyoxylate. Furthermore, untargeted metabolomics discovered two unusual metabolites, alanine (Ala)-meso-diaminopimelic acid (mDAP) and Ala-mDAP-Ala, each over 45-fold higher in the ecm overexpressing strain. These two peptides were also found to be highly produced in a dose-dependent manner when glyoxylate was added to the control strain. Overall, this work has explained a direct association of ecm overexpression with glyoxylate accumulation up to a toxic level, which inhibits cell growth on methanol. Lastly, this research provides useful insight for manipulating the EMC pathway for efficiently producing high-value chemicals in M. extorquens.« less

Optimal CV-22 Centralized Intermediate Repair Facility Locations and Parts Repair

DTIC Science & Technology

2009-06-01

and Reorder Point for TEWS ............................ 36 Table 8. Excel Model for Safety Stock and Reorder Point for FADEC ...Digital Engine Control ( FADEC ) Main Wheel Assembly Blade Fold System Landing Gear Control Panel Drive System Interface Unit Main Landing Gear...Radar 4 Forward Looking Infrared System (FLIR) 4 Tactical Electronic Warfare System (TEWS) 1 Full Authority Digital Engine Control ( FADEC ) 2 Blade

77 FR 72920 - Federal Housing Administration (FHA) Section 232 Healthcare Mortgage Insurance Program: Partial...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-07

...This rule amends the regulations governing FHA's Section 232 Healthcare Mortgage Insurance program (Section 232 program) by establishing the criteria and process by which FHA will accept and pay a partial payment of a claim under the FHA mortgage insurance contract. The Section 232 program insures mortgage loans to facilitate the construction, substantial rehabilitation, purchase, and refinancing of nursing homes, intermediate care facilities, board and care homes, and assisted-living facilities. Through acceptance and payment of a partial payment of claim, FHA pays the lender a portion of the unpaid principal balance and recasts a portion of the mortgage under terms and conditions determined by FHA, as an alternative to the lender assigning the entire mortgage to HUD. Partial payment of claim also allows FHA- insured healthcare projects to continue operating and providing services.

DNA packaging intermediates of bacteriophage Φ174

PubMed Central

Music, Cynthia L; Cheng, R Holland; Bowen, Zorina; McKenna, Robert; Rossmann, Michael G; Baker, Timothy S; Incardona, Nino L

2014-01-01

Background Like many viruses, bacteriophage ΦX174 packages its I)NA genome into a procapsid that is assembled from structural intermediates and scaffolding proteins. The procapsid contains the structural proteins F, G and H, as well as the scaffolding proteins B and D. Provirions are formed by packaging of DNA together with the small internal J proteins, while losing at least some of the B scaffolding proteins. Eventually, loss of the I) scaffolding proteins and the remaining B proteins leads to the formation of mature virions. Results ΦX174 108S 'procapsids' have been purified in milligram quantities by removing 114S (mature virion) and 70S (abortive capsid) particles from crude lysates by differential precipitation with polyethylene glycol. 132S 'provirions' were purified on sucrose gradients in the presence of EDTA. Cryo-electron microscopy (cryo-EM) was used to obtain reconstructions of procapsids and provirions. Although these are very similar to each other, their structures differ greatly from that of the virion. The F and G proteins, whose atomic structures in virions were previously determined from X-ray crystallography, were fitted into the cryo-EM reconstructions. This showed that the pentamer of G proteins on each five-fold vertex changes its conformation only slightly during DNA packaging and maturation, whereas major tertiary and quaternary structural changes occur in the F protein. The procapsids and provirions were found to contain 120 copies of the I) protein arranged as tetramers on the twofold axes. IDNA might enter procapsids through one of the 30 Å diameter holes on the icosahedral three-fold axes. Conclusions Combining cryo-EM image reconstruction and X-ray crystallography has revealed the major conformational changes that can occur in viral assembly. The function of the scaffolding proteins may be, in part, to support weak interactions between the structural proteins in the procapsids and to cover surfaces that are subsequently required for subunit–subunit interaction in the virion. The structures presented here are, therefore, analogous to chaperone proteins complexed with folding intermediates of a substrate. PMID:7613866

Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins

PubMed Central

De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

2017-01-01

The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination. PMID:23035660

An Examination of the Validity of Social Subtypes in Autism.

ERIC Educational Resources Information Center

Borden, M. Christopher; Ollendick, Thomas H.

1994-01-01

Examination of 53 children with autism supported the validity of the subtypes "aloof" and "active-but-odd" as predictors of behavior across language/communication, reciprocal social interaction, and stereotyped behavior/restricted interest domains. Partial support for an intermediate, "passive" subtype was garnered. (Author/JDD)

1000-fold enhancement in proton conductivity of a MOF using post-synthetically anchored proton transporters

PubMed Central

Shalini, Sorout; Dhavale, Vishal M.; Eldho, Kavalakal M.; Kurungot, Sreekumar; Ajithkumar, Thallaseril G.; Vaidhyanathan, Ramanathan

2016-01-01

Pyridinol, a coordinating zwitter-ionic species serves as stoichiometrically loadable and non-leachable proton carrier. The partial replacement of the pyridinol by stronger hydrogen bonding, coordinating guest, ethylene glycol (EG), offers 1000-fold enhancement in conductivity (10−6 to 10−3 Scm−1) with record low activation energy (0.11 eV). Atomic modeling coupled with 13C-SSNMR provides insights into the potential proton conduction pathway functionalized with post-synthetically anchored dynamic proton transporting EG moieties. PMID:27577681

Folding-unfolding transitions of Rv3221c on the pressure-temperature plane

NASA Astrophysics Data System (ADS)

Somkuti, Judit; Jain, Sriyans; Ramachandran, Srinivasan; ászló Smeller, L.

2013-06-01

Rv3221c is a biotin-binding protein found in Mycobacterium tuberculosis. It has been reported that an elevated temperature is needed for it to adopt a folded conformation. We determined the complete pressure-temperature phase diagram, and determined the thermodynamical parameters of the denaturation. The phase diagram follows well the Hawley theory. The secondary structure of the protein was found to contain predominantly beta sheet. The pressure unfolding was partially reversible, resulting in pressure-sensitive aggregates, besides the correctly refolded and biotin-bound fraction of proteins.

Dispersion relation for hadronic light-by-light scattering: two-pion contributions

DOE PAGES

Colangelo, Gilberto; Hoferichter, Martin; Procura, Massimiliano; ...

2017-04-27

In our third paper of a series dedicated to a dispersive treatment of the hadronic light-by-light (HLbL) tensor, we derive a partial-wave formulation for two-pion intermediate states in the HLbL contribution to the anomalous magnetic moment of the muon (g - 2) μ, including a detailed discussion of the unitarity relation for arbitrary partial waves. We show that obtaining a final expression free from unphysical helicity partial waves is a subtle issue, which we thoroughly clarify. As a by-product, we obtain a set of sum rules that could be used to constrain future calculations of γ*γ* → ππ. We validate the formalism extensively using the pion-box contribution, defined by two-pion intermediate states with a pion-pole left-hand cut, and demonstrate how the full known result is reproduced when resumming the partial waves. Using dispersive fits to high-statistics data for the pion vector form factor, we provide an evaluation of the full pion box, amore » $$π-box\\atop{μ}$$ =-15.9(2) × 10 -11. As an application of the partial-wave formalism, we present a first calculation of ππ-rescattering effects in HLbL scattering, with γ*γ* → ππ helicity partial waves constructed dispersively using ππ phase shifts derived from the inverse-amplitude method. In this way, the isospin-0 part of our calculation can be interpreted as the contribution of the f0(500) to HLbL scattering in (g - 2) μ. We also argue that the contribution due to charged-pion rescattering implements corrections related to the corresponding pion polarizability and show that these are moderate. Our final result for the sum of pion-box contribution and its S-wave rescattering corrections reads a$$π-box\\atop{μ}$$ + a$$ππ, π-pole LHC\\atop{μ, J=0}$$ = -24(1) × 10 -11.« less

