Abstract: A question of fundamental importance concerning protein folding in vivo is whether the kinetics of translation or the thermodynamics of the ribosome nascent chain (RNC) complex is the major determinant of cotranslational folding behavior. This is because translation rates can reduce the probability of cotranslational folding below that associated with arrested ribosomes, whose behavior is determined by the equilibrium thermodynamics of the RNC complex. Here, we combine a chemical kinetic equation with genomic and proteomic data to predict domain folding probabilities as a function of nascent chain length for Escherichia coli cytosolic proteins synthesized on both arrested and continuously translating ribosomes. Our results indicate that, at in vivo translation rates, about one-third of the Escherichia coli cytosolic proteins exhibit cotranslational folding, with at least one domain in each of these proteins folding into its stable native structure before the fulllength protein is released from the ribosome. The majority of these cotranslational folding domains are influenced by translation kinetics which reduces their probability of cotranslational folding and consequently increases the nascent chain length at which they fold into their native structures. For about 20% of all cytosolic proteins this delay in folding can exceed the length of the completely synthesized protein, causing one or more of their domains to switch from co- to posttranslational folding solely as a result of the in vivo translation rates. These kinetic effects arise from the difference in time scales of folding and amino-acid addition, and they represent as ource of metastability inEscherichia coli’ sp roteome.

Abstract: Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.

Abstract: Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage's exact folding mechanism is still a matter of debate. Here we investigate Trp-cage's relaxation dynamics in the amide I' spectral region (1530-1700 cm(-1)) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label ((13)C═(18)O) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 310-helical residue. The folding-unfolding equilibrium is perturbed using a nanosecond temperature-jump (T-jump), and the subsequent re-equilibration is probed by observing the time-dependent vibrational response in the amide I' region. We observe bimodal relaxation kinetics with time constants of 100 ± 10 and 770 ± 40 ns at 322 K, suggesting that the folding involves an intermediate state, the character of which can be determined from the time- and frequency-resolved data. We find that the relaxation dynamics close to the melting temperature involve fast fluctuations in the polyproline II region, whereas the slower process can be attributed to conformational rearrangements due to the global (un)folding transition of the protein. Combined analysis of our T-jump data and molecular dynamics simulations indicates that the formation of a well-defined α-helix precedes the rapid formation of the hydrophobic cage structure, implying a native-like folding intermediate, that mainly differs from the folded conformation in the orientation of the C-terminal polyproline II helix relative to the N-terminal part of the backbone. We find that the main free-energy barrier is positioned between the folding intermediate and the unfolded state ensemble, and that it involves the formation of the α-helix, the 310-helix, and the Asp9-Arg16 salt bridge. Our results suggest that at low temperature (T ≪ Tm) a folding path via formation of α-helical contacts followed by hydrophobic clustering becomes more important.

Abstract: 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 metastable 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...

Abstract: In the funneled landscape, proteins fold to their native states through a stochastic process in which the free energy decreases spontaneously and unfolded, transition, native, and possible intermediate states correspond to local minima or saddle points. Atomic description of the folding pathway appears therefore to be essential for a deep comprehension of the folding mechanism. In metallo-proteins, characterization of the folding pathways becomes even more complex, and therefore, despite their fundamental role in critical biological processes, little is known about their folding and assembly. The study of the mechanisms through which a cofactor influences the protein folding/unfolding reaction has been the rationale of the present study aimed at contributing to the search for cofactors’ general roles in protein folding reactions. In particular, we have investigated the folding pathway of two homologous proteins, Ros87, which contains a prokaryotic zinc finger domain, and Ml452–151, lacking the zinc ion. U...

Abstract: Analysis of effects of denaturants and temperature on folding and unfolding rate constants of 13 globular proteins yields the amount and composition of the surface buried in folding to and from the high–free-energy transition state (TS), and thereby provides information about folding mechanisms and early unstable folding intermediates. All 13 proteins preferentially bury amide surface in folding to TS; amounts of amide and hydrocarbon surface buried in folding to TS generally exceed those buried in forming the native secondary structure. From this, we conclude that most native secondary structure forms in early unstable folding intermediates and that conversion of these intermediates to TS involves nucleation of tertiary interactions, allowing preferential burial of hydrocarbon surface as TS folds.

