Complete change of the protein folding transition state upon circular permutation

Reversing the loop lengths of the small protein S6 by circular permutation has a dramatic effect on the transition state structure: it changes from globally diffuse to locally condensed. The phenomenon arises from a biased dispersion of the contact energies. Stability data derived from point mutations throughout the S6 structure show that interactions between residues that are far apart in sequence are stronger than those that are close. This entropy compensation drives all parts of the protein to fold simultaneously and produces the diffuse transition-state structure typical for two-state proteins. In the circular permutant, where strong contacts and short sequence separations are engineered to concur, the transition state becomes atypically condensed and polarized. Taken together with earlier findings that S6 may also fold by a 'collapsed' trajectory with an intermediate, the results suggest that this protein may fold by a multiplicity of mechanisms. The observations indicate that the diffuse transition state of S6 is not required for folding but could be an evolutionary development to optimize cooperativity.     

Systematic circular permutation of an entire protein reveals essential folding elements

The importance of chain connectivity in determining protein function and stability can be examined by breaking the peptide backbone using a technique such as circular permutation. Cleavage at certain positions results in a complete loss of the ability of the protein to fold. When such cleavage sites occur sequentially in the primary structure, we call the region a 'folding element', a new concept that could assist in our understanding of the protein folding problem. The folding elements of dihydrofolate reductase have been assigned by conducting a systematic circular permutation analysis in which the peptide backbone was sequentially broken between every pair of residues in the protein. The positions of folding elements do not appear to correspond to secondary structure motifs, substrate or coenzyme binding sites, or accessible surface area. However, almost all of the amino acid residues known to be involved in early folding events are located within the folding elements.     

Alteration of the disulfide-coupled folding pathway of BPTI by circular permutation

The kinetics of disulfide-coupled folding and unfolding of four circularly permuted forms of bovine pancreatic trypsin inhibitor (BPTI) were studied and compared with previously published results for both wild-type BPTI and a cyclized form. Each of the permuted proteins was found to be less stable than either the wild-type or circular proteins, by 3-8 kcal/mole. These stability differences were used to estimate effective concentrations of the chain termini in the native proteins, which were 1 mM for the wild-type protein and 2.5 to 4000 M for the permuted forms. The circular permutations increased the rates of unfolding and caused a variety of effects on the kinetics of refolding. For two of the proteins, the rates of a direct disulfide-formation pathway were dramatically increased, making this process as fast or faster than the competing disulfide rearrangement mechanism that predominates in the folding of the wild-type protein. These two permutations break the covalent connectivity among the beta-strands of the native protein, and removal of these constraints appears to facilitate direct formation and reduction of nearby disulfides that are buried in the folded structure. The effects on folding kinetics and mechanism do not appear to be correlated with relative contact order, a measure of overall topological complexity. These observations are consistent with the results of other recent experimental and computational studies suggesting that circular permutation may generally influence folding mechanisms by favoring or disfavoring specific interactions that promote alternative pathways, rather than through effects on the overall topology of the native protein.     

Prediction of protein folding pathways.

Recent 1H nuclear magnetic resonance (n.m.r.) hydrogen exchange experiments on five different proteins have delineated the secondary structures formed in trapped, partially folded intermediates. The early forming structural elements are identifiable through a technique described in this work to predict folding pathways. The method assumes that the sequential selection of structural fragments such as alpha-helices and beta-strands involved in the folding process is founded upon the maximal burial of solvent accessible surface from both the formation of internal structure and substructure association. The substructural elements were defined objectively by major changes in main-chain direction. The predicted folding pathways are in complete correspondence with the n.m.r. results in that the formed structural fragments found in the folding intermediates are those predicted earliest in the pathways. The technique was also applied to proteins of known tertiary structure and with fold similar to one of the five proteins examined by 1H n.m.r. The pathways for these structures also showed general consistency with the n.m.r. observations, suggesting conservation of a secondary structural framework or molten globule about which folding nucleates and proceeds.     