Dispersion relation for hadronic light-by-light scattering: two-pion contributions

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

Colangelo, Gilberto; Hoferichter, Martin; Procura, Massimiliano

In our third paper of a series dedicated to a dispersive treatment of the hadronic light-by-light (HLbL) tensor, we derive a partial-wave formulation for two-pion intermediate states in the HLbL contribution to the anomalous magnetic moment of the muon (g - 2) μ, including a detailed discussion of the unitarity relation for arbitrary partial waves. We show that obtaining a final expression free from unphysical helicity partial waves is a subtle issue, which we thoroughly clarify. As a by-product, we obtain a set of sum rules that could be used to constrain future calculations of γ*γ* → ππ. We validate the formalism extensively using the pion-box contribution, defined by two-pion intermediate states with a pion-pole left-hand cut, and demonstrate how the full known result is reproduced when resumming the partial waves. Using dispersive fits to high-statistics data for the pion vector form factor, we provide an evaluation of the full pion box, amore » $$π-box\\atop{μ}$$ =-15.9(2) × 10 -11. As an application of the partial-wave formalism, we present a first calculation of ππ-rescattering effects in HLbL scattering, with γ*γ* → ππ helicity partial waves constructed dispersively using ππ phase shifts derived from the inverse-amplitude method. In this way, the isospin-0 part of our calculation can be interpreted as the contribution of the f0(500) to HLbL scattering in (g - 2) μ. We also argue that the contribution due to charged-pion rescattering implements corrections related to the corresponding pion polarizability and show that these are moderate. Our final result for the sum of pion-box contribution and its S-wave rescattering corrections reads a$$π-box\\atop{μ}$$ + a$$ππ, π-pole LHC\\atop{μ, J=0}$$ = -24(1) × 10 -11.« less

Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations

PubMed Central

Stadlbauer, Petr; Krepl, Miroslav; Cheatham, Thomas E.; Koča, Jaroslav; Šponer, Jiří

2013-01-01

Explicit solvent molecular dynamics simulations have been used to complement preceding experimental and computational studies of folding of guanine quadruplexes (G-DNA). We initiate early stages of unfolding of several G-DNAs by simulating them under no-salt conditions and then try to fold them back using standard excess salt simulations. There is a significant difference between G-DNAs with all-anti parallel stranded stems and those with stems containing mixtures of syn and anti guanosines. The most natural rearrangement for all-anti stems is a vertical mutual slippage of the strands. This leads to stems with reduced numbers of tetrads during unfolding and a reduction of strand slippage during refolding. The presence of syn nucleotides prevents mutual strand slippage; therefore, the antiparallel and hybrid quadruplexes initiate unfolding via separation of the individual strands. The simulations confirm the capability of G-DNA molecules to adopt numerous stable locally and globally misfolded structures. The key point for a proper individual folding attempt appears to be correct prior distribution of syn and anti nucleotides in all four G-strands. The results suggest that at the level of individual molecules, G-DNA folding is an extremely multi-pathway process that is slowed by numerous misfolding arrangements stabilized on highly variable timescales. PMID:23700306

Etoxazole resistance in predatory mite Phytoseiulus persimilis A.-H. (Acari: Phytoseiidae): Cross-resistance, inheritance and biochemical resistance mechanisms.

PubMed

Yorulmaz Salman, Sibel; Aydınlı, Fatma; Ay, Recep

2015-07-01

Phytoseiulus persimilis of the family Phytoseiidae is an effective predatory mite species that is used to control pest mites. The LC50 and LC60 values of etoxazole were determined on P. persimilis using a leaf-disc method and spraying tower. A laboratory selection population designated ETO6 was found to have a 111.63-fold resistance to etoxazole following 6 selection cycles. This population developed low cross-resistance to spinosad, spiromesifen, acetamiprid, indoxacarb, chlorantraniliprole, milbemectin and moderate cross-resistance to deltamethrin. PBO, IBP and DEM synergised resistance 3.17-, 2.85- and 3.60-fold respectively. Crossing experiments revealed that etoxazole resistance in the ETO6 population was an intermediately dominant and polygenic. In addition, detoxifying enzyme activities were increased 2.71-fold for esterase, 3.09-fold for glutathione S-transferase (GST) and 2.76-fold for cytochrome P450 monooxygenase (P450) in the ETO6 population. Selection for etoxazole under laboratory conditions resulted in the development of etoxazole resistance in the predatory mite P. persimilis that are resistant to pesticides are considered valuable for use in resistance management programmes within integrated pest control strategies. Copyright © 2014 Elsevier Inc. All rights reserved.

Partial molar volumes of proteins: amino acid side-chain contributions derived from the partial molar volumes of some tripeptides over the temperature range 10-90 degrees C.

PubMed

Häckel, M; Hinz, H J; Hedwig, G R

1999-11-15

The partial molar volumes of tripeptides of sequence glycyl-X-glycine, where X is one of the amino acids alanine, leucine, threonine, glutamine, phenylalanine, histidine, cysteine, proline, glutamic acid, and arginine, have been determined in aqueous solution over the temperature range 10-90 degrees C using differential scanning densitometry . These data, together with those reported previously, have been used to derive the partial molar volumes of the side-chains of all 20 amino acids. The side-chain volumes are critically compared with literature values derived using partial molar volumes for alternative model compounds. The new amino acid side-chain volumes, along with that for the backbone glycyl group, were used to calculate the partial specific volumes of several proteins in aqueous solution. The results obtained are compared with those observed experimentally. The new side-chain volumes have also been used to re-determine residue volume changes upon protein folding.

Association between partial-volume corrected SUVmax and Oncotype DX recurrence score in early-stage, ER-positive/HER2-negative invasive breast cancer.

PubMed

Lee, Su Hyun; Ha, Seunggyun; An, Hyun Joon; Lee, Jae Sung; Han, Wonshik; Im, Seock-Ah; Ryu, Han Suk; Kim, Won Hwa; Chang, Jung Min; Cho, Nariya; Moon, Woo Kyung; Cheon, Gi Jeong

2016-08-01

Oncotype DX, a 21-gene expression assay, provides a recurrence score (RS) which predicts prognosis and the benefit from adjuvant chemotherapy in patients with early-stage, estrogen receptor-positive (ER-positive), and human epidermal growth factor receptor 2-negative (HER2-negative) invasive breast cancer. However, Oncotype DX tests are expensive and not readily available in all institutions. The purpose of this study was to investigate whether metabolic parameters on (18)F-FDG PET/CT are associated with the Oncotype DX RS and whether (18)F-FDG PET/CT can be used to predict the Oncotype DX RS. The study group comprised 38 women with stage I/II, ER-positive/HER2-negative invasive breast cancer who underwent pretreatment (18)F-FDG PET/CT and Oncotype DX testing. On PET/CT, maximum (SUVmax) and average standardized uptake values, metabolic tumor volume, and total lesion glycolysis were measured. Partial volume-corrected SUVmax (PVC-SUVmax) determined using the recovery coefficient method was also evaluated. Oncotype DX RS (0 - 100) was categorized as low (<18), intermediate (18 - 30), or high (≥31). The associations between metabolic parameters and RS were analyzed. Multivariate logistic regression was used to identify significant independent predictors of low versus intermediate-to-high RS. Of the 38 patients, 22 (58 %) had a low RS, 13 (34 %) had an intermediate RS, and 3 (8 %) had a high RS. In the analysis with 38 index tumors, PVC-SUVmax was higher in tumors in patients with intermediate-to-high RS than in those with low RS (5.68 vs. 4.06; P = 0.067, marginally significant). High PVC-SUVmax (≥4.96) was significantly associated with intermediate-to-high RS (odds ratio, OR, 10.556; P = 0.004) in univariate analysis. In multivariate analysis with clinicopathologic factors, PVC-SUVmax ≥4.96 (OR 8.459; P = 0.013) was a significant independent predictor of intermediate-to-high RS. High PVC-SUVmax on (18)F-FDG PET/CT was significantly associated with an intermediate-to-high Oncotype DX RS. PVC metabolic parameters on (18)F-FDG PET/CT can be used to predict the Oncotype DX RS in patients with early-stage, ER-positive/HER2-negative breast cancer.

Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

PubMed Central

Iswari, S.; Palta, Jiwan P.

1989-01-01

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

Novel surface-modified nanostructured lipid carriers with partially deacetylated water-soluble chitosan for efficient ocular delivery.

PubMed

Tian, Baocheng; Luo, Qiuhua; Song, Shuangshuang; Liu, Dandan; Pan, Hao; Zhang, Wenji; He, Ling; Ma, Shilin; Yang, Xinggang; Pan, Weisan

2012-03-01

The objective of this study was to propose novel surface-modified nanostructured lipid carriers with partially deacetylated water-soluble chitosan (NLC-PDSC) as an efficient ocular delivery system to improve its transcorneal penetration and precorneal retention. PDSC with a deacetylation degree of around 50% was synthesized using an improved method. NLC loaded with flurbiprofen (FB) were prepared by melt emulsification method. They presented spherical morphology under both transmission electron microscope and scanning electron microscope. After coating with 0.15% (w/v) PDSC solution, the NLC showed a core-shell structure and a reversed zeta potential. The enhanced transcorneal penetration of the coated NLC was evaluated using isolated rabbit corneas, with significantly increased apparent permeability coefficient being 1.40- and 1.75-fold of the NLC and FB phosphate solution (FB-sol; p < 0.05), respectively. Precorneal retention assessed by gamma scintigraphy in vivo showed that the area under the remaining activity-time curve of the PDSC-coated formulation was 1.3-fold of the NLC and 2.4-fold of FB-sol. Moreover, in vivo ocular tolerance study indicated that there was no difference in irritation between the coated and noncoated NLC. In conclusion, novel NLC demonstrate high potential for ocular drug delivery. Copyright © 2011 Wiley Periodicals, Inc.

Neurotrophic factor - Characterization and partial purification

NASA Technical Reports Server (NTRS)

Popiela, H.; Ellis, S.

1981-01-01

Recent evidence suggests that neurotrophic activity is required for the normal proliferation and development of muscle cells. The present paper reports a study of the purification and characterization of a neurotrophic factor (NTF) from adult chicken ischiatic-peroneal nerves using two independent quantitative in vitro assay systems. The assays were performed by the measurement of the incorporation of tritiated thymidine or the sizes of single-cell clones by chick muscle cells grown in culture. The greatest amount of neutrotrophic activity is found to be extracted at a pH of 8; aqueous suspensions of the activity are stable to long-term storage at room temperature. The specific activity of the substance is doubled upon precipitation with ammonium sulfate or after gel filtration, and increase 4 to 5 fold after salt gradient elution from DEAE cellulose columns. The active fraction obtained after gel filtration and rechromatography on DEAE cellulose exhibits a 7 to 10-fold increase in specific activity. Electrophoresis of the most highly purified material yields a greatly concentrated band at around 80,000 daltons. Although NTF is purified almost 10-fold as indicated by the increase in specific activity, the maximum activity of the partially purified material is greatly reduced, possibly due to a requirement for a cofactor for the expression of maximum activity.

Examination of key intermediates in the catalytic cycle of aspartate-beta-semialdehyde dehydrogenase from a gram-positive infectious bacteria.

PubMed

Faehnle, Christopher R; Le Coq, Johanne; Liu, Xuying; Viola, Ronald E

2006-10-13

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes a critical branch point transformation in amino acid bio-synthesis. The products of the aspartate pathway are essential in microorganisms, and this entire pathway is absent in mammals, making this enzyme an attractive target for antibiotic development. The first structure of an ASADH from a Gram-positive bacterium, Streptococcus pneumoniae, has now been determined. The overall structure of the apoenzyme has a similar fold to those of the Gram-negative and archaeal ASADHs but contains some interesting structural variations that can be exploited for inhibitor design. Binding of the coenzyme NADP, as well as a truncated nucleotide analogue, into an alternative conformation from that observed in Gram-negative ASADHs causes an enzyme domain closure that precedes catalysis. The covalent acyl-enzyme intermediate was trapped by soaking the substrate into crystals of the coenzyme complex, and the structure of this elusive intermediate provides detailed insights into the catalytic mechanism.

Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

PubMed Central

Sapra, K. Tanuj; Balasubramanian, G. Prakash; Labudde, Dirk; Bowie, James U.; Muller, Daniel J.

2009-01-01

Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wild-type and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others. PMID:18191146

Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin

NASA Astrophysics Data System (ADS)

Fogl, Claudia; Mohammed, Fiyaz; Al-Jassar, Caezar; Jeeves, Mark; Knowles, Timothy J.; Rodriguez-Zamora, Penelope; White, Scott A.; Odintsova, Elena; Overduin, Michael; Chidgey, Martyn

2016-03-01

Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks.

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope

PubMed Central

Otsuka, Shotaro; Bui, Khanh Huy; Schorb, Martin; Hossain, M Julius; Politi, Antonio Z; Koch, Birgit; Eltsov, Mikhail; Beck, Martin; Ellenberg, Jan

2016-01-01

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. DOI: http://dx.doi.org/10.7554/eLife.19071.001 PMID:27630123

Apparatus for Teaching Physics.

ERIC Educational Resources Information Center

Gottlieb, Herbert H., Ed.

1978-01-01

Discusses the adaptation of some familiar equipment for use in physics demonstrations: demonstrating waves using a folding slinky, measuring the partial pressure of water vapor, lengthening the time constant on a rate meter, measuring "g" with a phonograph turntable, and constructing a cheap signal generator. (GA)

Method of identifying hairpin DNA probes by partial fold analysis

DOEpatents

Miller, Benjamin L [Penfield, NY; Strohsahl, Christopher M [Saugerties, NY

2009-10-06

Method of identifying molecular beacons in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within a target gene having the requisite hairpin structure. Isolated oligonucleotides, molecular beacons prepared from those oligonucleotides, and their use are also disclosed.

Method of identifying hairpin DNA probes by partial fold analysis

DOEpatents

Miller, Benjamin L.; Strohsahl, Christopher M.

2008-10-28

Methods of identifying molecular beacons in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within a target gene having the requisite hairpin structure. Isolated oligonucleotides, molecular beacons prepared from those oligonucleotides, and their use are also disclosed.

Chaperonin GroE-facilitated refolding of disulfide-bonded and reduced Taka-amylase A from Aspergillus oryzae.

