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Results for “Protein Folding”

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At least 19 recordsLinked to original sources

PubMed2001

From folding theories to folding proteins: a review and assessment of simulation studies of protein folding and unfolding.

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.

Models, Molecular↗

PubMed2001

Integration of genome data and protein structures: prediction of protein folds, protein interactions and "molecular phenotypes" of single nucleotide polymorphisms.

With the massive amount of sequence and structural data being produced, new avenues emerge for exploiting the information therein for applications in several fields. Fold distributions can be mapped onto entire genomes to learn about the nature of the protein universe and many of the interactions between proteins can now be predicted solely on the basis of the genomic context of their genes. Furthermore, by utilising the new incoming data on single nucleotide polymorphisms by mapping them onto three-dimensional structures of proteins, problems concerning population, medical and evolutionary genetics can be addressed.

Apolipoproteins E↗

PubMed2004

Theory of protein folding.

Protein folding should be complex. Proteins organize themselves into specific three-dimensional structures, through a myriad of conformational changes. The classical view of protein folding describes this process as a nearly sequential series of discrete intermediates. In contrast, the energy landscape theory of folding considers folding as the progressive organization of an ensemble of partially folded structures through which the protein passes on its way to the natively folded structure. As a result of evolution, proteins have a rugged funnel-like landscape biased toward the native structure. Connecting theory and simulations of minimalist models with experiments has completely revolutionized our understanding of the underlying mechanisms that control protein folding.

Computer Simulation↗

PubMed1994

Cold denaturation of the molten globule states of apomyoglobin and a profile for protein folding.

Protein folding is a process in which an extended polypeptide chain acquires compact packing through the formation of specific secondary and tertiary structures and hydrophobic interactions. Although much attention has been paid to secondary and tertiary structures, there is no definitive view about the relationship between these structures, compactness, and hydrophobic interactions during the process of protein folding. We show here that the molten globule intermediates of horse apomyoglobin exhibit cold denaturation in addition to heat denaturation, which indicates that the heat capacity change upon unfolding is positive and significant. This demonstrates a small but distinct contribution of hydrophobic interactions to the stability of the molten globule state. We determined the radius of gyration of the various conformational states of horse apomyoglobin and holomyoglobin by measuring small angle X-ray scattering. By comparing the conformational states in terms of secondary structure, radius of gyration, and change in heat capacity upon unfolding, we constructed a folding profile. The profile shows that the protein becomes more compact with formation of the secondary structure, but does not form substantial hydrophobic interactions until a later rate-limiting stage when tight packing of the protein side chains occurs. A very similar profile was also obtained with horse cytochrome c. We propose that the folding profile obtained with these proteins will be common to many globular proteins.

Apoproteins↗

PubMed2000

Group II chaperonins as mediators of cytosolic protein folding.

Protein folding and assembly in the cell requires the assistance of molecular chaperones. These components prevent off-pathway folding reactions that lead to aggregation. They are also critical factors in organismal stress physiology, protecting cells against heat shock and providing thermotolerance. Among this important protein family are chaperonins. They form large cylindrical double ring complexes with a central cavity where protein binding and folding takes place in an ATP-dependent manner. Recently, components functionally related to the eubacterial and organellar chaperonins have been found in the cytosol of archaebacteria and of eukaryotic cells. Based on their sequences and structural features, they have been classified as group II chaperonins, to distinguish them from the group I chaperonins occurring in bacteria. Of particular interest in the group II family is the eukaryotic CCT complex, whose function in protein folding and assembly has been demonstrated mainly for the cytoskeletal proteins tubulin and actin. Together with the Hsp70 chaperone system, it can be considered as an essential helper factor to facilitate the folding of native proteins in the eukaryotic cytosol. Recent structural data have opened the path to a molecular understanding of group II chaperonins and have helped to define their role in cellular protein folding.

Animals↗

PubMed1996

X-ray solution scattering studies of protein folding.

