Gatekeepers in the ribosomal protein s6: thermodynamics, kinetics, and folding pathways revealed by a minimalist protein model

We investigate the effect of structural gatekeepers on the folding of the ribosomal protein S6. Folding thermodynamics and early refolding kinetics are studied for this system utilizing computer simulations of a minimalist protein model. When gatekeepers are eliminated, the thermodynamic signature of a folding intermediate emerges, and a marked decrease in folding efficiency is observed. We explain the prerequisites that determine the "strength" of a given gatekeeper. The investigated gatekeepers are found to have distinct functions, and to guide the folding and time-dependent packing of non-overlapping secondary structure elements in the protein. Gatekeepers avoid kinetic traps during folding by favoring the formation of "productive topologies" on the way to the native state. The trends in folding rates in the presence/absence of gatekeepers observed for our minimalist model of S6 are in very good agreement with experimental data on this protein.     

Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify “non-silent” synonymous mutations in other genes.     

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).     

Secondary structure prediction and folding of globular protein: refolding of ferredoxin

The physicochemical mechanism of protein folding has been elucidated by the island model, describing a growth type of folding. The folding pathway is closely related with nucleation on the polypeptide chain and thus the formation of small local structures or secondary structures at the earliest stage of folding is essential to all following steps. The island model is applicable to any protein, but a high precision of secondary structure prediction is indispensable to folding simulation. The secondary structures formed at the earliest stage of folding are supposed to be of standard form, but they are usually deformed during the folding process, especially at the last stage, although the degree of deformation is different for each protein. Ferredoxin is an example of a protein having this property. According to X-ray investigation (1FDX), ferredoxin is not supposed to have secondary structures. However, if we assumed that in ferredoxin all the residues are in a coil state, we could not attain the correct structure similar to the native one. Further, we found that some parts of the chain are not flexible, suggesting the presence of secondary structures, in agreement with the recent PDB data (1DUR). Assuming standard secondary structures (-helices and -strands) at the nonflexible parts at the early stage of folding, and deforming these at the final stage, a structure similar to the native one was obtained. Another peculiarity of ferredoxin is the absence of disulfide bonds, in spite of its having eight cysteines. The reason cysteines do not form disulfide bonds became clear by applying the lampshade criterion, but more importantly, the two groups of cysteines are ready to make iron complexes, respectively, at a rather later stage of folding. The reason for poor prediction accuracy of secondary structure with conventional methods is discussed.     

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.     

Loop mutations can cause a substantial conformational change in the carboxy terminus of the ferritin protein.

Although some protein folding theories sustain that the peptides (loops) that connect elements of more compact secondary structure may be important in the folding process, most of the data accumulated until now seems to contradict this notion. To approach this problem we have isolated and characterized a number of mutants in which the amino acid sequence of the peptide that connects helix D and helix E in the H-chain of human ferritin has been randomized. Our results indicate that, though no single loop residue is absolutely required for ferritin to attain the native conformation, most of the mutants that we have obtained by random regional mutagenesis, affect its folding/assembly process. This conclusion was reached utilizing a sensitive test that associates the color formed by a colony synthesizing a hybrid ferritin-beta-galactosidase protein to the ability of the ferritin domain to fold and assemble as the native protein. The characterization of the folding/assembly properties of our collection of mutants and the comparison of the mutant loop sequences, have allowed us to draw the following conclusions. Mutants that have positively charged residues at position 159, 160 or 161 fail to assemble into the native protein shell and form an insoluble aggregate. Interestingly some loop amino acid sequences cause the E-helix to reverse direction and to expose its COOH group, normally hidden inside the protein cavity, to the solvent. The propensity of a given ferritin mutant to fold into this "non-native" conformation can be attenuated by the introduction of Gly at position 159 and 164, as in the natural ferritin.     

NMR identification of local structural preferences in HIV-1 protease tethered heterodimer in 6 M guanidine hydrochloride.

Understanding protein folding requires complete characterization of all the states of the protein present along the folding pathways. For this purpose nuclear magnetic resonance (NMR) has proved to be a very powerful technique because of the great detail it can unravel regarding the structure and dynamics of protein molecules. We report here NMR identification of local structural preferences in human immunodeficiency virus-1 protease in the 'unfolded state'. Analyses of the chemical shifts revealed the presence of local structural preferences many of which are native-like, and there are also some non-native structural elements. Three-bond H(N)-H(alpha) coupling constants that could be measured for some of the N-terminal and C-terminal residues are consistent with the native-like beta-structure. Unusually shifted 15N and amide proton chemical shifts of residues adjacent to some prolines and tryptophans also indicate the presence of some structural elements. These conclusions are supported by amide proton temperature coefficients and nuclear Overhauser enhancement data. The locations of the residues exhibiting preferred structural propensities on the crystal structure of the protein, give useful insights into the folding mechanism of this protein.     

