A sequence function reveals new features in beta-protein folding

When amino acid residues are represented by parameters describing their side chain lengths and polarities, a sequence function defined as the sum of the first two sequence autocorrelation functions is found to be negatively and linearly correlated with the logarithms of folding rates of beta-proteins. The new function reveals new features in beta-protein folding: larger residues slow down the folding while alternative distribution of polar-non-polar residues accelerates the folding.     

Competition between folding and glycosylation in the endoplasmic reticulum.

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new acceptor sites was analysed by pulse-labelling under two sets of conditions that are known to reduce the rate of folding: (i) addition of dithiothreitol to the growth medium and (ii) introduction of deletions in the propeptide. A variety of effects was observed, depending on the position of the new acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation reactions can compete in vivo and that glycosylation does not necessarily precede folding. The approach described may be generally applicable for the analysis of protein folding in vivo.     

Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells

Subcellular compartments in which folding and assembly of proteins occur seem to have a set of PCB proteins capable of mediating these and related processes, such as translocation across membranes. When a domain of a polypeptide chain emerges from a ribosome during synthesis or from the distal side of a membrane during translocation, successive segments of the chain are incrementally exposed to solvent and yet are unlikely to be able to fold. This topological restriction on folding likely mandates a need for PCB proteins to prevent aggregation. Catalysis of topologically restricted folding by PCB proteins is likely to involve both an antifolding activity that postpones folding until entire domains are available and, more speculatively, a folding activity resulting from a programmed stepwise release that employs the energy of ATP hydrolysis to ensure a favorable pathway. We are left with a new set of problems. How do proteins fold in cells? What are the sequences or structural signals that dictate folding pathways? The new challenge will be to understand folding as a combination of physical chemistry, enzymology, and cell biology.     

Folding of tandem-linked domains

One of the factors, which influences protein folding in vivo, is a linkage of protein domains into multidomain tandems. However, relatively little is known about the impact of domain connectivity on protein folding mechanisms. In this article, we use coarse grained models of proteins to explore folding of tandem-linked domains (TLD). We found TLD folding to follow two scenarios. In the first, the tandem connectivity produces relatively minor impact on folding and the mechanisms of folding of tandem-linked and single domains remain similar. The second scenario involves qualitative changes in folding mechanism because of tandem linkage. As a result, protein domains, which fold via two-state mechanism as single isolated domains, may form new stable intermediates when inserted into tandems. The new intermediates are created by topological constraints imposed by the linkers between domains. In both cases tandem linkage slows down folding. We propose that the impact of tandem connectivity can be minimized, if the terminal secondary structure elements (SSEs) are flexible. In particular, two factors appear to facilitate TLD folding: (1) the interactions between terminal SSE are poorly ordered in the folding transition state, whereas nonterminal SSE are better structured, (2) the interactions between terminal SSE are weak in the native state. We apply these findings to wild-type proteins by examining experimental phi-value data and by performing all-atom molecular dynamics simulations. We show that immunoglobulin-like domains appear to utilize the factors, which minimize the impact of tandem connectivity on their folding. Several single domain proteins, which are likely to misfold in tandems, are also identified.     

Pathways in two-state protein folding

Thermodynamic measurements of proteins indicate that the folding to the native state takes place either through stable intermediates or through a two-state process without intermediates. The rather short folding times of proteins indicate that folding is guided through some sequence of contact bindings. We discuss the possibility of reconciling a two-state folding event with a sequential folding process in a schematic model of protein folding. We propose a new dynamical transition temperature that is lower than the temperature at which proteins in equilibrium unfold. This is in qualitative agreement with observations of in vivo protein folding activity quantified by chaperone concentration in Escherichia coli. Finally, we discuss our framework in connection with the unfolding of proteins at low temperatures.     

Identification and characterization of folding inhibitors of hen egg lysozyme: an example of a new paradigm of drug design

Studies of protein folding indicate the presence of native contacts in the denatured state, giving rise to folding elements which contribute to the accomplishment of the native state. The possibility of finding molecules which can interact with specific folding elements of a target protein preventing it from reaching its native state, and hence from becoming biologically active, is particularly attractive. The notion that folding elements not only provide molecular recognition directing the folding process, but also have conserved sequence, implies that targeting such elements will make protein folding inhibitors less susceptible to mutations which, in many cases, abrogate drug effects. The folding-inhibition strategy can lead to a truly novel and rational approach to drug design, aside from providing new insight into folding. This is illustrated in the case of hen egg lysozyme.     