PubMed

Kawata, Y; Hongo, K; Mizobata, T; Nagai, J

1998-12-01

The refolding characteristics of Taka-amylase A (TAA) from Aspergillus oryzae in the presence of the chaperonin GroE were studied in terms of activity and fluorescence. Disulfide-bonded (intact) TAA and non-disulfide-bonded (reduced) TAA were unfolded in guanidine hydrochloride and refolded by dilution into buffer containing GroE. The intermediates of both intact and reduced enzymes were trapped by GroEL in the absence of nucleotide. Upon addition of nucleotides such as ATP, ADP, CTP or UTP, the intermediates were released from GroEL and recovery of activity was detected. In both cases, the refolding yields in the presence of GroEL and ATP were higher than spontaneous recoveries. Fluorescence studies of intrinsic tryptophan and a hydrophobic probe, 8-anilinonaphthalene-1-sulfonate, suggested that the intermediates trapped by GroEL assumed conformations with different hydrophobic properties. The presence of protein disulfide isomerase or reduced and oxidized forms of glutathione in addition to GroE greatly enhanced the refolding reaction of reduced TAA. These findings suggest that GroE has an ability to recognize folding intermediates of TAA protein and facilitate refolding, regardless of the existence or absence of disulfide bonds in the protein.

High-resolution nuclear magnetic resonance studies of proteins.

PubMed

Jonas, Jiri

2002-03-25

The combination of advanced high-resolution nuclear magnetic resonance (NMR) techniques with high-pressure capability represents a powerful experimental tool in studies of protein folding. This review is organized as follows: after a general introduction of high-pressure, high-resolution NMR spectroscopy of proteins, the experimental part deals with instrumentation. The main section of the review is devoted to NMR studies of reversible pressure unfolding of proteins with special emphasis on pressure-assisted cold denaturation and the detection of folding intermediates. Recent studies investigating local perturbations in proteins and the experiments following the effects of point mutations on pressure stability of proteins are also discussed. Ribonuclease A, lysozyme, ubiquitin, apomyoglobin, alpha-lactalbumin and troponin C were the model proteins investigated.

Alpha-glucosidase folding during urea denaturation: enzyme kinetics and computational prediction.

PubMed

Wu, Xue-Qiang; Wang, Jun; Lü, Zhi-Rong; Tang, Hong-Min; Park, Daeui; Oh, Sang-Ho; Bhak, Jong; Shi, Long; Park, Yong-Doo; Zou, Fei

2010-03-01

In this study, we investigated structural changes in alpha-glucosidase during urea denaturation. Alpha-glucosidase was inactivated by urea in a dose-dependent manner. The inactivation was a first-order reaction with a monophase process. Urea inhibited alpha-glucosidase in a mixed-type reaction. We found that an increase in the hydrophobic surface of this enzyme induced by urea resulted in aggregation caused by unstable folding intermediates. We also simulated the docking between alpha-glucosidase and urea. The docking simulation suggested that several residues, namely THR9, TRP14, LYS15, THR287, ALA289, ASP338, SER339, and TRP340, interact with urea. Our study provides insights into the alpha-glucosidase unfolding pathway and 3D structure of alpha-glucosidase.

Protein folding by NMR.

PubMed

Zhuravleva, Anastasia; Korzhnev, Dmitry M

2017-05-01

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

Domain atrophy creates rare cases of functional partial protein domains.

PubMed

Prakash, Ananth; Bateman, Alex

2015-04-30

Protein domains display a range of structural diversity, with numerous additions and deletions of secondary structural elements between related domains. We have observed a small number of cases of surprising large-scale deletions of core elements of structural domains. We propose a new concept called domain atrophy, where protein domains lose a significant number of core structural elements. Here, we implement a new pipeline to systematically identify new cases of domain atrophy across all known protein sequences. The output of this pipeline was carefully checked by hand, which filtered out partial domain instances that were unlikely to represent true domain atrophy due to misannotations or un-annotated sequence fragments. We identify 75 cases of domain atrophy, of which eight cases are found in a three-dimensional protein structure and 67 cases have been inferred based on mapping to a known homologous structure. Domains with structural variations include ancient folds such as the TIM-barrel and Rossmann folds. Most of these domains are observed to show structural loss that does not affect their functional sites. Our analysis has significantly increased the known cases of domain atrophy. We discuss specific instances of domain atrophy and see that there has often been a compensatory mechanism that helps to maintain the stability of the partial domain. Our study indicates that although domain atrophy is an extremely rare phenomenon, protein domains under certain circumstances can tolerate extreme mutations giving rise to partial, but functional, domains.

Retention of intermediate polarization states in ferroelectric materials enabling memories for multi-bit data storage

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

Zhao, Dong; Asadi, Kamal; Blom, Paul W. M.

A homogeneous ferroelectric single crystal exhibits only two remanent polarization states that are stable over time, whereas intermediate, or unsaturated, polarization states are thermodynamically instable. Commonly used ferroelectric materials however, are inhomogeneous polycrystalline thin films or ceramics. To investigate the stability of intermediate polarization states, formed upon incomplete, or partial, switching, we have systematically studied their retention in capacitors comprising two classic ferroelectric materials, viz. random copolymer of vinylidene fluoride with trifluoroethylene, P(VDF-TrFE), and Pb(Zr,Ti)O{sub 3}. Each experiment started from a discharged and electrically depolarized ferroelectric capacitor. Voltage pulses were applied to set the given polarization states. The retention wasmore » measured as a function of time at various temperatures. The intermediate polarization states are stable over time, up to the Curie temperature. We argue that the remarkable stability originates from the coexistence of effectively independent domains, with different values of polarization and coercive field. A domain growth model is derived quantitatively describing deterministic switching between the intermediate polarization states. We show that by using well-defined voltage pulses, the polarization can be set to any arbitrary value, allowing arithmetic programming. The feasibility of arithmetic programming along with the inherent stability of intermediate polarization states makes ferroelectric materials ideal candidates for multibit data storage.« less

29 CFR 1918.31 - Hatch coverings.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 29 Labor 7 2011-07-01 2011-07-01 false Hatch coverings. 1918.31 Section 1918.31 Labor Regulations...) SAFETY AND HEALTH REGULATIONS FOR LONGSHORING Working Surfaces § 1918.31 Hatch coverings. (a) No cargo... partially opened intermediate deck unless either the hatch at that deck is sufficiently covered or an...

29 CFR 1918.31 - Hatch coverings.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 7 2010-07-01 2010-07-01 false Hatch coverings. 1918.31 Section 1918.31 Labor Regulations...) SAFETY AND HEALTH REGULATIONS FOR LONGSHORING Working Surfaces § 1918.31 Hatch coverings. (a) No cargo... partially opened intermediate deck unless either the hatch at that deck is sufficiently covered or an...

29 CFR 1918.31 - Hatch coverings.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 29 Labor 7 2013-07-01 2013-07-01 false Hatch coverings. 1918.31 Section 1918.31 Labor Regulations...) SAFETY AND HEALTH REGULATIONS FOR LONGSHORING Working Surfaces § 1918.31 Hatch coverings. (a) No cargo... partially opened intermediate deck unless either the hatch at that deck is sufficiently covered or an...

29 CFR 1918.31 - Hatch coverings.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 29 Labor 7 2014-07-01 2014-07-01 false Hatch coverings. 1918.31 Section 1918.31 Labor Regulations...) SAFETY AND HEALTH REGULATIONS FOR LONGSHORING Working Surfaces § 1918.31 Hatch coverings. (a) No cargo... partially opened intermediate deck unless either the hatch at that deck is sufficiently covered or an...

29 CFR 1918.31 - Hatch coverings.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 29 Labor 7 2012-07-01 2012-07-01 false Hatch coverings. 1918.31 Section 1918.31 Labor Regulations...) SAFETY AND HEALTH REGULATIONS FOR LONGSHORING Working Surfaces § 1918.31 Hatch coverings. (a) No cargo... partially opened intermediate deck unless either the hatch at that deck is sufficiently covered or an...

