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Folding Pathway (folding + pathway)
Selected AbstractsStabilization of Taq DNA Polymerase at High Temperature by Protein Folding Pathways From a Hyperthermophilic Archaeon, Pyrococcus furiosusBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Pongpan Laksanalamai Abstract Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 100°C, encodes three protein chaperones, a small heat shock protein (sHsp), a prefoldin (Pfd), and a chaperonin (Cpn). In this study, we report that the passive chaperones sHsp and Pfd from P. furiosus can boost the protein refolding activity of the ATP-dependent Cpn from the same hyperthermophile. The thermo-stability of Taq polymerase was significantly improved by combinations of P. furiosus chaperones, showing ongoing protein folding activity at elevated temperatures and during thermal cycling. Based on these results, we propose that the protein folding apparatus in the hyperthermophilic archaeon, P. furiosus can be utilized to enhance the durability and cost effectiveness of high temperature biocatalysts. © 2005 Wiley Periodicals, Inc. [source] The elusive intermediate on the folding pathway of the prion proteinFEBS JOURNAL, Issue 6 2008David C. Jenkins A key molecular event in prion diseases is the conversion of the cellular conformation of the prion protein (PrPC) to an altered disease-associated form, generally denoted as scrapie isoform (PrPSc). The molecular details of this conformational transition are not fully understood, but it has been suggested that an intermediate on the folding pathway of PrPC may be recruited to form PrPSc. In order to investigate the folding pathway of PrP we designed and expressed two mutants, each possessing a single strategically located tryptophan residue. The secondary structure and folding properties of the mutants were examined. Using conventional analyses of folding transition data determined by fluorescence and CD, and novel phase-diagram analyses, we present compelling evidence for the presence of an intermediate species on the folding pathway of PrP. The potential role of this intermediate in prion conversion is discussed. [source] Physico-chemical properties of molten dimer ascorbate oxidaseFEBS JOURNAL, Issue 22 2006Eleonora Nicolai The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 × 108 Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (,V , ,200 mL·mol,1) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (,G , 2 kcal·mol,1) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes. [source] Influence of temperature, friction, and random forces on folding of the B-domain of staphylococcal protein A: All-atom molecular dynamics in implicit solventJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 6 2007Anna Jagielska Abstract The influences of temperature, friction, and random forces on the folding of protein A have been analyzed. A series of all-atom molecular dynamics folding simulations with the Amber ff99 potential and Generalized Born solvation, starting from the fully extended chain, were carried out for temperatures from 300 to 500 K, using (a) the Berendsen thermostat (with no explicit friction or random forces) and (b) Langevin dynamics (with friction and stochastic forces explicitly present in the system). The simulation temperature influences the relative time scale of the major events on the folding pathways of protein A. At lower temperatures, helix 2 folds significantly later than helices 1 and 3. However, with increasing temperature, the folding time of helix 2 approaches the folding times of helices 1 and 3. At lower temperatures, the complete formation of secondary and tertiary structure is significantly separated in time whereas, at higher temperatures, they occur simultaneously. These results suggest that some earlier experimental and theoretical observations of folding events, e.g., the order of helix formation, could depend on the temperature used in those studies. Therefore, the differences in temperature used could be one of the reasons for the discrepancies among published experimental and computational studies of the folding of protein A. Friction and random forces do not change the folding pathway that was observed in the simulations with the Berendsen thermostat, but their explicit presence in the system extends the folding time of protein A. © 2007 Wiley Periodicals, Inc. J Comput Chem 2007 [source] Controlling the mass action of ,-synuclein in Parkinson's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Changyoun Kim Abstract Parkinson's disease (PD) is an age-related neurodegenerative disease with unknown etiology. Growing evidence from genetic, pathologic, animal modeling, and biochemical studies strongly support the theory that abnormal aggregation of ,-synuclein plays a critical role in the pathogenesis of PD. Protein aggregation is an alternative folding process that competes with the native folding pathway. Whether or not a protein is subject to the aggregation process is determined by the concentration of the protein as well as thermodynamic properties inherent to each polypeptide. An increase in cellular concentration of ,-synuclein has been associated with the disease in both familial and sporadic forms of PD. Thus, maintenance of the intraneuronal steady state levels of ,-synuclein below the critical concentration is a key challenge neuronal cells are facing. Expression of the ,-synuclein gene is under the control of environmental factors and aging, the two best-established risk factors for PD. Studies also suggest that the degradation of this protein is mediated by proteasomal and autophagic pathways, which are two mechanisms that are related to the pathogenesis of PD. Recently, vesicle-mediated exocytosis has been suggested as a novel mechanism for disposal of neuronal ,-synuclein. Relocalization of the protein to specific compartments may be another method for increasing its local concentration. Regulation of the neuronal steady state levels of ,-synuclein has significant implications in the development of PD, and understanding the mechanism