Protein folding is a reaction in which an extended polypeptide chain acquires maximal packing through formation of secondary and tertiary structures. Compactness and shape are, therefore, critical properties characterizing the process of protein folding. Because the stability of the native state is determined by the subtle free energy balance between the native and denatured states, the characterization of the denatured state is also essential to understand the conformational stability of the native state. We show that solution X-ray scattering is the best technique available today to address these problems. Although the structural resolution of the unfolded or compact denatured states elucidated from solution X-ray scattering is low, it provides a variety of information complementary to that obtained by NMR or X-ray crystallography.

Animals↗

PubMed2007

Application of single molecule Förster resonance energy transfer to protein folding.

Protein folding is a process characterized by a large degree of conformational heterogeneity. In such cases, classical experimental methods yield only mean values, averaged over large ensembles of molecules. The microscopic distributions of conformations, trajectories, or sequences of events often remain unknown, and with them the underlying molecular mechanisms. Signal averaging can be avoided by observing individual molecules. A particularly versatile method is highly sensitive fluorescence detection. In combination with Förster resonance energy transfer, distances and conformational dynamics can be investigated in single molecules. This chapter introduces the practical aspects of applying this method to protein folding.

Chromatography, High Pressure Liquid↗

PubMed1996

Influence of molecular and chemical chaperones on protein folding.

Protein folding inside the cell involves the participation of accessory components known as molecular chaperones. In addition to their active participation in the folding process, molecular chaperones serve as a type of 'quality control system', recognizing, retaining and targeting misfolded proteins for their eventual degradation. It is now known that a number of human diseases arise as a consequence of specific point mutations or deletions within genes encoding essential proteins. In many cases these mutations/deletions are not so severe as to totally destroy the biological activity of the particular gene product. Rather, the mutations often result in only subtle folding abnormalities which lead to the newly synthesized protein being retained at the endoplasmic reticulum by the actions of the cellular quality control system. In this short review article we discuss our recent studies showing that the protein folding defect associated with the most common mutation in patients with cystic fibrosis can be overcome by a novel strategy. As shown in the paper by Brown et al in this issue (Brown et al 1996), a number of different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in rescuing the processing defect of the mutant cystic fibrosis transmembrane conductance regulator protein. We then discuss how these same compounds, which we now call chemical chaperones, also may prove to be effective in correcting a number of other protein folding abnormalities which constitute the underlying basis of a large number of different human diseases.

Humans↗

PubMed2006

Atom-by-atom analysis of global downhill protein folding.

Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.

Entropy↗

PubMed1995

Molecular chaperones in cellular protein folding.

Protein folding in the cell requires molecular chaperones. The chaperone proteins of the hsp70 and hsp60 (chaperonin) classes stabilize unfolded or partially folded polypeptides, thereby preventing aggregation, and mediate folding to the native state in ATP-dependent reactions. Recent advances include a more detailed understanding of the mechanistic principles of hsp70 and hsp60 action, the solution of the crystal structure of the chaperonin GroEL, and the definition of pathways of chaperone-mediated protein folding.

Chaperonins↗

PubMed2001

Secondary-structure-favored hydrophobic-polar lattice model of protein folding.

Protein folding is studied using a two-dimensional lattice model with the Hamiltonian including both hydrophobic interactions and main chain hydrogen bond interactions of amino acids. Since compact conformations have different designabilities and only highly designable conformations can act as native structural candidates [H. Li, R. Helling, C. Tang, and N. Wingreen, Science 273, 666 (1996)], it is shown that hydrophobic interaction alone is insufficient to explain the appearance of a high proportion of regular secondary structures, especially beta sheets whose content decreases with increasing designability, but interactions of main chain hydrogen bonds can account for this. Thus the emergence of only a small number of structure types (folds) among all possible structures can be understood to some extent.

Hydrogen Bonding↗

PubMed2001

Conformational propagation with prion-like characteristics in a simple model of protein folding.