Abstract: When an amino-acid sequence cannot be optimized for both folding and function, folding can get compromised in favor of function. To understand this tradeoff better, we devise a novel method for extracting the "function-less" folding-motif of a protein fold from a set of structurally similar but functionally diverse proteins. We then obtain the β-trefoil folding-motif, and study its folding using structure-based models and molecular dynamics simulations. CompariA protein sequence serves two purpson with the folding of wild-type β-trefoil proteins shows that function affects folding in two ways: In the slower folding interleukin-1β, binding sites make the fold more complex, increase contact order and slow folding. In the faster folding hisactophilin, residues which could have been part of the folding-motif are used for function. This reduces the density of native contacts in functional regions and increases folding rate. The folding-motif helps identify subtle structural deviations which perturb folding. These may then be used for functional annotation. Further, the folding-motif could potentially be used as a first step in the sequence design of function-less scaffold proteins. Desired function can then be engineered into these scaffolds.

Abstract: Protein folding is evidently not a random process given the speed and reproducibility of folding in vivo;1 yet how a given polypeptide sequence translates into the globular structure of a fully folded protein remains unclear,2 particularly with respect to the role that water plays in this process.3 One frequently occurring folding pattern in proteins is the β-turn,4 where the amino acid sequences that give rise to these turns are thought to nucleate folding.5 The question remains however, if it is the mere presence of certain amino acids which initiate the formation of β-turns or if water plays a fundamental role in this process.

Abstract: There is continued interest in predicting the structure of proteins either at the simplest level of identifying their fold class or persevering all the way to an atomic resolution structure. Protei...

Abstract: Protein folding is a process in which a polypeptide folds into a specific, stable, functional, three-dimensional structure. It is the process by which a protein structure assumes its functional shape or conformation. Proteins are comprised of amino acids with various types of side chains, which may be hydrophobic, hydrophilic or electrically charged. It is now well known that under physiological conditions, proteins normally spontaneously fold into their native conformations but there are some exterior factors which help polypeptide chain finding its natural shape. Different levels of folding a protein after amino acid sequence or primary structure consist of secondary, tertiary and quaternary structures. Protein folding pathway or mechanism is the typical sequence of structural changes; in which protein find its native structure. 3D structure of proteins is studied by scientists using different methods and there are many types of software to survey this. Many factors control protein folding, interior and exterior factors. Creation of natural folded proteins by these factors and protein translation are simultaneous. The main objective of this review is to unveil the fact; despite there are many factors controlling protein folding, mainspring is amino acids sequence which itself rises from chemo-physical laws.

Abstract: Theory predicts that folding free energy landscapes are intrinsically malleable and as such are expected to respond to perturbations in topographically complex ways. Structural changes upon perturbation have been observed experimentally for unfolded ensembles, folding transition states, and fast downhill folding proteins. However, the native state of proteins that fold in a two-state fashion is conventionally assumed to be structurally invariant during unfolding. Here we investigate how the native and unfolded states of the chicken α-spectrin SH3 domain (a well characterized slow two-state folder) change in response to chemical denaturants and/or temperature. We can resolve the individual properties of the two end-states across the chemical unfolding transition employing single-molecule fluorescence spectroscopy (SM-FRET) and across the thermal unfolding transition by NMR because SH3 folds-unfolds in the slow chemical exchange regime. Our results demonstrate that α-spectrin SH3 unfolds in a canonical way ...

Abstract: Although many proteins are recognized to undergo folding via an intermediate, the microscopic nature of folding intermediates is less understood. In this study, ¹⁹F NMR and near-UV circular dichroism (CD) are used to characterize a transition to a thermal folding intermediate of calmodulin, a water-soluble protein, which is biosynthetically enriched with 3-fluorophenylalanine (3F-Phe). ¹⁹F NMR solvent isotope shifts, resulting from replacing H₂O with D₂O, and paramagnetic shifts arising from dissolved O₂ are used to monitor changes in the water accessibility and hydrophobicity of the protein interior as the protein progresses from a native state to an unfolded state along a heat-denaturation pathway. In comparison to the native state, the solvent isotope shifts reveal the decreased presence of water in the hydrophobic core, whereas the paramagnetic shifts show the increased hydrophobicity of this folding intermediate. ¹⁵N, ¹H and methyl ¹³C,¹H HSQC NMR spectra demonstrate that this folding intermediate retains a near-native tertiary structure whose hydrophobic interior is highly dynamic. ¹⁹F NMR CPMG relaxation dispersion measurements suggest the near-native state is transiently adopted well below the temperature associated with its onset.