Creative Educational Leadership (A Partial Evaluation).

ERIC Educational Resources Information Center

Kralik, Daniel J.

The purpose of the project was to demonstrate the role and function of the new intermediate school district and to show that it could creatively initiate needed changes in education within three years in Pierce County, Washington. Three specific projects are delineated: (1) guidance training for teachers and administrators; (2) development of…

Computerized algorithms for partial cuts

Treesearch

R.L. Ernst; S.L. Stout

1991-01-01

Stand density, stand structure (diameter distribution), and species composition are all changed by intermediate treatments in forest stands. To use computer stand-growth simulators to assess the effects of different treatments on stand growth and development, users must be able to duplicate silviculturally realistic treatments in the simulator. In this paper, we review...

Partial Immersion Program for Saudi Bilinguals

ERIC Educational Resources Information Center

Alsulami, Sumayyah Qaed

2017-01-01

English is taught as a foreign language in Kingdom of Saudi Arabia. Although the government tries gradually to integrate teaching English in all grades: secondary, intermediate and elementary, learning English is still limited and need more developing. This essay is a brief review about bilingualism in Saudi education. This essay will be divided…

38 CFR 3.350 - Special monthly compensation ratings.

Code of Federal Regulations, 2010 CFR

2010-07-01

... constant inability to communicate by speech or, in the case of a woman veteran, loss of 25% or more of tissue from a single breast or both breasts in combination (including loss by mastectomy or partial... intermediate or next higher rate provisions outlined above, additional single permanent disability or...

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

PubMed

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

2008-01-22

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

Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

NASA Astrophysics Data System (ADS)

Iacovache, Ioan; de Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F. Gisou; Zuber, Benoît

2016-07-01

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

Protein S-sulfenylation is a fleeting molecular switch that regulates non-enzymatic oxidative folding

NASA Astrophysics Data System (ADS)

Beedle, Amy E. M.; Lynham, Steven; Garcia-Manyes, Sergi

2016-08-01

The post-translational modification S-sulfenylation functions as a key sensor of oxidative stress. Yet the dynamics of sulfenic acid in proteins remains largely elusive due to its fleeting nature. Here we use single-molecule force-clamp spectroscopy and mass spectrometry to directly capture the reactivity of an individual sulfenic acid embedded within the core of a single Ig domain of the titin protein. Our results demonstrate that sulfenic acid is a crucial short-lived intermediate that dictates the protein's fate in a conformation-dependent manner. When exposed to the solution, sulfenic acid rapidly undergoes further chemical modification, leading to irreversible protein misfolding; when cryptic in the protein's microenvironment, it readily condenses with a neighbouring thiol to create a protective disulfide bond, which assists the functional folding of the protein. This mechanism for non-enzymatic oxidative folding provides a plausible explanation for redox-modulated stiffness of proteins that are physiologically exposed to mechanical forces, such as cardiac titin.

Exploring the folding free energy landscape of a β-hairpin miniprotein, chignolin, using multiscale free energy landscape calculation method.

PubMed

Harada, Ryuhei; Kitao, Akio

2011-07-14

The folding process for a β-hairpin miniprotein, chignolin, was investigated by free energy landscape (FEL) calculations using the recently proposed multiscale free energy landscape calculation method (MSFEL). First, coarse-grained molecular dynamics simulations searched a broad conformational space, then multiple independent, all-atom molecular dynamics simulations with explicit solvent determined the detailed local FEL using massively distributed computing. The combination of the two models enabled efficient calculation of the free energy landscapes. The MSFEL analysis showed that chignolin has an intermediate state as well as a misfolded state. The folding process is initiated by the formation of a β-hairpin turn, followed by the formation of contacts in the hydrophobic core between Tyr2 and Trp9. Furthermore, mutation of Tyr2 shifts the population to the misfolded conformation. The results indicate that the hydrophobic core plays an important role in stabilizing the native state of chignolin. © 2011 American Chemical Society

Structure/function relationships of calcitonin analogues as agonists, antagonists, or inverse agonists in a constitutively activated receptor cell system.

PubMed

Pozvek, G; Hilton, J M; Quiza, M; Houssami, S; Sexton, P M

1997-04-01

The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

Bradykinin-induced relaxation of coronary microarteries: S-nitrosothiols as EDHF?

PubMed Central

Batenburg, Wendy W; Popp, Rüdiger; Fleming, Ingrid; Vries, René de; Garrelds, Ingrid M; Saxena, Pramod R; Danser, A H Jan

2004-01-01

To investigate whether S-nitrosothiols, in addition to NO, mediate bradykinin-induced vasorelaxation, porcine coronary microarteries (PCMAs) were mounted in myographs. Following preconstriction, concentration–response curves (CRCs) were constructed to bradykinin, the NO donors S-nitroso-N-penicillamine (SNAP) and diethylamine NONOate (DEA-NONOate) and the S-nitrosothiols L-S-nitrosocysteine (L-SNC) and D-SNC. All agonists relaxed PCMAs. L-SNC was ≈5-fold more potent than D-SNC. The guanylyl cyclase inhibitor ODQ and the NO scavenger hydroxocobalamin induced a larger shift of the bradykinin CRC than the NO synthase inhibitor L-NAME, although all three inhibitors equally suppressed bradykinin-induced cGMP responses. Complete blockade of bradykinin-induced relaxation was obtained with L-NAME in the presence of the large- and intermediate-conductance Ca2+-activated K+-channel (BKCa, IKCa) blocker charybdotoxin and the small-conductance Ca2+-activated K+-channel (SKCa) channel blocker apamin, but not in the presence of L-NAME, apamin and the BKCa channel blocker iberiotoxin. Inhibitors of cytochrome P450 epoxygenase, cyclooxygenase, voltage-dependent K+ channels and ATP-sensitive K+ channels did not affect bradykinin-induced relaxation. SNAP-, DEA-NONOate- and D-SNC-induced relaxations were mediated entirely by the NO-guanylyl cyclase pathway. L-SNC-induced relaxations were partially blocked by charybdotoxin+apamin, but not by iberiotoxin+apamin, and this blockade was abolished following endothelium removal. ODQ, but not hydroxocobalamin, prevented L-SNC-induced increases in cGMP, and both drugs shifted the L-SNC CRC 5–10-fold to the right. L-SNC hyperpolarized intact and endothelium-denuded coronary arteries. Our results support the concept that bradykinin-induced relaxation is mediated via de novo synthesized NO and a non-NO, endothelium-derived hyperpolarizing factor (EDHF). S-nitrosothiols, via stereoselective activation of endothelial IKCa and SKCa channels, and through direct effects on smooth muscle cells, may function as an EDHF in porcine coronary microarteries. PMID:15066907

Corticosterone-Induced Lipogenesis Activation and Lipophagy Inhibition in Chicken Liver Are Alleviated by Maternal Betaine Supplementation.