Protein refolding/misfolding to an alternative form plays an aetiologic role in many diseases in humans, including Alzheimer's disease, the systemic amyloidoses, and the prion diseases. Here we have discovered that such refolding can occur readily for a simple lattice model of proteins in a propagatable manner without designing for any particular alternative native state. The model uses a simple contact energy function for interactions between residues and does not consider the peculiarities of polypeptide geometry. In this model, under conditions where the normal (N) native state is marginally stable or unstable, two chains refold from the N native state to an alternative multimeric energetic minimum comprising a single refolded conformation that can then propagate itself to other protein chains. The only requirement for efficient propagation is that a two-faced mode of packing must be in the ground state as a dimer (a higher-energy state for this packing leads to less efficient propagation). For random sequences, these ground-state dimeric configurations tend to have more beta-sheet-like extended structure than almost any other sort of dimeric ground-state assembly. This implies that propagating states (such as for prions) are beta-sheet rich because the only likely propagating forms are beta-sheet rich. We examine the details of our simulations to see to what extent the observed properties of prion propagation can be predicted by a simple protein folding model. The formation of the alternative state in the present model shows several distinct features of amyloidogenesis and of prion propagation. For example, an analog of the phenomenon of conformationally distinct strains in prions is observed. We find a parallel between 'glassy' behavior in liquids and the formation of a propagatable state in proteins. This is the first report of simulation of conformational propagation using any heteropolymer model. The results imply that some (but not most) small protein sequences must maintain a sequence signal that resists refolding to propagatable alternative native states and that the ability to form such states is not limited to polypeptides (or reliant on regular hydrogen bonding per se) but can occur for other protein-like heteropolymers.

Amino Acid Sequence↗

PubMed1996

Emergence of preferred structures in a simple model of protein folding.

Protein structures in nature often exhibit a high degree of regularity (for example, secondary structure and tertiary symmetries) that is absent from random compact conformations. With the use of a simple lattice model of protein folding, it was demonstrated that structural regularities are related to high "designability" and evolutionary stability. The designability of each compact structure is measured by the number of sequences that can design the structure-that is, sequences that possess the structure as their nondegenerate ground state. Compact structures differ markedly in terms of their designability; highly designable structures emerge with a number of associated sequences much larger than the average. These highly designable structures possess "proteinlike" secondary structure and even tertiary symmetries. In addition, they are thermodynamically more stable than other structures. These results suggest that protein structures are selected in nature because they are readily designed and stable against mutations, and that such a selection simultaneously leads to thermodynamic stability.

Amino Acid Sequence↗

PubMed2004

Structural examination of phi-value analysis in protein folding.

Protein folding intermediates and transition states are commonly characterized using a protein engineering procedure (Phi-value analysis) based on several assumptions, including (1) intermediates and transition states have native-like conformations and (2) single mutations from larger hydrophobic residues to smaller ones do not perturb their structures. Although Phi-value analysis has been widely used, these assumptions have not been tested to date because of the lack of high-resolution structures of intermediates and transition states. We recently have determined the structure of a folding intermediate for a four-helix bundle protein (Rd-apocytochrome b(562)) using NMR. The intermediate has the N-terminal helix unfolded. The other three helices fold in a native-like topology with extensive non-native hydrophobic interactions. Here, we have determined the Phi values for 14 hydrophobic core residues, including those with significant non-native interactions. All of the Phi values are in the normal range from 0 to 1, indicating that these non-native interactions cannot be identified by the common Phi-value analysis, and therefore, the first assumption is not valid for this intermediate. We also determined the structure of a mutant (F65A) of the intermediate and found that the structure of the intermediate is not perturbed by the mutation, supporting the second assumption. Together, these results suggest that Phi-value analysis may be valid for characterizing the energetics of the interactions between the mutated residue and others, but not for determining the detailed structures of intermediates and transition states because non-native interactions may exist and may not be identifiable by the common Phi-value analysis.

Alanine↗

PubMed2004

A high-resolution probe of protein folding.

Protein folding is a central problem in the biological sciences. To generate residue-specific information on the equilibrium folding of cytochrome c, we have semisynthesized the protein with specifically deuterated residues. The C-D bonds may be easily visualized in an otherwise transparent region of the IR spectra, even at high protein and denaturant concentrations. Plotted as a function of added guanidine hydrochloride denaturant, the absorption intensities reveal that the protein undergoes a conformational change at the protein-based ligand, Met80, which is then followed by a more global unfolding at 2.3 M denaturant. Deuteration and characterization of other residues in cytochrome c, or other protein of interest, should provide complete views of folding with residue specific detail that is capable of resolving even the most rapidly interconverting intermediates.

