A cytoplasmic chaperonin that catalyzes beta-actin folding.

We have isolated a cytoplasmic chaperonin based on its ability to catalyze the folding of denatured beta-actin. The cytoplasmic chaperonin is organized as a multisubunit toroid and requires Mg2+ and ATP for activity. The folding reaction proceeds via the rapid ATP-independent formation of a binary complex, followed by a slower ATP-dependent release of the native product. Electron microscopic observations reveal a striking structural change that occurs upon addition of Mg2+ and ATP. The eukaryotic cytoplasm thus contains a chaperonin that is functionally analagous to its prokaryotic, mitochondrial, and chloroplastic counterparts.     

Heat capacity change for ribonuclease A folding.

The change in heat capacity deltaCp for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, deltaCp = 1.15+/-0.08 kcal mol(-1) K(-1). Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method.     

Solvent accessibility, protein surfaces, and protein folding.

Studies of the native structures of proteins, together with measurements of the thermodynamic properties of the transition between unfolded and native states, have defined the major components of the forces that stabilize native protein structures. However, the nature of the intermediates in the folding process remains largely hypothetical. It is a fairly widespread and not implausible assumption that the intermediates in the folding of a monomeric protein contain the same kinds of secondary and tertiary structures that appear in the native conformation, and that, although unstable, their lifetimes are prolonged by forces similar to those that stabilize the native structure. We wished to examine what happens if, during the folding of a monomeric protein, regions of secondary structure come together to form an intermediate of reduced instability. We applied calculations of accessible surface area (a measure of hydrophobic stabilization) and parameterized nonbonded energy calculations (measuring the strengths of van der Waals forces) to identify the kinds of stabilizing interactions that might be available to such an intermediate. First, we analyzed the total buried surface area of two types of proteins into contributions from formation of secondary structure alone, interaction of pairs of secondary-structural elements, the formation of the structure alone, interaction of pairs of secondary-structural elements, the formation of the complete secondary structure without the turns, and the complete native structure. The formation of secondary structure alone, without tertiary-structural interactions, buries roughly half the surface that the complete structure does. We then analyzed in more detail the approach of two alpha-helices to form a complex, as an illustrative example of the nature of the interaction between compact structural units which remain fairly rigid during their interaction. Many features of the results are not limited to the interaction of alpha-helices. (The results therefore neither confirm nor refute the hypothesis that alpha-helices are intermediates in the folding proteins). We find that the first forces to be felt upon approach arise from solvent conditions on the relative position and orientation of the two helices as does the close packing which optimizes the van der Waals interactions at shorter distances apart. Therefore there appears to be a range of distances in which hydrophobic interactions could create a nonspecific complex between two helices in which the side chains might have sufficient time to seek the proper interdigitation observed in the native structure, where the two helices are in intimate contact. Indeed, we find that only in the final stages of approach is the native geometry the most stable; in the region in which solvent-exclusion forces predominate, the conformation with helix axes parallel is more stable than the native conformation, in the cases we examined...     

Monte carlo simulations of protein folding. II. Application to protein A,ROP, and crambin

The hierarchy of lattice Monte Carlo models described in the accompanying paper (Kolinski, A., Skolnick, J. Monte Carlo simulations of protein folding. I. Lattice model and interaction scheme. Proteins 18:338–352, 1994) is applied to the simulation of protein folding and the prediction of 3-dimensional structure. Using sequence information alone, three proteins have been successfully folded: the B domain of staphylococcal protein A, a 120 residue, monomeric version of ROP dimer, and crambin. Starting from a random expanded conformation, the model proteins fold along relatively well-defined folding pathways. These involve a collection of early intermediates, which are followed by the final (and rate-determining) transition from compact intermediates closely resembling the molten globule state to the native-like state. The predicted structures are rather unique, with native-like packing of the side chains. The accuracy of the predicted native conformations is better than those obtained in previous folding simulations. The best (but by no means atypical) folds of protein A have a coordinate rms of 2.25 Å from the native Cα trace, and the best coordinate rms from crambin is 3.18 Å. For ROP monomer, the lowest coordinate rms from equivalent Cαs of ROP dimer is 3.65 Å. Thus, for two simple helical proteins and a small α/β protein, the ability to predict protein structure from sequence has been demonstrated. © 1994 John Wiley & Sons, Inc.     

