Protein folding

The problem of protein folding is that how proteins acquire their native unique three‐dimensional structure in the physiological milieu. To solve the problem, the following key questions should be answered: do proteins fold co‐ or post‐translationally, i.e. during or after biosynthesis, what is the mechanism of protein folding, and what is the explanation for fast folding of proteins? The two first questions are discussed in the current review. The general lines are to show that the opinion, that proteins fold after they are synthesized is hardly substantiated and suitable for solving the problem of protein folding and why proteins should fold cotranslationally. A possible tentative model for the mechanism of protein folding is also suggested. To this end, a thorough analysis is made of the biosynthesis, delivery to the folding compartments, and the rates of the biosynthesis, translocation and folding of proteins. A cursory attention is assigned to the role of GroEL/ES‐like chaperonins in protein folding.     

Mechanism of induced folding: Both folding before binding and binding before folding can be realized in staphylococcal nuclease mutants

A considerable number of functional proteins are unstructured under physiological condition. These "intrinsically disordered" proteins exhibit induced folding when they bind their targets. The induced folding comprises two elementary processes: folding and binding. Two mechanisms are possible for the induced folding: either folding before binding or binding before folding. We found that these two mechanisms can be distinguished by the target-concentration dependence of folding kinetics. We also created two types of mutants of staphylococcal nuclease showing the different inhibitor-concentration dependence of induced folding kinetics. One mutant obeys the scheme of binding before folding, while the other the folding before binding. This is the first experimental evidence demonstrating that both mechanisms are realized for a single protein. Binding before folding is possible, when the protein lacks essential nonlocal interaction to stabilize the native conformation. The results cast light on the protein folding mechanism involved in the intrinsically disordered proteins.     

Rapid protein-folding assay using green fluorescent protein.

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.     

The dynamical contact order: Protein folding rate parameters based on quantum conformational transitions

Protein folding is regarded as a quantum transition between the torsion states of a polypeptide chain. According to the quantum theory of conformational dynamics, we propose the dynamical contact order (DCO) defined as a characteristic of the contact described by the moment of inertia and the torsion potential energy of the polypeptide chain between contact residues. Consequently, the protein folding rate can be quantitatively studied from the point of view of dynamics. By comparing theoretical calculations and experimental data on the folding rate of 80 proteins, we successfully validate the view that protein folding is a quantum conformational transition. We conclude that (i) a correlation between the protein folding rate and the contact inertial moment exists; (ii) multi-state protein folding can be regarded as a quantum conformational transition similar to that of two-state proteins but with an intermediate delay. We have estimated the order of magnitude of the time delay; (iii) folding can be classified into two types, exergonic and endergonic. Most of the two-state proteins with higher folding rate are exergonic and most of the multi-state proteins with low folding rate are endergonic. The folding speed limit is determined by exergonic folding.     

Comparison between long-range interactions and contact order in determining the folding rate of two-state proteins: application of long-range order to folding rate prediction.

The contact order is believed to be an important factor for understanding protein folding mechanisms. In our earlier work, we have shown that the long-range interactions play a vital role in protein folding. In this work, we analyzed the contribution of long-range contacts to determine the folding rate of two-state proteins. We found that the residues that are close in space and are separated by at least ten to 15 residues in sequence are important determinants of folding rates, suggesting the presence of a folding nucleus at an interval of approximately 25 residues. A novel parameter "long-range order" has been proposed to predict protein folding rates. This parameter shows as good a relationship with the folding rate of two-state proteins as contact order. Further, we examined the minimum limit of residue separation to determine the long-range contacts for different structural classes. We observed an excellent correlation between long-range order and folding rate for all classes of globular proteins. We suggest that in mixed-class proteins, a larger number of residues can serve as folding nuclei compared to all-alpha and all-beta proteins. A simple statistical method has been developed to predict the folding rates of two-state proteins using the long-range order that produces an agreement with experimental results that is better or comparable to other methods in the literature.     