Abstract: The fold stabilities and folding dynamics of a series of mutants of a model hairpin, KTW-NPATGK-WTE (HP7), are reported. The parent system and the corresponding DPATGK loop species display submicrosecond folding time constants. The mutational studies revealed that ultrafast folding requires both some prestructuring of the loop and a favorable interaction between the chain termini in the transition state. In the case of YY-DPETGT-WY, another submicrosecond folding species [Davis, C. M., Xiao, S., Raleigh, D. P., and Dyer, R. B. (2012) J. Am. Chem. Soc. 134, 14476–14482], a hydrophobic cluster provides the latter. In the case of HP7, the Coulombic interaction between the terminal NH3+ and CO2– units provides this; a C-terminal Glu to amidated Ala mutation results in a 5-fold retardation of the folding rate. The effects of mutations within the reversing loop indicate the balance between loop flexibility (favoring fast conformational searching) and turn formation in the unfolded state is a major factor in det...

Abstract: Three structurally similar domains from α-spectrin have been shown to fold very differently. Firstly, there is a contrast in the folding mechanism, as probed by Φ-value analysis, between the R15 domain and the R16 and R17 domains. Secondly, there are very different contributions from internal friction to folding: the folding rate of the R15 domain was found to be inversely proportional to solvent viscosity, showing no apparent frictional contribution from the protein, but in the other two domains a large internal friction component was evident. Non-native misdocking of helices has been suggested to be responsible for this phenomenon. Here, I study the folding of these three proteins with minimalist coarse-grained models based on a funneled energy landscape. Remarkably, I find that, despite the absence of non-native interactions, the differences in folding mechanism of the domains are well captured by the model, and the agreement of the Φ-values with experiment is fairly good. On the other hand, within the context of this model, there are no significant differences in diffusion coefficient along the chosen folding coordinate, and the model cannot explain the large differences in folding rates between the proteins found experimentally. These results are nonetheless consistent with the expectations from the energy landscape perspective of protein folding: namely, that the folding mechanism is primarily determined by the native-like interactions present in the Gō-like model, with missing non-native interactions being required to explain the differences in “internal friction” seen in experiment.

Abstract: The folding of bacterial tRNAs with disparate sequences has been observed to proceed in distinct folding mechanisms despite their structural similarity. To explore the folding landscapes of tRNA, we performed ion concentration-dependent coarse-grained TIS model MD simulations of several E. coli tRNAs to compare their thermodynamic melting profiles to the classical absorbance spectra of Crothers and co-workers. To independently validate our findings, we also performed atomistic empirical force field MD simulations of tRNAs, and we compared the base-to-base distances from coarse-grained and atomistic MD simulations to empirical base-stacking free energies. We then projected the free energies to the secondary structural elements of tRNA, and we observe distinct, parallel folding mechanisms whose differences can be inferred on the basis of their sequence-dependent base-stacking stabilities. In some cases, a premature, nonproductive folding intermediate corresponding to the Ψ hairpin loop must backtrack to the unfolded state before proceeding to the folded state. This observation suggests a possible explanation for the fast and slow phases observed in tRNA folding kinetics.

Abstract: Internal friction arising from local steric hindrance and/or the excluded volume effect plays an important role in controlling not only the dynamics of protein folding but also conformational transitions occurring within the native state potential well. However, experimental assessment of such local friction is difficult because it does not manifest itself as an independent experimental observable. Herein, we demonstrate, using the miniprotein trp-cage as a testbed, that it is possible to selectively increase the local mass density in a protein and hence the magnitude of local friction, thus making its effect directly measurable via folding kinetic studies. Specifically, we show that when a helix cross-linker, m-xylene, is placed near the most congested region of the trp-cage it leads to a significant decrease in both the folding rate (by a factor of 3.8) and unfolding rate (by a factor of 2.5 at 35 °C) but has little effect on protein stability. Thus, these results, in conjunction with those obtained wit...

Abstract: One strategy for reaching the downhill folding regime, primarily exploited for the λ6–85 protein fragment, consists of cumulatively introducing mutations that speed up folding. This is an experimentally demanding process where chemical intuition usually serves as a guide for the choice of amino acid residues to mutate. Such an approach can be aided by computational methods that screen for protein engineering hot spots. Here we present one such method that involves sampling the energy landscape of the pseudo-wild-type protein and investigating the effect of point mutations on this landscape. Using a novel metric for the cooperativity, we identify those residues leading to the least cooperative folding. The folding dynamics of the selected mutants are then directly characterized and the differences in the kinetics are analyzed within a Markov-state model framework. Although the method is general, here we present results for a coarse-grained topology-based simulation model of λ-repressor, whose barrier is re...

