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.     

Simulated folding in polypeptides of diversified molecular tacticity: implications for protein folding and de novo design

Stereochemistry could be a powerful variable for conformational tune up of polypeptides for de novo design. It may be also useful probe of possible role of interamide energetics in selection and stabilization of conformation. The homopolypeptides Ac-Xxx30-NHMe, with Xxx = Ala, Val, and Leu, of diversified stereochemical structure are generated by simulated racemization with a modified GROMOS-96 force field. The polypeptides, and other systematic stereochemical variants, are folded by simulated annealing with another modified GROMOS-96 force field under the dielectric constant values 1, 4, and 10. The resultant 15,000 molecular folds of isotactic (poly-L-chiral), syndiotactic (alternating L,D-chiral), and heterotactic (random-L,D-chiral) stereochemical structure, belonging to three polypeptide series, achieved under three different folding conditions, are assessed statistically for structure-to-energy-to-conformation relationship. The results suggest that interamide electrostatics could be a major factor in secondary-structure selection in polypeptides while main-chain stereochemistry could dictate molecular packing and therefore the relative magnitude of hydrogen-bond and Lennard-Jones (LJ) contributions in conformational energy. A method for computational design of heterotactic molecular folds in polypeptide structure has been developed, and the first road map for a chiral tune up of polypeptide structure based on stereochemical engineering has been laid down. Broad implications for protein structure, folding, and de novo design are briefly discussed.     

Endoplasmic reticulum quality control and the unfolded protein response: insights from plants

Protein quality control (QC) within the endoplasmic reticulum and the related unfolded protein response (UPR) pathway of signal transduction are major regulators of the secretory pathway, which is involved in virtually any aspect of development and reproduction. The study of plant-specific processes such as pathogen response, seed development and the synthesis of seed storage proteins and of particular toxins is providing novel insights, with potential implications for the general recognition events and mechanisms of action of QC and UPR.     

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.     

On the origin of the cooperativity of protein folding: Implications from model simulations

There is considerable experimental evidence that the cooperativity of protein folding resides in the transition from the molten globule to the native state. The objective of this study is to examine whether simplified models can reproduce this cooperativity and if so, to identify its origin. In particular, the thermodynamics of the conformational transition of a previously designed sequence (A. Kolinski, W. Galazka, and J. Skolnick, J. Chem. Phys. 103: 10286–10297, 1995), which adopts a very stable Greek-key β-barrel fold has been investigated using the entropy Monte Carlo sampling (ESMC) technique of Hao and Scheraga (M.-H. Hao and H.A. Scheraga, J. Phys. Chem. 98: 9882–9883, 1994). Here, in addition to the original potential, which includes one body and pair interactions between side chains, the force field has been supplemented by two types of multi-body potentials describing side chain interactions. These potentials facilitate the proteinlike pattern of side chain packing and consequently increase the cooperativity of the folding process. Those models that include an explicit cooperative side chain packing term exhibit a well-defined all-or-none transition from a denatured, random coil state to a high-density, well-defined, nativelike low-energy state. By contrast, models lacking such a term exhibit a conformational transition that is essentially continuous. Finally, an examination of the conformations at the free-energy barrier between the native and denatured states reveals that they contain a substantial amount of native-state secondary structure, about 50% of the native contacts, and have an average root mean square radius of gyration that is about 15% larger than native. © 1996 Wiley-Liss, Inc.     

A computer model to dynamically simulate protein folding: studies with crambin

The current work describes a simplified representation of protein structure with uses in the simulation of protein folding. The model assumes that a protein can be represented by a freely rotating rigid chain with a single atom approximating the effect of each side chain. Potentials describing the attraction or repulsion between different types of amino acids are determined directly from the distribution of amino acids in the database of known protein structures. The optimization technique of simulated annealing has been used to dynamically sample the conformations available to this simple model, allowing the protein to evolve from an extended, random coil into a compact globular structure. Many characteristics expected of true proteins, such as the sequence-dependent formation of secondary structure, the partitioning of hydrophobic residues, and specific disulfide pairing, are reproduced by the simulation, suggesting the model may accurately simulate the folding process.     

Fast and slow intermediate accumulation and the initial barrier mechanism in protein folding

Do stable intermediates form very early in the protein folding process? New results and a quantity of literature that bear on this issue are examined here. Results available provide little support for early intermediate accumulation before an initial search-dependent nucleation barrier.     

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone.

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

Hydrophobic folding units at protein-protein interfaces: implications to protein folding and to protein-protein association.