may disclose potential therapeutic targets for PD and other related diseases. [source] Kinetic barriers and the role of topology in protein and RNA foldingPROTEIN SCIENCE, Issue 8 2008Tobin R. Sosnick Abstract This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events including collapse, properties of denatured states, pathway heterogeneity, and the influence of the mode of initiation on the folding pathway. [source] Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDSPROTEIN SCIENCE, Issue 7 2006Junghwa Kim Abstract Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS,gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers. [source] Substitutions of prolines examine their role in kinetic trap formation of the caspase recruitment domain (CARD) of RICKPROTEIN SCIENCE, Issue 3 2006Yun-Ru Chen Abstract Caspase recruitment domains (CARDs) are small helical protein domains that adopt the Greek key fold. For the two CARDs studied to date, RICK-CARD and caspase-1-CARD (CP1-CARD), the proteins unfold by an apparent two-state process at equilibrium. However, the folding kinetics are complex for both proteins and may contain kinetically trapped species on the folding pathway. In the case of RICK-CARD, the time constants of the slow refolding phases are consistent with proline isomerism. RICK-CARD contains three prolines, P47 in turn 3, and P85 and P87. The latter two prolines constitute a nonconserved PxP motif in helix 6. To examine the role of the prolines in the complex folding kinetics of RICK-CARD, we generated seven proline-to-alanine mutants, including three single mutants, three double mutants, and one triple mutant. We examined the spectroscopic properties, equilibrium folding, binding to CP1-CARD, and folding kinetics. The results show that P85 is critical for maintaining the function of the protein and that all mutations decrease the stability. Results from single mixing and sequential mixing stopped-flow studies strongly suggest the presence of parallel folding pathways consisting of at least two unfolded populations. The mutations affect the distribution of the two unfolded species, thereby affecting the population that folds through each channel. The two conformations also are present in the triple mutant, demonstrating that interconversion between them is not due to prolyl isomerism. Overall, the data show that the complex folding pathway, especially formation of kinetically trapped species, is not due to prolyl isomerism. [source] Alteration of the disulfide-coupled folding pathway of BPTI by circular permutationPROTEIN SCIENCE, Issue 5 2004Grzegorz Bulaj BPTI, bovine pancreatic trypsin inhibitor; cBPTI, a circular form of BPTI generated by forming a peptide bond between the natural termini; cpBPTI, circularly permuted BPTI. Abstract The kinetics of disulfide-coupled folding and unfolding of four circularly permuted forms of bovine pancreatic trypsin inhibitor (BPTI) were studied and compared with previously published results for both wild-type BPTI and a cyclized form. Each of the permuted proteins was found to be less stable than either the wild-type or circular proteins, by 3,8 kcal/mole. These stability differences were used to estimate effective concentrations of the chain termini in the native proteins, which were 1 mM for the wild-type protein and 2.5 to 4000 M for the permuted forms. The circular permutations increased the rates of unfolding and caused a variety of effects on the kinetics of refolding. For two of the proteins, the rates of a direct disulfide-formation pathway were dramatically increased, making this process as fast or faster than the competing disulfide rearrangement mechanism that predominates in the folding of the wild-type protein. These two permutations break the covalent connectivity among the ,-strands of the native protein, and removal of these constraints appears to facilitate direct formation and reduction of nearby disulfides that are buried in the folded structure. The effects on folding kinetics and mechanism do not appear to be correlated with relative contact order, a measure of overall topological complexity. These observations are consistent with the results of other recent experimental and computational studies suggesting that circular permutation may generally influence folding mechanisms by favoring or disfavoring specific interactions that promote alternative pathways, rather than through effects on the overall topology of the native protein. [source] The dual role of a loop with low loop contact distance in folding and domain swappingPROTEIN SCIENCE, Issue 7 2002Apichart Linhananta Abstract , helices, , strands, and loops are the basic building blocks of protein structure. The folding kinetics of , helices and , strands have been investigated extensively. However, little is known about the formation of loops. Experimental studies show that for some proteins, the formation of a single loop is the rate-determining step for folding, whereas for others, a loop (or turn) can misfold to serve as the hinge loop region for domain-swapped species. Computer simulations of an all-atom model of fragment B of Staphylococcal protein A found that the formation of a single loop initiates the dominant folding pathway. On the other hand, the stability analysis of intermediates suggests that the same loop is a likely candidate to serve as a hinge loop for domain swapping. To interpret the simulation result, we developed a simple structural parameter: the loop contact distance (LCD), or the sequence distance of contacting residues between a loop and the rest of the protein. The parameter is applied to a number of other proteins, including SH3 domains and prion protein. The results suggest that a locally interacting loop (low LCD) can either promote folding or serve as the hinge region for domain swapping. Thus, there is an intimate connection between folding and domain swapping, a possible cause of misfolding and aggregation. [source] Kinetic evidence of an on-pathway intermediate in the folding of lysozymePROTEIN SCIENCE, Issue 1 2000Yawen Bai Abstract By means of a kinetic