Abstract: After decades of research, the nature of the rate-limiting step in the folding of globular proteins still elicits a range of opinions (1). The properties of the associated transition state (TS) provide critical insights into possible folding mechanisms. However, the characterization of the TS is challenging because atomic level methods cannot be applied to this minimally populated state, and lower resolution methods have produced divergent views. Even the existence of a generalized TS remains actively debated. In PNAS, Guinn et al. (2) dissect the burial properties of the TS for 13 proteins by analyzing the denaturant and temperature dependence of folding rates to distinguish the burial of hydrophobic surface from that of amide groups. With this capability, they propose that the TSs generally are very advanced and often contain the native 2° structure, a level higher than most prior methods have indicated.

Abstract: As one of the most valuable methods for drug design, homology modeling shows that protein structures are more conserved than protein sequences, that is, the proteins with high sequence identity have high structural similarity, but protein pairs G(A)88/G(B)88 and G(A)95/G(B)95 prove the opposite The pairs G(A)88 and G(B)88 shares the 88% sequence identity, but display different structures, and the pair G(A)95 and G(B)95 with 95% sequence identity yet presents different structures The research on these proteins provides an opportunity of complementary study In the process of protein folding, at which stage the protein final structure was determined and which residues were important for folding to a given structure were still unknown Here we used OPLS all-atom force field for molecular dynamics simulations to study the unfolding of G(A)88, G(B)88, G(A)95 and G(B)95 at high temperatures, and used the process of protein unfolding to reverse the process of protein folding G(B)88 and G(B)95 folded to the α+β structure, but G(A)88 and G(A)95 folded to the all-α-helix structure In the process of G(A)88 and G(A)95 folding, the helices folded earlier than the formation of tertiary interactions In the process of folding to G(B)88 and G(B)95, the α-helix formed earlier We showed that early along the folding pathway, the final protein structure was confirmed, and very small differences between protein sequences determined the protein structure

Abstract: WW domain proteins are usually regarded as simple models for understanding the folding mechanism of β-sheet. CC45 is an artificial protein that is capable of folding into the same structure as WW domain. In this article, the replica exchange molecular dynamics simulations are performed to investigate the folding mechanism of CC45. The analysis of thermal stability shows that β-hairpin 1 is more stable than β-hairpin 2 during the unfolding process. Free energy analysis shows that the unfolding of this protein substantially proceeds through solvating the smaller β-hairpin 2, followed by the unfolding of β-hairpin 1. We further propose the unfolding process of CC45 and the folding mechanism of two β-hairpins. These results are similar to the previous folding studies of formin binding protein 28 (FBP28). Compared with FBP28, it is found that CC45 has more aromatic residues in N-terminal loop, and these residues contact with C-terminal loop to form the outer hydrophobic core, which increases the stability of C...

Abstract: In order to carry out their biological functions, most polypeptide chains must fold into stable three-dimensional structures, for it is the precise spatial distribution of chemical groups within a protein that gives the molecule its ability to interact specifically with other molecules and, in the case of an enzyme, catalyze a chemical reaction. In many cases, individual polypeptide chains must also assemble into larger structures containing additional proteins or nucleic acids. The folding of many proteins is reversible, so that the native structure can be disrupted by a change in temperature or addition of a chemical denaturant, and the unfolded protein can then be induced to refold and assemble by returning it to physiological conditions. Experiments of this type demonstrate that the information specifying the native structure of a protein resides in its amino acid sequence, and in vitro studies have provided important insights into the energetic factors that drive folding and assembly and the kinetic mechanisms of these processes. Folding in vivo is often facilitated by transient interactions with other proteins, molecular chaperones. Folding may also compete with the formation of aberrant aggregates in vivo , sometimes leading to pathological conditions such as amyloid diseases.

Abstract: An intrinsically disordered protein is one that does not spontaneously fold in physiological conditions but only folds when it binds to a target protein. Computer simulation of this coupled folding and binding is one of the central subjects of computational biophysics. Computing the free energy landscape is helpful in understanding coupled folding and binding. For this reason, we developed an Ising-like protein model and a multicanonical simulation in an energy-entropy space. The calculated free energy landscape indicates that coupled folding and binding induces rapid structural switching of the bound target protein.