A hydrophobic folding unit cutting algorithm, originally developed for dissecting single-chain proteins, has been applied to a dataset of dissimilar two-chain protein-protein interfaces. Rather than consider each individual chain separately, the two-chain complex has been treated as a single chain. The two-chain parsing results presented in this work show hydrophobicity to be a critical attribute of two-state versus three-state protein-protein complexes. The hydrophobic folding units at the interfaces of two-state complexes suggest that the cooperative nature of the two-chain protein folding is the outcome of the hydrophobic effect, similar to its being the driving force in a single-chain folding. In analogy to the protein-folding process, the two-chain, two-state model complex may correspond to the formation of compact, hydrophobic nuclei. On the other hand, the three-state model complex involves binding of already folded monomers, similar to the association of the hydrophobic folding units within a single chain. The similarity between folding entities in protein cores and in two-state protein-protein interfaces, despite the absence of some chain connectivities in the latter, indicates that chain linkage does not necessarily affect the native conformation. This further substantiates the notion that tertiary, non-local interactions play a critical role in protein folding. These compact, hydrophobic, two-chain folding units, derived from structurally dissimilar protein-protein interfaces, provide a rich set of data useful in investigations of the role played by chain connectivity and by tertiary interactions in studies of binding and of folding. Since they are composed of non-contiguous pieces of protein backbones, they may also aid in defining folding nuclei.     

Kinetic studies of folding of the B-domain of staphylococcal protein A with molecular dynamics and a united-residue (UNRES) model of polypeptide chains

Langevin dynamics is used with our physics-based united-residue (UNRES) force field to study the folding pathways of the B-domain of staphylococcal protein A (1BDD (alpha; 46 residues)). With 400 trajectories of protein A started from the extended state (to gather meaningful statistics), and simulated for more than 35 ns each, 380 of them folded to the native structure. The simulations were carried out at the optimal folding temperature of protein A with this force field. To the best of our knowledge, this is the first simulation study of protein-folding kinetics with a physics-based force field in which reliable statistics can be gathered. In all the simulations, the C-terminal alpha-helix forms first. The ensemble of the native basin has an average RMSD value of 4 A from the native structure. There is a stable intermediate along the folding pathway, in which the N-terminal alpha-helix is unfolded; this intermediate appears on the way to the native structure in less than one-fourth of the folding pathways, while the remaining ones proceed directly to the native state. Non-native structures persist until the end of the simulations, but the native-like structures dominate. To express the kinetics of protein A folding quantitatively, two observables were used: (i) the average alpha-helix content (averaged over all trajectories within a given time window); and (ii) the fraction of conformations (averaged over all trajectories within a given time window) with Calpha RMSD values from the native structure less than 5 A (fraction of completely folded structures). The alpha-helix content grows quickly with time, and its variation fits well to a single-exponential term, suggesting fast two-state kinetics. On the other hand, the fraction of folded structures changes more slowly with time and fits to a sum of two exponentials, in agreement with the appearance of the intermediate, found when analyzing the folding pathways. This observation demonstrates that different qualitative and quantitative conclusions about folding kinetics can be drawn depending on which observable is monitored.     

A common landscape for membrane‐active peptides

Three families of membrane‐active peptides are commonly found in nature and are classified according to their initial apparent activity. Antimicrobial peptides are ancient components of the innate immune system and typically act by disruption of microbial membranes leading to cell death. Amyloid peptides contribute to the pathology of diverse diseases from Alzheimer's to type II diabetes. Preamyloid states of these peptides can act as toxins by binding to and permeabilizing cellular membranes. Cell‐penetrating peptides are natural or engineered short sequences that can spontaneously translocate across a membrane. Despite these differences in classification, many similarities in sequence, structure, and activity suggest that peptides from all three classes act through a small, common set of physical principles. Namely, these peptides alter the Brownian properties of phospholipid bilayers, enhancing the sampling of intrinsic fluctuations that include membrane defects. A complete energy landscape for such systems can be described by the innate membrane properties, differential partition, and the associated kinetics of peptides dividing between surface and defect regions of the bilayer. The goal of this review is to argue that the activities of these membrane‐active families of peptides simply represent different facets of what is a shared energy landscape.     

Translation‐coupled protein folding assay using a protease to monitor the folding status

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large‐scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli‐based cell‐free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation‐prone fractions. We translated ～90 E. coli proteins in the protease‐inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large‐scale analysis.     