Random circular permutation of DsbA reveals segments that are essential for protein folding and stability

One of the key questions in protein folding is whether polypeptide chains require unique nucleation sites to fold to the native state. In order to identify possible essential polypeptide segments for folding, we have performed a complete circular permutation analysis of a protein in which the natural termini are in close proximity. As a model system, we used the disulfide oxidoreductase DsbA from Escherichia coli, a monomeric protein of 189 amino acid residues. To introduce new termini at all possible positions in its polypeptide chain, we generated a library of randomly circularly permuted dsbA genes and screened for active circularly permuted variants in vivo. A total of 51 different active variants were identified. The new termini were distributed over about 70 % of the polypeptide chain, with the majority of them occurring within regular secondary structures. New termini were not found in approximately 30 % of the DsbA sequence which essentially correspond to four alpha-helices of DsbA. Introduction of new termini into these "forbidden segments" by directed mutagenesis yielded proteins with altered overall folds and strongly reduced catalytic activities. In contrast, all active variants analysed so far show structural and catalytic properties comparable with those of DsbA wild-type. We suggest that random circular permutation allows identification of contiguous structural elements in a protein that are essential for folding and stability.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

Structural features of protein folding nuclei

A crucial event of protein folding is the formation of a folding nucleus. We demonstrate the presence of a considerable coincidence between the location of folding nuclei and the location of so-called "root structural motifs", which have unique overall folds and handedness. In the case of proteins with a single root structural motif, the involvement in the formation of a folding nucleus is in average significantly higher for amino acids residues that are in root structural motifs, compared to residues in other parts of the protein. The tests carried out revealed that the observed difference is statistically reliable. Thus, a structural feature that corresponds to the protein folding nucleus is now found.     

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg²⁺ ion concentrations are low, K⁺ concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo–like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg²⁺ (0.5–2 mM) and K⁺ (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.     

An analysis of protein folding pathways

We have developed a model of the protein folding process based on three primary assumptions: that burying of hydrophobic area is the dominant contribution to the relative free energy of a conformation, that a record of the folding process is largely preserved in the final structure, and that the denatured state is a random coil. Detailed folding pathways are identified for 19 protein structures. The picture of the folding process that emerges from this analysis is one of nucleation by regions of 8-16 residues. Nucleation sites then lead to larger structures by two mechanisms: propagation and diffusion/collision. A Monte Carlo simulation is used to follow the folding pathway when propagation is the dominant mechanism. Because detailed pathways are derived for each protein, the models are susceptible to experimental verification.     

Calculation of the free energy and cooperativity of protein folding

Calculation of the free energy of protein folding and delineation of its pre-organization are of foremost importance for understanding, predicting and designing biological macromolecules. Here, we introduce an energy smoothing variant of parallel tempering replica exchange Monte Carlo (REMS) that allows for efficient configurational sampling of flexible solutes under the conditions of molecular hydration. Its usage to calculate the thermal stability of a model globular protein, Trp cage TC5b, achieves excellent agreement with experimental measurements. We find that the stability of TC5b is attained through the coupled formation of local and non-local interactions. Remarkably, many of these structures persist at high temperature, concomitant with the origin of native-like configurations and mesostates in an otherwise macroscopically disordered unfolded state. Graph manifold learning reveals that the conversion of these mesostates to the native state is structurally heterogeneous, and that the cooperativity of their formation is encoded largely by the unfolded state ensemble. In all, these studies establish the extent of thermodynamic and structural pre-organization of folding of this model globular protein, and achieve the calculation of macromolecular stability ab initio, as required for ab initio structure prediction, genome annotation, and drug design.     

Changes of protein stiffness during folding detect protein folding intermediates

Single-molecule force-quench atomic force microscopy (FQ-AFM) is used to detect folding intermediates of a simple protein by detecting changes of molecular stiffness of the protein during its folding process. Those stiffness changes are obtained from shape and peaks of an autocorrelation of fluctuations in end-to-end length of the folding molecule. The results are supported by predictions of the equipartition theorem and agree with existing Langevin dynamics simulations of a simplified model of a protein folding. In the light of the Langevin simulations the experimental data probe an ensemble of random-coiled collapsed states of the protein, which are present both in the force-quench and thermal-quench folding pathways.     

Using motion planning to study protein folding pathways.