The imprint of codons on protein structure

The "central dogma" of biology outlines the unidirectional flow of interpretable data from genetic sequence to protein sequence. This has led to the idea that a protein's structure is dependent only on its amino acid sequence and not its genetic sequence. Recently, however, a more than transient link between the coding genetic sequence and the protein structure has become apparent. The two interact at the ribosome via the process of co-translational protein folding. Evidence for co-translational folding is growing rapidly, but the influence of codons on the protein structure attained is still highly contentious. It is theorised that the speed of codon translation modulates the time available for protein folding and hence the protein structure. Here, past and present research regarding synonymous codons and codon translation speed are reviewed within the context of protein structure attainment.     

Multi-State Proteins: Approach Allowing Experimental Determination of the Formation Order of Structure Elements in the Green Fluorescent Protein

The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named μ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are “tested” by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an μ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.     

The importance of explicit chain representation in protein folding models: an examination of Ising-like models

A class of models that represents a protein chain as a sequence of "folded" and "unfolded" residues has recently been used to correlate rates and mechanisms of protein folding with the protein native structure. In order to better understand the conditions under which these "Ising-like" models apply, we compare results from this model to those obtained from an off-lattice model which uses the same potential function. We find that Ising-like models by construction impose folding via a highly sequential nucleation-condensation mechanism, which in turn leads to more rugged energy landscapes, fewer "pathways" to the native state, and in the specific case examined here, the cold shock protein A from Escherichia coli, a qualitative difference in the most likely order of events in folding.     

Autonomous subdomains in protein folding.

Proteolytic dissection of native trp repressor and horse heart cytochrome c has been used to infer some of the steps in the folding pathways of the intact proteins. For both proteins, small fragments are capable of undergoing spontaneous noncovalent association to form subdomains with native-like secondary and/or tertiary structural features, suggesting that dissection/reassembly may be a general method to gain insight into the structures of folding intermediates. The importance of this approach is its simplicity and potential applicability to studying the folding pathways of a wide range of proteins. The proteases report on the structure and dynamics of the native state, circumventing the need for prior knowledge of the structures of folding intermediates. The observation that small fragments of proteins can associated noncovalently suggests that protein folding can be viewed as an intramolecular "recognition" process. The results imply that substantial information about protein structure and folding is encoded at the level of subdomains, and that chain connectivity has only a minor role in determining the fold.     

An evolutionary strategy for all-atom folding of the 60-amino-acid bacterial ribosomal protein l20

We have investigated an evolutionary algorithm for de novo all-atom folding of the bacterial ribosomal protein L20. We report results of two simulations that converge to near-native conformations of this 60-amino-acid, four-helix protein. We observe a steady increase of "native content" in both simulated ensembles and a large number of near-native conformations in their final populations. We argue that these structures represent a significant fraction of the low-energy metastable conformations, which characterize the folding funnel of this protein. These data validate our all-atom free-energy force field PFF01 for tertiary structure prediction of a previously inaccessible structural family of proteins. We also compare folding simulations of the evolutionary algorithm with the basin-hopping technique for the Trp-cage protein. We find that the evolutionary algorithm generates a dynamic memory in the simulated population, which leads to faster overall convergence.     

Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase

The unfolded state of a protein is an ensemble of a large number of conformations ranging from fully extended to compact structures. To investigate the effects of the difference in the unfolded-state ensemble on protein folding, we have studied the structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond, and compared the results with those of disulfide-reduced "linear" DHFR. Equilibrium studies by circular dichroism, difference absorption spectra, solution X-ray scattering, and size-exclusion chromatography show that whereas the native structures of both proteins are essentially the same, the unfolded state of circular DHFR adopts more compact conformations than the unfolded state of the linear form, even with the absence of secondary structure. Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization. Kinetic refolding measurements by stopped-flow circular dichroism and fluorescence show that under the native conditions both proteins accumulate a burst-phase intermediate having the same structures and both fold by the same complex folding mechanism with the same folding rates. Thus, the effects of the difference in the unfolded state of circular and linear DHFRs on the refolding reaction are not observed after the formation of the intermediate. This suggests that for the proteins with close termini in the native structure, early compaction of a protein molecule to form a specific folding intermediate with the N and C-terminal regions in close proximity is a crucial event in folding. If there is an enhancement in the folding reflecting the reduction in the breadth of the unfolded-state ensemble for circular DHFR, this acceleration must occur in the sub-millisecond time-range.     

The ins and outs of GroEL-mediated protein folding.

Two papers recently published in Cell investigate the role of protein encapsulation by GroEL in assisting folding. The first shows how encapsulation can "smooth" the folding landscape, accelerating folding of some proteins. The second defines a confinement-independent pathway, which allows GroEL to assist folding of substrates too large to be encapsulated.     

The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state

Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.     