PubMed

Hu, Yun; Sun, Qinwei; Hu, Yan; Hou, Zhen; Zong, Yibo; Omer, Nagmeldin A; Abobaker, Halima; Zhao, Ruqian

2018-03-01

We have shown previously that in ovo betaine injection can prevent nonalcoholic fatty liver induced by glucocorticoid exposure in chickens; yet it remains unknown whether feeding betaine to laying hens may exert similar effects in their progeny. In this study, we fed laying hens a betaine-supplemented diet, and the progeny were later exposed chronically to corticosterone (CORT) to test hepatoprotective effects and further elucidate underlying mechanisms. Rugao yellow-feathered laying hens (n = 120) were fed a basal (control, C) diet or a 0.5% betaine-supplemented (B) diet for 28 d before their eggs were collected for incubation. At 49 d of age, male chickens selected from each group were daily injected subcutaneously with solvent (15% ethanol; vehicle, VEH) or CORT (4.0 mg/kg body mass) for 7 d to establish a fatty liver model. Chickens in the 4 groups (C-VEH, C-CORT, B-VEH, and B-CORT) were killed at day 57. Plasma and hepatic triglyceride (TG) concentrations, as well as the hepatic expression of genes involved in lipogenesis and lipophagy, were determined. CORT induced a 1.6-fold increase in the plasma TG concentration (P < 0.05) and a 1.8-fold increment in the hepatic TG concentration (P < 0.05), associated with activation of lipogenic genes (70-780%). In contrast, lipophagy and mitochondrial β-oxidation genes were inhibited by 30-60% (P < 0.05) in CORT-treated chickens. These CORT-induced changes were completely normalized by maternal betaine supplementation or were partially normalized to intermediate values that were significantly different from those in the C-VEH and C-CORT groups. These effects were accompanied by modifications in CpG methylation and glucocorticoid receptor binding to the promoters of major lipogenic and lipophagic genes (P < 0.05). These results indicate that maternal betaine supplementation protects male juvenile chickens from CORT-induced TG accumulation in the liver via epigenetic modulation of lipogenic and lipophagic genes.

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

Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA*

PubMed Central

Greenfeld, Max; Solomatin, Sergey V.; Herschlag, Daniel

2011-01-01

RNA folding landscapes have been described alternately as simple and as complex. The limited diversity of RNA residues and the ability of RNA to form stable secondary structures prior to adoption of a tertiary structure would appear to simplify folding relative to proteins. Nevertheless, there is considerable evidence for long-lived misfolded RNA states, and these observations have suggested rugged energy landscapes. Recently, single molecule fluorescence resonance energy transfer (smFRET) studies have exposed heterogeneity in many RNAs, consistent with deeply furrowed rugged landscapes. We turned to an RNA of intermediate complexity, the P4-P6 domain from the Tetrahymena group I intron, to address basic questions in RNA folding. P4-P6 exhibited long-lived heterogeneity in smFRET experiments, but the inability to observe exchange in the behavior of individual molecules led us to probe whether there was a non-conformational origin to this heterogeneity. We determined that routine protocols in RNA preparation and purification, including UV shadowing and heat annealing, cause covalent modifications that alter folding behavior. By taking measures to avoid these treatments and by purifying away damaged P4-P6 molecules, we obtained a population of P4-P6 that gave near-uniform behavior in single molecule studies. Thus, the folding landscape of P4-P6 lacks multiple deep furrows that would trap different P4-P6 molecules in different conformations and contrasts with the molecular heterogeneity that has been seen in many smFRET studies of structured RNAs. The simplicity of P4-P6 allowed us to reliably determine the thermodynamic and kinetic effects of metal ions on folding and to now begin to build more detailed models for RNA folding behavior. PMID:21478155

3D geometry and kinematic evolution of the Wadi Mayh sheath fold, Oman, using detailed mapping from high-resolution photography

NASA Astrophysics Data System (ADS)

Cornish, Sam; Searle, Mike

2017-08-01

The Wadi Mayh sheath fold in north-eastern Oman is one of the largest and best-exposed sheath folds known, and presents a unique opportunity to better understand this somewhat enigmatic style of deformation. We undertook high-resolution photographic surveying along Wadi Mayh to document the sheath fold in 61 georeferenced panoramic photomerges. Here we present ten such images that provide a structural interpretation of the sheath fold and surrounding structure. We resolve this structure in a simplified three-dimensional model and in two orthogonal cross sections, and propose a kinematic evolution to explain the geometry. The Wadi Mayh sheath fold is the most prominent example within what we suggest is a composite sequence of sheath folds, which is itself enclosed within a SSW-closing recumbent syncline at the base of the major Saih Hatat nappe. Sheath folding is accommodated within Permian Saiq Formation limestones showing carpholite assemblages (6-8 kbar; 275-375 °C). A major discontinuity separates this sequence from enveloping older rock units. The sequence formed during progressive top-to-north, ductile shearing as the overlying nappe migrated northwards with respect to the underthrusting Hulw unit. This process occurred during SSW-directed exhumation of partially subducted continental crust in NE Oman, approximately 15 Ma after obduction of the Oman ophiolite initiated.

Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation.

PubMed

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

1998-08-15

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

Equation Chapter 1 Section 1Sequence-To-Conformation Relationships of Disordered Regions Tethered to Folded Domains of Proteins.

PubMed

Mittal, Anuradha; Holehouse, Alex S; Cohan, Megan C; Pappu, Rohit V

2018-05-12

Intrinsically disordered proteins and regions (IDPs / IDRs) are characterized by well-defined sequence-to-conformation relationships (SCRs). These relationships refer to the sequence-specific preferences for average sizes, shapes, residue-specific secondary structure propensities, and amplitudes of multiscale conformational fluctuations. SCRs are discerned from the sequence-specific conformational ensembles of IDPs. A vast majority of IDPs are actually tethered to folded domains (FDs). This raises the question of whether or not SCRs inferred for IDPs are applicable to IDRs tethered to folded domains. Here, we use atomistic simulations based on a well-established forcefield paradigm and an enhanced sampling method to obtain comparative assessments of SCRs for thirteen archetypal IDRs modeled as autonomous units, as C-terminal tails connected to folded domains, and as linkers between pairs of folded domains. Our studies uncover a set of general observations regarding context-independent versus context-dependent SCRs of IDRs. SCRs are minimally perturbed upon tethering to folded domains if the IDRs are deficient in charged residues and for polyampholytic IDRs where the oppositely charged residues within the sequence of the IDR are separated into distinct blocks. In contrast, the interplay between IDRs and tethered folded domains has a significant modulatory effect on SCRs if the IDRs have intermediate fractions of charged residues or if they have sequence-intrinsic conformational preferences for canonical random coils. Our findings suggest that IDRs with context-independent SCRs might be independent evolutionary modules whereas IDRs with context-dependent intrinsic SCRs might co-evolve with the FDs to which they are tethered. Copyright © 2018. Published by Elsevier Ltd.

Reversible thermal denaturation of a 60-kDa genetically engineered beta-sheet polypeptide.

PubMed

Lednev, Igor K; Ermolenkov, Vladimir V; Higashiya, Seiichiro; Popova, Ludmila A; Topilina, Natalya I; Welch, John T

2006-11-15

A de novo 687-amino-acid residue polypeptide with a regular 32-amino-acid repeat sequence, (GA)(3)GY(GA)(3)GE(GA)(3)GH(GA)(3)GK, forms large beta-sheet assemblages that exhibit remarkable folding properties and, as well, form fibrillar structures. This construct is an excellent tool to explore the details of beta-sheet formation yielding intimate folding information that is otherwise difficult to obtain and may inform folding studies of naturally occurring materials. The polypeptide assumes a fully folded antiparallel beta-sheet/turn structure at room temperature, and yet is completely and reversibly denatured at 125 degrees C, adopting a predominant polyproline II conformation. Deep ultraviolet Raman spectroscopy indicated that melting/refolding occurred without any spectroscopically distinct intermediates, yet the relaxation kinetics depend on the initial polypeptide state, as would be indicative of a non-two-state process. Thermal denaturation and refolding on cooling appeared to be monoexponential with characteristic times of approximately 1 and approximately 60 min, respectively, indicating no detectable formation of hairpin-type nuclei in the millisecond timescale that could be attributed to nonlocal "nonnative" interactions. The polypeptide folding dynamics agree with a general property of beta-sheet proteins, i.e., initial collapse precedes secondary structure formation. The observed folding is much faster than expected for a protein of this size and could be attributed to a less frustrated free-energy landscape funnel for folding. The polypeptide sequence suggests an important balance between the absence of strong nonnative contacts (salt bridges or hydrophobic collapse) and limited repulsion of charged side chains.