We present a framework for studying protein folding pathways and potential landscapes which is based on techniques recently developed in the robotics motion planning community. Our focus in this work is to study the protein folding mechanism assuming we know the native fold. That is, instead of performing fold prediction, we aim to study issues related to the folding process, such as the formation of secondary and tertiary structure, and the dependence of the folding pathway on the initial denatured conformation. Our work uses probabilistic roadmap (PRM) motion planning techniques which have proven successful for problems involving high-dimensional configuration spaces. A strength of these methods is their efficiency in rapidly covering the planning space without becoming trapped in local minima. We have applied our PRM technique to several small proteins (~60 residues) and validated the pathways computed by comparing the secondary structure formation order on our paths to known hydrogen exchange experimental results. An advantage of the PRM framework over other simulation methods is that it enables one to easily and efficiently compute folding pathways from any denatured starting state to the (known) native fold. This aspect makes our approach ideal for studying global properties of the protein's potential landscape, most of which are difficult to simulate and study with other methods. For example, in the proteins we study, the folding pathways starting from different denatured states sometimes share common portions when they are close to the native fold, and moreover, the formation order of the secondary structure appears largely independent of the starting denatured conformation. Another feature of our technique is that the distribution of the sampled conformations is correlated with the formation of secondary structure and, in particular, appears to differentiate situations in which secondary structure clearly forms first and those in which the tertiary structure is obtained more directly. Overall, our results applying PRM techniques are very encouraging and indicate the promise of our approach for studying proteins for which experimental results are not available.     

Amino acid substitutions influencing intracellular protein folding pathways.

Though an increasing variety of chaperonins are emerging as important factors in directing polypeptide chain folding off the ribosome, the primary amino acid sequence remains the major determinant of final conformation. The ability to identify cytoplasmic folding intermediates in the formation of the tailspike endorhamnosidase of phage P22 has made it possible to isolate two classes of mutations influencing folding intermediates-temperature-sensitive folding mutations and global suppressors of tsf mutants. These and related amino acid substitutions in eukaryotic proteins are discussed in the context of inclusion body formation and problems in the recovery of correctly folded proteins.     

Constructing templates for protein structure prediction by simulation of protein folding pathways

How a one-dimensional protein sequence folds into a specific 3D structure remains a difficult challenge in structural biology. Many computational methods have been developed in an attempt to predict the tertiary structure of the protein; most of these employ approaches that are based on the accumulated knowledge of solved protein structures. Here we introduce a novel and fully automated approach for predicting the 3D structure of a protein that is based on the well accepted notion that protein folding is a hierarchical process. Our algorithm follows the hierarchical model by employing two stages: the first aims to find a match between the sequences of short independently-folding structural entities and parts of the target sequence and assigns the respective structures. The second assembles these local structural parts into a complete 3D structure, allowing for long-range interactions between them. We present the results of applying our method to a subset of the targets from CASP6 and CASP7. Our results indicate that for targets with a significant sequence similarity to known structures we are often able to provide predictions that are better than those achieved by two leading servers, and that the most significant improvements in comparison with these methods occur in regions of a gapped structural alignment between the native structure and the closest available structural template. We conclude that in addition to performing well for targets with known homologous structures, our method shows great promise for addressing the more general category of comparative modeling targets, which is our next goal.     

Copper binding to the octarepeats of the prion protein. Affinity,specificity, folding,and cooperativity: insights from circular dichroism

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein. There is increasing evidence that PrP functions as a copper transporter. In addition, strains of prion disease have been linked with copper binding. We present here CD spectroscopic studies of Cu(2+) binding to various fragments of the octarepeat region of the prion protein. We show that glycine and l-histidine will successfully compete for all Cu(2+) ions bound to the PrP octapeptide region, suggesting Cu(2+) coordinates with a lower affinity for PrP than the fm dissociation constant reported previously. We show that each of the octarepeats do not form an isolated Cu(2+) binding motif but fold up cooperatively within multiple repeats. In addition to the coordinating histidine side chain residues, we show that the glycine residues and the proline within each octarepeat are also necessary to maintain the coordination geometry. The highly conserved octarepeat region in mammals is a hexarepeat in birds that also binds copper but with different coordination geometry. Finally, in contrast to other reports, we show that Mn(2+) does not bind to the octarepeat region of PrP.     

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.     

The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.     

Structure and folding of potato type II proteinase inhibitors: circular permutation and intramolecular domain swapping

Potato type II serine proteinase inhibitors are proteins that consist of multiple sequence repeats, and exhibit a multidomain structure. The structural domains are circular permutations of the repeat sequence, as a result of intramolecular domain swapping. Structural studies give indications for the origins of this folding behaviour, and the evolution of the inhibitor family.