Folding studies of immunoglobulin-like beta-sandwich proteins suggest that they share a common folding pathway.

BACKGROUND: Are folding pathways conserved in protein families? To test this explicitly and ask to what extent structure specifies folding pathways requires comparison of proteins with a common fold. Our strategy is to choose members of a highly diverse protein family with no conservation of function and little or no sequence identity, but with structures that are essentially the same. The immunoglobulin-like fold is one of the most common structural families, and is subdivided into superfamilies with no detectable evolutionary or functional relationship. RESULTS: We compared the folding of a number of immunoglobulin-like proteins that have a common structural core and found a strong correlation between folding rate and stability. The results suggest that the folding pathways of these immunoglobulin-like proteins share common features. CONCLUSIONS: This study is the first to compare the folding of structurally related proteins that are members of different superfamilies. The most likely explanation for the results is that interactions that are important in defining the structure of immunoglobulin-like proteins are also used to guide folding.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Oxidative protein folding in the plant endoplasmic reticulum

For most of the proteins synthesized in the endoplasmic reticulum (ER), disulfide bond formation accompanies protein folding in a process called oxidative folding. Oxidative folding is catalyzed by a number of enzymes, including the family of protein disulfide isomerases (PDIs), as well as other proteins that supply oxidizing equivalents to PDI family proteins, like ER oxidoreductin 1 (Ero1). Oxidative protein folding in the ER is a basic vital function, and understanding its molecular mechanism is critical for the application of plants as protein production tools. Here, I review the recent research and progress related to the enzymes involved in oxidative folding in the plant ER. Firstly, nine groups of plant PDI family proteins are introduced. Next, the enzymatic properties of plant Ero1 are described. Finally, the cooperative folding by multiple PDI family proteins and Ero1 is described.     

A rapid protein folding assay for the bacterial periplasm

An array of genetic screens and selections has been developed for reporting protein folding and solubility in the cytoplasm of living cells. However, there are currently no analogous folding assays for the bacterial periplasm, despite the significance of this compartment for the expression of recombinant proteins, especially those requiring important posttranslational modifications (e.g., disulfide bond formation). Here, we describe an engineered genetic selection for monitoring protein folding in the periplasmic compartment of Escherichia coli cells. In this approach, target proteins are sandwiched between an N‐terminal signal recognition particle (SRP)‐dependent signal peptide and a C‐terminal selectable marker, TEM‐1 β‐lactamase. The resulting chimeras are localized to the periplasmic space via the cotranslational SRP pathway. Using a panel of native and heterologous proteins, we demonstrate that the folding efficiency of various target proteins correlates directly with in vivo β‐lactamase activity and thus resistance to ampicillin. We also show that this reporter is useful for the discovery of extrinsic periplasmic factors (e.g., chaperones) that affect protein folding and for obtaining folding‐enhanced proteins via directed evolution. Collectively, these data demonstrate that our periplasmic folding reporter is a powerful tool for screening and engineering protein folding in a manner that does not require any structural or functional information about the target protein.     

Folding superfunnel to describe cooperative folding of interacting proteins

This paper proposes a generalization of the well‐known folding funnel concept of proteins. In the funnel model the polypeptide chain is treated as an individual object not interacting with other proteins. Since biological systems are considerably crowded, protein–protein interaction is a fundamental feature during the life cycle of proteins. The folding superfunnel proposed here describes the folding process of interacting proteins in various situations. The first example discussed is the folding of the freshly synthesized protein with the aid of chaperones. Another important aspect of protein–protein interactions is the folding of the recently characterized intrinsically disordered proteins, where binding to target proteins plays a crucial role in the completion of the folding process. The third scenario where the folding superfunnel is used is the formation of aggregates from destabilized proteins, which is an important factor in case of several conformational diseases. The folding superfunnel constructed here with the minimal assumption about the interaction potential explains all three cases mentioned above. Proteins 2016; 84:1009–1016. © 2016 Wiley Periodicals, Inc.     