Animals↗

PubMed1998

Directionality of polypeptide transfer in the mitochondrial pathway of chaperone-mediated protein folding.

Protein folding in mitochondria depends on the functional cooperation of the Hsp70 and Hsp60 chaperone systems, at least for a subset of mitochondrial polypeptides. As suggested previously, Hsp70 and Hsp60 act sequentially. However, recent proposals that the chaperonin Hsp60 functions by releasing substrate protein in an unfolded state would predict a lateral partitioning of folding intermediates between chaperone systems. Firefly luciferase, carrying a mitochondrial targeting signal, was used as a model protein to analyze the degree of coupling and the directionality of substrate transfer between the Hsp70 and Hsp60 chaperones. In vitro, Hsp60 binds unfolded luciferase with high affinity but is unable to promote its folding, whereas the Hsp70 system assists the folding of luciferase efficiently. Upon import into yeast mitochondria, luciferase interacted first with Hsp70. Surprisingly, most of the protein subsequently accumulated in a complex with Hsp60 and never reached the native state. Import into mitochondria that lack a functional Hsp60 did not result in increased folding, but in the aggregation of luciferase. Thus, in intact organelles the two chaperone systems do not function independently in de novo folding of aggregation-sensitive proteins but rather act in an ordered pathway with substrate transfer predominantly in the direction from Hsp70 to Hsp60.

Biological Transport↗

PubMed2004

Correspondence between anomalous m- and DeltaCp-values in protein folding.

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant LA30 has a curved unfolding limb, which is most consistent with TSE mobility. The kinetic m-values (derivatives of the rate constants with respect to denaturant concentration) in themselves depend on denaturant concentration. To obtain complementary information about putative mobile TSEs, we have carried out a thermodynamic analysis of the three proteins, based on data for refolding and unfolding over the range 10 degrees C to 70 degrees C. The data at all temperatures can be fitted to two-state model systems. Importantly, for all three proteins the activation heat capacities are, within error, identical to the heat capacities measured in independent experiments under equilibrium conditions. Although the equilibrium heat capacities are essentially invariant with regard to denaturant concentration, the activation heat capacities, similar to the structurally equivalent kinetic m-values, show marked denaturant dependence. Furthermore, the values of beta++ at different denaturant concentrations measured by m-values and by heat capacity values are very similar. These observations are consistent with significant transition state movements within the framework of two-state folding. The basis for TSE movement appears to be enthalpic rather than entropic, suggesting that the binding energy of denaturant-protein interactions is a major determinant of the response of energy landscape contours to changing environments.

Protein Denaturation↗

PubMed1991

Protein stability and protein folding.

Proteins show only marginal free energies of stabilization. Mutative adaptations to extremes of physical conditions (high temperature, pressure and salt concentration) tend to maintain 'corresponding states' regarding overall topology, flexibility and hydration. Since enhanced stability requires only minute local changes in the structure of a given protein, general strategies of adaptation cannot be established. Apart from alterations at the protein level, extrinsic factors such as ions, cofactors or specific ligands may serve to enhance in vivo and in vitro protein stability. Protein folding and association reflect the hierarchy of protein structure, with the formation of secondary/supersecondary structure, subdomains/domains and structured monomers as consecutive steps. The process requires highly specified environmental conditions; e.g. active mesophilic or halophilic proteins cannot be expressed in thermophilic and non-halophilic hosts. On the other hand, a given protein may tolerate extreme sequence variability without substantially altering its three-dimensional structure and stability. Significant rate-determining steps in the overall reaction, that is, formation of disulphide bridges, proline isomerization and oligomerization, are catalysed by specific enzymes or directed by 'helper proteins' (protein disulphide isomerase, peptidyl-prolyl cis-trans isomerase and chaperones). Physiological stress conditions, (site-directed) mutations, and in vitro studies may be used to unravel the significance of the three 'shuffling reactions'.

Amino Acid Isomerases↗