Local interactions in protein folding determined through an inverse folding model

We adopt a model of inverse folding in which folding stability results from the combination of the hydrophobic effect with local interactions responsible for secondary structure preferences. Site-specific amino acid distributions can be calculated analytically for this model. We determine optimal parameters for the local interactions by fitting the complete inverse folding model to the site-specific amino acid distributions found in the Protein Data Bank. This procedure reduces drastically the influence on the derived parameters of the preference of different secondary structures for buriedness, which affects local interaction parameters determined through the standard approach based on amino acid propensities. The quality of the fit is evaluated through the likelihood of the observed amino acid distributions given the model and the Bayesian Information Criterion, which indicate that the model with optimal local interaction parameters is strongly preferable to the model where local interaction parameters are determined through propensities. The optimal model yields a mean correlation coefficient r = 0.96 between observed and predicted amino acid distributions. The local interaction parameters are then tested in threading experiments, in combination with contact interactions, for their capacity to recognize the native structure and structures similar to the native against unrelated ones. In a challenging test, proteins structurally aligned with the Mammoth algorithm are scored with the effective free energy function. The native structure gets the highest stability score in 100% of the cases, a high recognition rate comparable to that achieved against easier decoys generated by gapless threading. We then examine proteins for which at least one highly similar template exists. In 61% of the cases, the structure with the highest stability score excluding the native belongs to the native fold, compared to 60% if we use local interaction parameters derived from the usual amino acid propensities and 52% if we use only contact interactions. A highly similar structure is present within the five best stability scores in 82%, 81%, and 76% of the cases, for local interactions determined through inverse folding, through propensity, and set to zero, respectively. These results indicate that local interactions improve substantially the performances of contact free energy functions in fold recognition, and that similar structures tend to get high stability scores, although they are often not high enough to discriminate them from unrelated structures. This work highlights the importance to apply more challenging tests, as the recognition of homologous structures, for testing stability scores for protein folding.     

The folding pathway of spectrin R17 from experiment and simulation: using experimentally validated MD simulations to characterize States hinted at by experiment

We present an experimental and computational analysis of the folding pathway of the 17th domain of chicken brain alpha-spectrin, R17. Wild-type R17 folds in a two-state manner and the chevron plot (plot of the logarithm of the observed rate constant against concentration of urea) shows essentially linear folding and unfolding arms. A number of mutant proteins, however, show a change in slope of the unfolding arm at high concentration of denaturant, hinting at complexity in the folding landscape. Through a combination of mutational studies and high temperature molecular dynamics simulations we show that the folding of R17 can be described by a model with two sequential transition states separated by an intermediate species. The rate limiting transition state for folding in water has been characterized both through experimental Phi-value analysis and by simulation. In contrast, a detailed analysis of the transition state predicted to dominate under highly denaturing conditions is only possible by simulation.     

Distinct protein interfaces in transmembrane domains suggest an in vivo folding model

Given the known high-resolution structures of alpha-helical transmembrane domains, we show that there are statistically distinct classes of transmembrane interfaces which relate to the folding and oligomerization of transmembrane domains. Distinct types of interfaces have been categorized and refer to those between: the same polypeptide chain, different polypeptide chains, helices that are sequential neighbors, and those that are nonsequential. These different interfaces may reflect different phases in the mechanism of transmembrane domain folding and are consistent with the current experimental evidence pertaining to the folding and oligomerization of transmembrane domains. The classes of helix-helix interfaces have been identified in terms of the numbers and different types of pairwise amino acid interactions. The specific measures used are interaction entropy, the information content of interacting partners compared to a random set of contacts, the amino acid composition of the classes and the abundances of specific amino acid pairs in close contact. Knowledge of the clear differences in the types of helix-helix contacts helps with the derivation of knowledge-based constraints which until now have focused on only the interiors of transmembrane domains as compared to the exterior. Taken together, an in vivo model for membrane protein folding is presented, which is distinct from the familiar two-stage model. The model takes into account the different interfaces of membrane helices defined herein, and the available data regarding folding in the translocation channel.     

Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.     

Multiscale simulations of protein folding: application to formation of secondary structures

A multiscale simulation method of protein folding is proposed, using atomic representation of protein and solvent, combing genetic algorithms to determine the key protein structures from a global view, with molecular dynamic simulations to reveal the local folding pathways, thus providing an integrated landscape of protein folding. The method is found to be superior to previously investigated global search algorithms or dynamic simulations alone. For secondary structure formation of a selected peptide, RN24, the structures and dynamics produced by this method agree well with corresponding experimental results. Three most populated conformations are observed, including hairpin, β-sheet and α-helix. The energetic barriers separating these three structures are comparable to the kinetic energy of the atoms of the peptide, implying that the transition between these states can be easily triggered by kinetic perturbations, mainly through electrostatic interactions between charged atoms. Transitions between α-helix and β-sheet should jump over at least two energy barriers and may stay in the energetic trap of hairpin. It is proposed that the structure of proteins should be jointly governed by thermodynamic and dynamic factors; free energy is not the exclusive dominant for stability of proteins.