Entropy capacity determines protein folding

Search and study of the general principles that govern kinetics and thermodynamics of protein folding generate a new insight into the factors controlling this process. Here, based on the known experimental data and using theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue-that is, an entropy capacity-for fast protein folding. Statistical analysis of conformational entropy and number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, alpha/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts (class alpha/beta has the largest number of contacts) and average conformational entropy per residue (class all-alpha has the largest number of rotatable angles phi, psi, and chi per residue). These class-specific features determine the folding rates: alpha proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones. Our result is in agreement with the experimental folding rates for 60 proteins. This suggests that structural and sequence properties are important determinants of protein folding rates.     

Single amino acid substitutions globally suppress the folding defects of temperature-sensitive folding mutants of phage P22 coat protein.

The amino acid sequence of a polypeptide defines both the folding pathway and the final three-dimensional structure of a protein. Eighteen amino acid substitutions have been identified in bacteriophage P22 coat protein that are defective in folding and cause their folding intermediates to be substrates for GroEL and GroES. These temperature-sensitive folding (tsf) substitutions identify amino acids that are critical for directing the folding of coat protein. Additional amino acid residues that are critical to the folding process of P22 coat protein were identified by isolating second site suppressors of the tsf coat proteins. Suppressor substitutions isolated from the phage carrying the tsf coat protein substitutions included global suppressors, which are substitutions capable of alleviating the folding defects of numerous tsf coat protein mutants. In addition, potential global and site-specific suppressors were isolated, as well as a group of same site amino acid substitutions that had a less severe phenotype than the tsf parent. The global suppressors were located at positions 163, 166, and 170 in the coat protein sequence and were 8-190 amino acid residues away from the tsf parent. Although the folding of coat proteins with tsf amino acid substitutions was improved by the global suppressor substitutions, GroEL remained necessary for folding. Therefore, we believe that the global suppressor sites identify a region that is critical to the folding of coat protein.     

Designing a High Throughput Refolding Array Using a Combination of the GroEL Chaperonin and Osmolytes

Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL–GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin–substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a ‘folding array’ to identify superior folding conditions. The stable chaperonin–substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (~mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.     

Protein Folding in the Cell: On the Mechanisms of Its Acceleration

The mechanisms responsible for protein folding in the cell can be divided in two groups. The ones in the first group would be those preventing the aggregation of unfolded polypeptide chains or of incompletely folded proteins, as well as the mechanisms which provide for the energy-consuming unfolding of incorrectly folded structures, giving them a chance to begin a new folding cycle. Mechanisms of this type do not affect the rate of folding (it occurs spontaneously), yet considerably increase the efficiency of the entire process. By contrast, the mechanisms belonging to second group actually accelerate protein folding by exerting a direct influence on the rate-limiting steps of the overall reaction. Although not a conventional one, such a classification helps define the topic of this review. Its main purpose is to discuss the ability of chaperonins (and that of some chaperones) to interact directly with substrate proteins in the course of their folding and thus accelerate the rate-limiting steps of that process. (Mechanisms of protein folding acceleration produced by the action of enzymes, e.g., peptidyl-prolyl cis/trans isomerase and protein disulfide isomerase, are not considered in this review.) Specific cases demonstrating an accelerated folding of some proteins encapsulated in the bacterial chaperonin GroEL cavity are considered, and the conditions favoring such acceleration are examined. Experimental data supporting the notion that the structure and functional properties of GroEL are not optimal for an effective folding of many of its substrate proteins is discussed. The current status of research on the mechanism behind the active participation of different subunits of eucaryotic cytosol chaperonin (CCT) in the final steps of the folding of actin and tubulin is reviewed. Particular attention is devoted to steric chaperones, which dramatically accelerate the formation of the native structure of their substrate proteins by stabilizing certain folding intermediates. The structural foundations underlying the effect of the subtilisin pro-domain on the folding of the mature enzyme are considered. The prospects of future studies into the mechanisms responsible for accelerating protein folding in the cell are commented upon.     