Mesoscale studies of ionic closed membranes with polyhedral geometries

DTIC Science & Technology

2016-07-25

assembled ionic amphiphiles.4 The most commonly observed polyhedral symmetry in self-organized homogeneous structures is the icosahedron, which has the...Possible buckled structures can be obtained considering components A, B with intermediate compositions f of the B component such that the stable shape...lines aids the faceting of the shell into a polyhedral structure often with three-fold vertices. Such vertices are joined together by sharp edges

A role for intermediate radial glia in the tangential expansion of the mammalian cerebral cortex.

PubMed

Reillo, Isabel; de Juan Romero, Camino; García-Cabezas, Miguel Ángel; Borrell, Víctor

2011-07-01

The cerebral cortex of large mammals undergoes massive surface area expansion and folding during development. Specific mechanisms to orchestrate the growth of the cortex in surface area rather than in thickness are likely to exist, but they have not been identified. Analyzing multiple species, we have identified a specialized type of progenitor cell that is exclusive to mammals with a folded cerebral cortex, which we named intermediate radial glia cell (IRGC). IRGCs express Pax6 but not Tbr2, have a radial fiber contacting the pial surface but not the ventricular surface, and are found in both the inner subventricular zone and outer subventricular zone (OSVZ). We find that IRGCs are massively generated in the OSVZ, thus augmenting the numbers of radial fibers. Fanning out of this expanding radial fiber scaffold promotes the tangential dispersion of radially migrating neurons, allowing for the growth in surface area of the cortical sheet. Accordingly, the tangential expansion of particular cortical regions was preceded by high proliferation in the underlying OSVZ, whereas the experimental reduction of IRGCs impaired the tangential dispersion of neurons and resulted in a smaller cortical surface. Thus, the generation of IRGCs plays a key role in the tangential expansion of the mammalian cerebral cortex.

A Simulated Intermediate State for Folding and Aggregation Provides Insights into ΔN6 β2-Microglobulin Amyloidogenic Behavior

PubMed Central

Estácio, Sílvia G.; Krobath, Heinrich; Vila-Viçosa, Diogo; Machuqueiro, Miguel; Shakhnovich, Eugene I.; Faísca, Patrícia F. N.

2014-01-01

A major component of ex vivo amyloid plaques of patients with dialysis-related amyloidosis (DRA) is a cleaved variant of β2-microglobulin (ΔN6) lacking the first six N-terminal residues. Here we perform a computational study on ΔN6, which provides clues to understand the amyloidogenicity of the full-length β2-microglobulin. Contrary to the wild-type form, ΔN6 is able to efficiently nucleate fibrillogenesis in vitro at physiological pH. This behavior is enhanced by a mild acidification of the medium such as that occurring in the synovial fluid of DRA patients. Results reported in this work, based on molecular simulations, indicate that deletion of the N-terminal hexapeptide triggers the formation of an intermediate state for folding and aggregation with an unstructured strand A and a native-like core. Strand A plays a pivotal role in aggregation by acting as a sticky hook in dimer assembly. This study further predicts that the detachment of strand A from the core is maximized at pH 6.2 resulting into higher aggregation efficiency. The structural mapping of the dimerization interface suggests that Tyr10, His13, Phe30 and His84 are hot-spot residues in ΔN6 amyloidogenesis. PMID:24809460

Exploring the mechanical behavior of single intermediate filaments.

PubMed

Kreplak, L; Bär, H; Leterrier, J F; Herrmann, H; Aebi, U

2005-12-02

Intermediate filaments (IFs) are structural elements of eukaryotic cells with distinct mechanical properties. Tissue integrity is severely impaired, in particular in skin and muscle, when IFs are either absent or malfunctioning due to mutations. Our knowledge on the mechanical properties of IFs is mainly based on tensile testing of macroscopic fibers and on the rheology of IF networks. At the single filament level, the only piece of data available is a measure of the persistence length of vimentin IFs. Here, we have employed an atomic force microscopy (AFM) based protocol to directly probe the mechanical properties of single cytoplasmic IFs when adsorbed to a solid support in physiological buffer environment. Three IF types were studied in vitro: recombinant murine desmin, recombinant human keratin K5/K14 and neurofilaments isolated from rat brains, which are composed of the neurofilament triplet proteins NF-L, NF-M and NF-H. Depending on the experimental conditions, the AFM tip was used to laterally displace or to stretch single IFs on the support they had been adsorbed to. Upon applying force, IFs were stretched on average 2.6-fold. The maximum stretching that we encountered was 3.6-fold. A large reduction of the apparent filament diameter was observed concomitantly. The observed mechanical properties therefore suggest that IFs may indeed function as mechanical shock absorbers in vivo.

The Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single Turnover Experiments†

PubMed Central

Roberts, Kenneth M.; Pavon, Jorge Alex; Fitzpatrick, Paul F.

2013-01-01

Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH4) and O2. A complete kinetic mechanism for PheH was determined by global analysis of single turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH4-phenylalanine complex begins with the rapid binding of BH4 (Kd = 65 µM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (Kd = 130 µM) is approximately ten-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O2 rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, detectable as a decrease in absorbance at 340 nm, with a rate constant of 140 s−1. Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is ten-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines kcat. Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation. PMID:23327364

Patient goal setting as a method for program improvement/development in partial hospitalization programs.

PubMed

Gates, A

1991-12-01

Data were collected from a study of 49 patients in 1990 and 106 patients in 1991 admitted into Country View Treatment Center and Green Country Counseling Center. Country View is a 30-bed chemical dependency residential center operating under St. John Medical Center in Tulsa, Oklahoma. Green Country is an evening partial hospital chemical dependency program operating under St. John Medical Center in Tulsa, Oklahoma, The tools used in this study were the Country View Patient Self-Reporting Questionnaire, the global Rating Scale, and the Model of Recovering Alcoholics Behavior Stages and Goal Setting (Wing, 1990). These assessments were specifically designed to measure the patient's perceptions of goal setting and the patient's perspective on treatment outcome. The study outcome resulted in program improvement (Green Country evening partial hospital program) and the development of the Country View Substance Abuse Intermediate Link (SAIL) Program (day partial hospital).

Absolute total and partial dissociative cross sections of pyrimidine at electron and proton intermediate impact velocities

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

Wolff, Wania, E-mail: wania@if.ufrj.br; Luna, Hugo; Sigaud, Lucas

Absolute total non-dissociative and partial dissociative cross sections of pyrimidine were measured for electron impact energies ranging from 70 to 400 eV and for proton impact energies from 125 up to 2500 keV. MOs ionization induced by coulomb interaction were studied by measuring both ionization and partial dissociative cross sections through time of flight mass spectrometry and by obtaining the branching ratios for fragment formation via a model calculation based on the Born approximation. The partial yields and the absolute cross sections measured as a function of the energy combined with the model calculation proved to be a useful toolmore » to determine the vacancy population of the valence MOs from which several sets of fragment ions are produced. It was also a key point to distinguish the dissociation regimes induced by both particles. A comparison with previous experimental results is also presented.« less

(Un)Folding Mechanisms of the FBP28 WW Domain in Explicit Solvent Revealed by Multiple Rare Event Simulation Methods

PubMed Central

Juraszek, Jarek; Bolhuis, Peter G.