test, it was demonstrated that one of the folding intermediates (I,) of hen lysozyme with ,-domain folded and ,-domain unfolded is on the folding pathway under the classical definition. l, folds to the native (N) state directly (unfolded (U) , I, , N) without having to unfold to U and then refold to N through alternative folding pathways as in I, , U , N. [source] Parallel protein folding with STAPLCONCURRENCY AND COMPUTATION: PRACTICE & EXPERIENCE, Issue 14 2005Shawna Thomas Abstract The protein-folding problem is a study of how a protein dynamically folds to its so-called native state,an energetically stable, three-dimensional conformation. Understanding this process is of great practical importance since some devastating diseases such as Alzheimer's and bovine spongiform encephalopathy (Mad Cow) are associated with the misfolding of proteins. We have developed a new computational technique for studying protein folding that is based on probabilistic roadmap methods for motion planning. Our technique yields an approximate map of a protein's potential energy landscape that contains thousands of feasible folding pathways. We have validated our method against known experimental results. Other simulation techniques, such as molecular dynamics or Monte Carlo methods, require many orders of magnitude more time to produce a single, partial trajectory. In this paper we report on our experiences parallelizing our method using STAPL (Standard Template Adaptive Parallel Library) that is being developed in the Parasol Lab at Texas A&M. An efficient parallel version will enable us to study larger proteins with increased accuracy. We demonstrate how STAPL enables portable efficiency across multiple platforms, ranging from small Linux clusters to massively parallel machines such as IBM's BlueGene/L, without user code modification. Copyright © 2005 John Wiley & Sons, Ltd. [source] Influence of temperature, friction, and random forces on folding of the B-domain of staphylococcal protein A: All-atom molecular dynamics in implicit solventJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 6 2007Anna Jagielska Abstract The influences of temperature, friction, and random forces on the folding of protein A have been analyzed. A series of all-atom molecular dynamics folding simulations with the Amber ff99 potential and Generalized Born solvation, starting from the fully extended chain, were carried out for temperatures from 300 to 500 K, using (a) the Berendsen thermostat (with no explicit friction or random forces) and (b) Langevin dynamics (with friction and stochastic forces explicitly present in the system). The simulation temperature influences the relative time scale of the major events on the folding pathways of protein A. At lower temperatures, helix 2 folds significantly later than helices 1 and 3. However, with increasing temperature, the folding time of helix 2 approaches the folding times of helices 1 and 3. At lower temperatures, the complete formation of secondary and tertiary structure is significantly separated in time whereas, at higher temperatures, they occur simultaneously. These results suggest that some earlier experimental and theoretical observations of folding events, e.g., the order of helix formation, could depend on the temperature used in those studies. Therefore, the differences in temperature used could be one of the reasons for the discrepancies among published experimental and computational studies of the folding of protein A. Friction and random forces do not change the folding pathway that was observed in the simulations with the Berendsen thermostat, but their explicit presence in the system extends the folding time of protein A. © 2007 Wiley Periodicals, Inc. J Comput Chem 2007 [source] Substitutions of prolines examine their role in kinetic trap formation of the caspase recruitment domain (CARD) of RICKPROTEIN SCIENCE, Issue 3 2006Yun-Ru Chen Abstract Caspase recruitment domains (CARDs) are small helical protein domains that adopt the Greek key fold. For the two CARDs studied to date, RICK-CARD and caspase-1-CARD (CP1-CARD), the proteins unfold by an apparent two-state process at equilibrium. However, the folding kinetics are complex for both proteins and may contain kinetically trapped species on the folding pathway. In the case of RICK-CARD, the time constants of the slow refolding phases are consistent with proline isomerism. RICK-CARD contains three prolines, P47 in turn 3, and P85 and P87. The latter two prolines constitute a nonconserved PxP motif in helix 6. To examine the role of the prolines in the complex folding kinetics of RICK-CARD, we generated seven proline-to-alanine mutants, including three single mutants, three double mutants, and one triple mutant. We examined the spectroscopic properties, equilibrium folding, binding to CP1-CARD, and folding kinetics. The results show that P85 is critical for maintaining the function of the protein and that all mutations decrease the stability. Results from single mixing and sequential mixing stopped-flow studies strongly suggest the presence of parallel folding pathways consisting of at least two unfolded populations. The mutations affect the distribution of the two unfolded species, thereby affecting the population that folds through each channel. The two conformations also are present in the triple mutant, demonstrating that interconversion between them is not due to prolyl isomerism. Overall, the data show that the complex folding pathway, especially formation of kinetically trapped species, is not due to prolyl isomerism. [source] Kinetic evidence of an on-pathway intermediate in the folding of lysozymePROTEIN SCIENCE, Issue 1 2000Yawen Bai Abstract By means of a kinetic test, it was demonstrated that one of the folding intermediates (I,) of hen lysozyme with ,-domain folded and ,-domain unfolded is on the folding pathway under the classical definition. l, folds to the native (N) state directly (unfolded (U) , I, , N) without having to unfold to U and then refold to N through alternative folding pathways as in I, , U , N. [source] | |||||||||||||