Optimal region of average side-chain entropy for fast protein folding

Search and study the general principles that govern kinetics and thermodynamics of protein folding generates new insight into the factors that control this process. Here, we demonstrate based on the known experimental data and using theoretical modeling of protein folding that side-chain entropy is one of the general determinants of protein folding. We show for proteins belonging to the same structural family that there exists an optimal relationship between the average side-chain entropy and the average number of contacts per residue for fast folding kinetics. Analysis of side-chain entropy for proteins that fold without additional agents demonstrates that there exists an optimal region of average side-chain entropy for fast folding. Deviation of the average side-chain entropy from the optimal region results in an anomalous protein folding process (prions, alpha-lytic protease, subtilisin, some DNA-binding proteins). Proteins with high or low side-chain entropy would have extended unfolded regions and would require some additional agents for complete folding. Such proteins are common in nature, and their structure properties have biological importance.     

GroEL binds a late folding intermediate of phage P22 coat protein

GroEL recognizes proteins that are folding improperly or that have aggregation-prone intermediates. Here we have used as substrates for GroEL, wildtype (WT) coat protein of phage P22 and 3 coat proteins that carry single amino acid substitutions leading to a temperature-sensitive folding (tsf) phenotype. In vivo, WT coat protein does not require GroEL for proper folding, whereas GroEL is necessary for the folding of the tsf coat proteins; thus, the single amino acid substitutions cause coat protein to become a substrate for GroEL. The conformation of WT and tsf coat proteins when in a binary complex with GroEL was investigated using tryptophan fluorescence, quenching of fluorescence, and accessibility of the coat proteins to proteolysis. WT coat protein and the tsf coat protein mutants were each found to be in a different conformation when bound to GroEL. As an additional measure of the changes in the bound conformation, the affinity of binding of WT and tsf coat proteins to GroEL was determined using a fluorescence binding assay. The tsf coat proteins were bound more tightly by GroEL than WT coat protein. Therefore, even though the proteins are identical except for a single amino acid substitution, GroEL did not bind these substrate polypeptides in the same conformation within its central cavity. Therefore, GroEL is likely to bind coat protein in a conformation consistent with a late folding intermediate, with substantial secondary and tertiary structure formed.     

Exploring folding free energy landscapes using computational protein design

Recent advances in computational protein design have allowed exciting new insights into the sequence dependence of protein folding free energy landscapes. Whereas most previous studies have examined the sequence dependence of protein stability and folding kinetics by characterizing naturally occurring proteins and variants of these proteins that contain a small number of mutations, it is now possible to generate and characterize computationally designed proteins that differ significantly from naturally occurring proteins in sequence and/or structure. These computer-generated proteins provide insights into the determinants of protein structure, stability and folding, and make it possible to disentangle the properties of proteins that are the consequence of natural selection from those that reflect the fundamental physical chemistry of polypeptide chains.     

Knotted fusion proteins reveal unexpected possibilities in protein folding

Proteins that contain a distinct knot in their native structure are impressive examples of biological self-organization. Although this topological complexity does not appear to cause a folding problem, the mechanisms by which such knotted proteins form are unknown. We found that the fusion of an additional protein domain to either the amino terminus, the carboxy terminus, or to both termini of two small knotted proteins did not affect their ability to knot. The multidomain constructs remained able to fold to structures previously thought unfeasible, some representing the deepest protein knots known. By examining the folding kinetics of these fusion proteins, we found evidence to suggest that knotting is not rate limiting during folding, but instead occurs in a denatured-like state. These studies offer experimental insights into when knot formation occurs in natural proteins and demonstrate that early folding events can lead to diverse and sometimes unexpected protein topologies.     

Cotranslational Folding of a Pentarepeat β-Helix Protein

It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum—a homolog of the fluoroquinolone resistance protein MfpA—using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is ~ 35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.