The influence of protein coding sequences on protein folding rates of all-β proteins

It is currently believed that the protein folding rate is related to the protein structures and its amino acid sequence. However, few studies have been done on the problem that whether the protein folding rate is influenced by its corresponding mRNA sequence. In this paper, we analyzed the possible relationship between the protein folding rates and the corresponding mRNA sequences. The content of guanine and cytosine (GC content) of palindromes in protein coding sequence was introduced as a new parameter and added in the Gromiha's model of predicting protein folding rates to inspect its effect in protein folding process. The multiple linear regression analysis and jack-knife test show that the new parameter is significant. The linear correlation coefficient between the experimental and the predicted values of the protein folding rates increased significantly from 0.96 to 0.99, and the population variance decreased from 0.50 to 0.24 compared with Gromiha's results. The results show that the GC content of palindromes in the corresponding protein coding sequence really influences the protein folding rate. Further analysis indicates that this kind of effect mostly comes from the synonymous codon usage and from the information of palindrome structure itself, but not from the translation information from codons to amino acids.     

Protein folding at single-molecule resolution

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Characterisation of the transition states for protein folding: towards a new level of mechanistic detail in protein engineering analysis

The field of protein folding now offers considerable excitement. Comparative studies of the transition-state structures for a series of protein families with analogous structures have helped to uncover the overall rules for protein folding. In addition, new protein engineering experiments that continuously follow the growth of the folding nucleus have started to fill in the missing details.     

Native disulfide bond formation in proteins

Native disulfide bond formation is critical for the proper folding of many proteins. Recent studies using newly identified protein oxidants, folding catalysts, and mutant cells provide insight into the mechanism of oxidative protein folding in vivo. This insight promises new strategies for more efficient protein production.     

基于序列疏水值震荡的折叠速率预测

蛋白质折叠速率的正确预测对理解蛋白质的折叠机理非常重要。本文从伪氨基酸组成的方法出发,提出利用序列疏水值震荡的方法来提取蛋白质氨基酸的序列顺序信息,建立线性回归模型进行折叠速率预测。该方法不需要蛋白质的任何二级结构、三级结构信息或结构类信息,可直接从序列对蛋白质折叠速率进行预测。对含有62个蛋白质的数据集,经过Jack．knife交互检验验证,相关系数达到0．804,表示折叠速率预测值与实验值有很好的相关性,说明了氨基酸序列信息对蛋白质折叠速率影响重要。同其他方法相比,本文的方法具有计算简单,输入参数少等特点。     

Testing the relationship between foldability and the early folding events of dihydrofolate reductase from Escherichia coli

A "folding element" is a contiguous peptide segment crucial for a protein to be foldable and is a new concept that could assist in our understanding of the protein-folding problem. It is known that the presence of the complete set of folding elements of dihydrofolate reductase (DHFR) from Escherichia coli is essential for the protein to be foldable. Since almost all of the amino acid residues known to be involved in the early folding events of DHFR are located within the folding elements, a close relationship between the folding elements and early folding events is hypothesized. In order to test this hypothesis, we have investigated whether or not the early folding events are preserved in circular permutants and topological mutants of DHFR, in which the order of the folding elements is changed but the complete set of folding elements is present. The stopped-flow circular dichroism (CD) measurements show that the CD spectra at the early stages of folding are similar among the mutants and the wild-type DHFR, indicating that the presence of the complete set of folding elements is sufficient to preserve the early folding events. We have further examined whether or not sequence perturbation on the folding elements by a single amino acid substitution affects the early folding events of DHFR. The results show that the amino acid substitutions inside of the folding elements can affect the burst-phase CD spectra, whereas the substitutions outside do not. Taken together, these results indicate that the above hypothesis is true, suggesting a close relationship between the foldability of a protein and the early folding events. We propose that the folding elements interact with each other and coalesce to form a productive intermediate(s) early in the folding, and these early folding events are important for a protein to be foldable.     

Redesigning the hydrophobic core of a model beta-sheet protein: destabilizing traps through a threading approach

An off-lattice 46-bead model of a small all-beta protein has been recently criticized for possessing too many traps and long-lived intermediates compared with the folding energy landscape predicted for real proteins and models using the principle of minimal frustration. Using a novel sequence design approach based on threading for finding beneficial mutations for destabilizing traps, we proposed three new sequences for folding in the beta-sheet model. Simulated annealing on these sequences found the global minimum more reliably, indicative of a smoother energy landscape, and simulated thermodynamic variables found evidence for a more cooperative collapse transition, lowering of the collapse temperature, and higher folding temperatures. Folding and unfolding kinetics were acquired by calculating first-passage times, and the new sequences were found to fold significantly faster than the original sequence, with a concomitant lowering of the glass temperature, although none of the sequences have highly stable native structures. The new sequences found here are more representative of real proteins and are good folders in the T(f) > T(g) sense, and they should prove useful in future studies of the details of transition states and the nature of folding intermediates in the context of simplified folding models. These results show that our sequence design approach using threading can improve models possessing glasslike folding dynamics.     

Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit.

There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin beta subunit (hCG-beta), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG-beta fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the beta subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects.     

A comprehensive database of verified experimental data on protein folding kinetics

Insights into protein folding rely increasingly on the synergy between experimental and theoretical approaches. Developing successful computational models requires access to experimental data of sufficient quantity and high quality. We compiled folding rate constants for what initially appeared to be 184 proteins from 15 published collections/web databases. To generate the highest confidence in the dataset, we verified the reported lnk_f value and exact experimental construct and conditions from the original experimental report(s). The resulting comprehensive database of 126 verified entries, ACPro, will serve as a freely accessible resource ( https://www.ats.amherst.edu/protein/ ) for the protein folding community to enable confident testing of predictive models. In addition, we provide a streamlined submission form for researchers to add new folding kinetics results, requiring specification of all the relevant experimental information according to the standards proposed in 2005 by the protein folding consortium organized by Plaxco. As the number and diversity of proteins whose folding kinetics are studied expands, our curated database will enable efficient and confident incorporation of new experimental results into a standardized collection. This database will support a more robust symbiosis between experiment and theory, leading ultimately to more rapid and accurate insights into protein folding, stability, and dynamics.     

Growing up in a dangerous environment: a network of multiple targeting and folding pathways for nascent polypeptides in the cytosol

The first events in the lives of proteins are the most hazardous. Starting at the ribosome, nascent polypeptides undergo complex folding processes endangered by aggregation reactions. Proteins with organellar destinations require correct targeting to the translocation machineries and prevention from premature folding. The high precision and speed of these processes is ensured by a cystosolic system consisting of molecular chaperones, folding catalysts and targeting factors. This review focuses on the interactions of this system with nascent polypeptides and discusses new concepts for protein folding in the cytosol. It is proposed that folding and targeting are promoted by a flexible network of multiple unassisted and assisted pathways.     

Folding mechanism of all-beta globular proteins

Elucidating the mechanism for the fast folding of proteins is a challenging task. In our earlier work, we introduced the concept of "long-range order" and related it successfully to protein folding rates. In this article, we propose a new hypothesis for the folding of two-state all-beta proteins. The mechanism is based on the formation of a hydrophobic core, propagation of beta-strands, and the establishment of hydrogen bonds. Our hypothesis has been strengthened by the observation of a folding nucleus in beta-strands and the hydrogen-bonding network between residues in beta-strands. Our insights on protein folding show an excellent agreement with experimental observations.     

Taming the complexity of protein folding

Protein folding is an important problem in structural biology with significant medical implications, particularly for misfolding disorders like Alzheimer's disease. Solving the folding problem will ultimately require a combination of theory and experiment, with theoretical models providing a comprehensive view of folding and experiments grounding these models in reality. Here we review progress towards this goal over the past decade, with an emphasis on recent theoretical advances that are empowering chemically detailed models of folding and the new results these technologies are providing. In particular, we discuss new insights made possible by Markov state models (MSMs), including the role of non-native contacts and the hub-like character of protein folded states.     

A New Computational Approach for Mechanical Folding Kinetics of RNA Hairpins

Based on an ensemble of kinetically accessible conformations, we propose a new analytical model for RNA folding kinetics. The model gives populational kinetics, kinetic rates, transition states, and pathways from the rate matrix. Applications of the new kinetic model to mechanical folding of RNA hairpins such as trans-activation-responsive RNA reveal distinct kinetic behaviors in different force regimes, from zero force to forces much stronger than the critical force for the folding-unfolding transition. In the absence of force or a low force, folding can be initiated (nucleated) at any position by forming the first base stack and there exist many pathways for the folding process. In contrast, for a higher force, the folding/unfolding would predominantly proceed along a single zipping/unzipping pathway. Studies for different hairpin-forming sequences indicate that depending on the nucleotide sequence, a kinetic intermediate can emerge in the low force regime but disappear in high force regime, and a new kinetic intermediate, which is absent in the low and high force regimes, can emerge in the medium force range. Variations of the force lead to changes in folding cooperativity and rate-limiting steps. The predicted network of pathways for trans-activation-responsive RNA suggests two parallel dominant pathways. The rate-limiting folding steps (at f = 8 pN) are the formation of specific basepairs that are 2-4 basepairs away from the loop. At a higher force (f = 11 pN), the folding rate is controlled by the formation of the bulge loop. The predicted rates and transition states are in good agreement with the experimental data for a broad force regime.