2010-01-01

Abstract We report a numerical study of the (un)folding routes of the truncated FBP28 WW domain at ambient conditions using a combination of four advanced rare event molecular simulation techniques. We explore the free energy landscape of the native state, the unfolded state, and possible intermediates, with replica exchange molecular dynamics. Subsequent application of bias-exchange metadynamics yields three tentative unfolding pathways at room temperature. Using these paths to initiate a transition path sampling simulation reveals the existence of two major folding routes, differing in the formation order of the two main hairpins, and in hydrophobic side-chain interactions. Having established that the hairpin strand separation distances can act as reasonable reaction coordinates, we employ metadynamics to compute the unfolding barriers and find that the barrier with the lowest free energy corresponds with the most likely pathway found by transition path sampling. The unfolding barrier at 300 K is ∼17 kBT ≈ 42 kJ/mol, in agreement with the experimental unfolding rate constant. This work shows that combining several powerful simulation techniques provides a more complete understanding of the kinetic mechanism of protein folding. PMID:20159161

Tat transport of a Sec passenger leads to both completely translocated as well as membrane-arrested passenger proteins.

PubMed

Dittmar, Julia; Schlesier, René; Klösgen, Ralf Bernd

2014-02-01

We have studied the membrane transport of the chimeric precursor protein 16/33, which is composed of the Tat(1)-specific transport signal of OEC16 and the Sec passenger protein OEC33, both subunits of the oxygen-evolving system associated with photosystem II. Protein transport experiments performed with isolated pea thylakoids show that the 16/33 chimera is transported in a strictly Tat-dependent manner into the thylakoid vesicles yielding mature OEC33 (mOEC33) in two different topologies. One fraction accumulates in the thylakoid lumen and is thus resistant to externally added protease. A second fraction is arrested during transport in an N-in/C-out topology within the membrane. Chase experiments demonstrate that this membrane-arrested mOEC33 moiety does not represent a translocation intermediate but instead an alternative end product of the transport process. Transport arrest of mOEC33, which is embedded in the membrane with a mildly hydrophobic protein segment, requires more than 26 additional and predominantly hydrophilic residues C-terminal of the membrane-embedded segment. Furthermore, it is stimulated by mutations which potentially affect the conformation of mOEC33 suggesting that at least partial folding of the passenger protein is required for complete membrane translocation. Copyright © 2013 Elsevier B.V. All rights reserved.

Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

PubMed Central

Ganguly, Debabani; Zhang, Weihong; Chen, Jianhan

2013-01-01

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

Electrostatic lipid-protein interactions sequester the curli amyloid fold on the lipopolysaccharide membrane surface.

PubMed

Swasthi, Hema M; Mukhopadhyay, Samrat

2017-12-01

Curli is a functional amyloid protein in the extracellular matrix of enteric Gram-negative bacteria. Curli is assembled at the cell surface and consists of CsgA, the major subunit of curli, and a membrane-associated nucleator protein, CsgB. Oligomeric intermediates that accumulate during the lag phase of amyloidogenesis are generally toxic, but the underlying mechanism by which bacterial cells overcome this toxicity during curli assembly at the surface remains elusive. Here, we elucidated the mechanism of curli amyloidogenesis and provide molecular insights into the strategy by which bacteria can potentially bypass the detrimental consequences of toxic amyloid intermediates. Using a diverse range of biochemical and biophysical tools involving circular dichroism, fluorescence, Raman spectroscopy, and atomic force microscopy imaging, we characterized the molecular basis of the interaction of CsgB with a membrane-mimetic anionic surfactant as well as with lipopolysaccharide (LPS) constituting the outer leaflet of Gram-negative bacteria. Aggregation studies revealed that the electrostatic interaction of the positively charged C-terminal region of the protein with a negatively charged head group of surfactant/LPS promotes a protein-protein interaction that results in facile amyloid formation without a detectable lag phase. We also show that CsgB, in the presence of surfactant/LPS, accelerates the fibrillation rate of CsgA by circumventing the lag phase during nucleation. Our findings suggest that the electrostatic interactions between lipid and protein molecules play a pivotal role in efficiently sequestering the amyloid fold of curli on the membrane surface without significant accumulation of toxic oligomeric intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Further optimization of a hybrid united-atom and coarse-grained force field for folding simulations: Improved backbone hydration and interactions between charged side chains

PubMed Central

Han, Wei; Schulten, Klaus

2012-01-01

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

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

Void-Free Lid for Food Packaging

NASA Technical Reports Server (NTRS)

Watson, C. D.; Farris, W. P.

1986-01-01

Flexible cover eliminates air pockets in sealed container. Universal food-package lid formed from flexible plastic. Partially folded, lid unfolded by depressing center portion. Height of flat portion of lid above flange thereby reduced. Pressure of food against central oval depression pops it out, forming dome that provides finger grip for mixing contents with water or opening lid. Therefore food stays fresh, allows compact stacking of partially filled containers, and resists crushing. Originally developed for packaging dehydrated food for use in human consumption on Space Shuttle missions. Other uses include home canning and commercial food packaging.

STS-71 astronauts and cosmonauts during egress training

NASA Image and Video Library

1994-10-18

S94-47079 (18 Oct 1994) --- Astronaut Robert L. Gibson, (arms folded, near center) STS-71 mission commander, joins several crew mates during a briefing preceding emergency egress training in the Systems Integration Facility at the Johnson Space Center (JSC). Astronauts Bonnie J. Dunbar and Gregory J. Harbaugh (partially obscured), along with cosmonaut Anatoliy Y. Solovyev, all mission specialists, are attired in training versions of the partial pressure launch and entry space suits. Astronaut Charles J. Precourt, pilot, is in center foreground, and Ellen S. Baker, mission specialist, is in left background.

Mechanism of nitrite oxidation by eosinophil peroxidase: implications for oxidant production and nitration by eosinophils

PubMed Central

van Dalen, Christine J.; Winterbourn, Christine C.; Kettle, Anthony J.

2005-01-01

Eosinophil peroxidase is a haem enzyme of eosinophils that is implicated in oxidative tissue injury in asthma. It uses hydrogen peroxide to oxidize thiocyanate and bromide to their respective hypohalous acids. Nitrite is also a substrate for eosinophil peroxidase. We have investigated the mechanisms by which the enzyme oxidizes nitrite. Nitrite was very effective at inhibiting hypothiocyanous acid (‘cyanosulphenic acid’) and hypobromous acid production. Spectral studies showed that nitrite reduced the enzyme to its compound II form, which is a redox intermediate containing FeIV in the haem active site. Compound II does not oxidize thiocyanate or bromide. These results demonstrate that nitrite is readily oxidized by compound I, which contains FeV at the active site. However, it reacts more slowly with compound II. The observed rate constant for reduction of compound II by nitrite was determined to be 5.6×103 M−1·s−1. Eosinophils were at least 4-fold more effective at promoting nitration of a heptapeptide than neutrophils. This result is explained by our finding that nitrite reacts 10-fold faster with compound II of eosinophil peroxidase than with the analogous redox intermediate of myeloperoxidase. Nitration by eosinophils was increased 3-fold by superoxide dismutase, which indicates that superoxide interferes with nitration. We propose that at sites of eosinophilic inflammation, low concentrations of nitrite will retard oxidant production by eosinophil peroxidase, whereas at higher concentrations nitrogen dioxide will be a major oxidant formed by these cells. The efficiency of protein nitration will be decreased by the diffusion-controlled reaction of superoxide with nitrogen dioxide. PMID:16336215