Intrinsically disordered protein from a pathogenic mesophile Mycobacterium tuberculosis adopts structured conformation at high temperature

Compared to eukaryotes, the occurrence of "intrinsically disordered" or "natively unfolded" proteins in prokaryotes has not been explored extensively. Here, we report the occurrence of an intrinsically disordered protein from the mesophilic human pathogen Mycobacterium tuberculosis. The Histidine-tagged recombinant Rv3221c biotin-binding protein is intrinsically disordered at ambient and physiological growth temperatures as revealed by circular dichroism and Fourier transform infrared (FTIR) spectroscopic studies. However, an increase in temperature induces a transition from disordered to structured state with a folding temperature of approximately 53 degrees C. Addition of a structure inducing solvent trifluoroethanol (TFE) causes the protein to fold at lower temperatures suggesting that TFE fosters hydrophobic interactions, which drives protein folding. Differential Scanning Calorimetry studies revealed that folding is endothermic and the transition from a disordered to structured state is continuous (higher-order), implying existence of intermediates during folding process. Secondary structure analysis revealed that the protein has propensity to form beta-sheets. This is in conformity with FTIR spectrum that showed an absorption peak at wave number of 1636 cm(-1), indicative of disordered beta-sheet conformation in the native state. These data suggest that although Rv3221c may be disordered under ambient or optimal growth temperature conditions, it has the potential to fold into ordered structure at high temperature driven by increased hydrophobic interactions. In contrast to the generally known behavior of other intrinsically disordered proteins folding at high temperature, Rv3221c does not appear to oligomerize or aggregate as revealed through numerous experiments including Congo red binding, Thioflavin T-binding, turbidity measurements, and examining molar ellipticity as a function of protein concentration. The amino acid composition of Rv3221c reveals that it has 24% charged and 54.9% hydrophobic amino acid residues. In this respect, this protein, although belonging to the class of intrinsically disordered proteins, has distinct features. The intrinsically disordered state and the biotin-binding feature of this protein suggest that it may participate in many biochemical processes requiring biotin as a cofactor and adopt suitable conformations upon binding other folded targets.     

The folding thermodynamics and kinetics of crambin using an all-atom Monte Carlo simulation

We present a novel Monte Carlo simulation of protein folding, in which all heavy atoms are represented as interacting hard spheres. This model includes all degrees of freedom relevant to folding, all side-chain and backbone torsions, and uses a Go potential. In this study, we focus on the 46 residue alpha/beta protein crambin and two of its structural components, the helix and helix hairpin. For a wide range of temperatures, we recorded multiple folding events of these three structures from random coils to native conformations that differ by less than 1 A C(alpha) dRMS from their crystal structure coordinates. The thermodynamics and kinetic mechanism of the helix-coil transition obtained from our simulation shows excellent agreement with currently available experimental and molecular dynamics data. Based on insights obtained from folding its smaller structural components, a possible folding mechanism for crambin is proposed. We observed that the folding occurs via a cooperative, first order-like process, and that many folding pathways to the native state exist. One particular sequence of events constitutes a "fast-folding" pathway where kinetic traps are avoided. At very low temperatures, a kinetic trap arising from the incorrect packing of side-chains was observed. These results demonstrate that folding to the native state can be observed in a reasonable amount of time on desktop computers even when an all-atom representation is used, provided the energetics sufficiently stabilize the native state.     

Hybridized distance- and contact-based hierarchical structure modeling for folding soluble and membrane proteins

Crystallography and NMR system (CNS) is currently a widely used method for fragment-free ab initio protein folding from inter-residue distance or contact maps. Despite its widespread use in protein structure prediction, CNS is a decade-old macromolecular structure determination system that was originally developed for solving macromolecular geometry from experimental restraints as opposed to predictive modeling driven by interaction map data. As such, the adaptation of the CNS experimental structure determination protocol for ab initio protein folding is intrinsically anomalous that may undermine the folding accuracy of computational protein structure prediction. In this paper, we propose a new CNS-free hierarchical structure modeling method called DConStruct for folding both soluble and membrane proteins driven by distance and contact information. Rigorous experimental validation shows that DConStruct attains much better reconstruction accuracy than CNS when tested with the same input contact map at varying contact thresholds. The hierarchical modeling with iterative self-correction employed in DConStruct scales at a much higher degree of folding accuracy than CNS with the increase in contact thresholds, ultimately approaching near-optimal reconstruction accuracy at higher-thresholded contact maps. The folding accuracy of DConStruct can be further improved by exploiting distance-based hybrid interaction maps at tri-level thresholding, as demonstrated by the better performance of our method in folding free modeling targets from the 12th and 13th rounds of the Critical Assessment of techniques for protein Structure Prediction (CASP) experiments compared to popular CNS- and fragment-based approaches and energy-minimization protocols, some of which even using much finer-grained distance maps than ours. Additional large-scale benchmarking shows that DConStruct can significantly improve the folding accuracy of membrane proteins compared to a CNS-based approach. These results collectively demonstrate the feasibility of greatly improving the accuracy of ab initio protein folding by optimally exploiting the information encoded in inter-residue interaction maps beyond what is possible by CNS.