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.     

Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates.

The hierarchical partition function formalism for protein folding developed earlier has been extended through the use of three-dimensional polar and apolar contact plots. For each amino acid residue in the protein, these plots indicate the apolar and polar surfaces that are buried from the solvent, the identity of all amino acid residues that contribute to this shielding, and the magnitude of their contributions. These contact plots are then used to examine the distribution of the free energy of stabilization throughout the protein molecule. Analysis of these data allows identification of co-operative folding units and their hierarchical levels, and the identification of partially folded intermediates with a significant probability of being populated. The overall folding/unfolding thermodynamics of 12 globular proteins, for which crystallographic and experimental thermodynamics are available, is predicted within error. An energetic classification of partially folded intermediates is presented and the results compared to those cases for which structural and thermodynamic experimental information is available. Four different types of partially folded states and their structural energies are considered. (1) Local intermediates, in which only a local region of the protein loses secondary and tertiary interactions, while the rest of the protein remains intact. (2) Global intermediates, corresponding to the standard molten globule definition, in which significant secondary structure is maintained but native-like tertiary structure contacts are disrupted. (3) Extended intermediates characterized by the existence of secondary structure elements (e.g. alpha-helices) exposed to solvent. (4) Folding intermediates in proteins with two structural domains. The structure and energetics of folding intermediates of apo-myoglobin, alpha-lactalbumin, phosphoglycerate kinase and arabinose-binding protein are considered in detail.     

GroEL-mediated protein folding.

I. Architecture of GroEL and GroES and the reaction pathway A. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding II. Polypeptide binding A. A parallel network of chaperones binding polypeptides in vivo B. Polypeptide binding in vitro 1. Role of hydrophobicity in recognition 2. Homologous proteins with differing recognition-differences in primary structure versus effects on folding pathway 3. Conformations recognized by GroEL a. Refolding studies b. Binding of metastable intermediates c. Conformations while stably bound at GroEL 4. Binding constants and rates of association 5. Conformational changes in the substrate protein associated with binding by GroEL a. Observations b. Kinetic versus thermodynamic action of GroEL in mediating unfolding c. Crossing the energy landscape in the presence of GroEL III. ATP binding and hydrolysis-driving the reaction cycle IV. GroEL-GroES-polypeptide ternary complexes-the folding-active cis complex A. Cis and trans ternary complexes B. Symmetric complexes C. The folding-active intermediate of a chaperonin reaction-cis ternary complex D. The role of the cis space in the folding reaction E. Folding governed by a "timer" mechanism F. Release of nonnative polypeptides during the GroEL-GroES reaction G. Release of both native and nonnative forms under physiologic conditions H. A role for ATP binding, as well as hydrolysis, in the folding cycle V. Concluding remarks.     

Catalysis, commitment and encapsulation during GroE-mediated folding.

The Escherichia coli GroE chaperones assist protein folding under conditions where no spontaneous folding occurs. To achieve this, the cooperation of GroEL and GroES, the two protein components of the chaperone system, is an essential requirement. While in many cases GroE simply suppresses unspecific aggregation of non-native proteins by encapsulation, there are examples where folding is accelerated by GroE.Using maltose-binding protein (MBP) as a substrate for GroE, it had been possible to define basic requirements for catalysis of folding. Here, we have analyzed key steps in the interaction of GroE and the MBP mutant Y283D during catalyzed folding. In addition to high temperature, high ionic strength was shown to be a restrictive condition for MBP Y283D folding. In both cases, the complete GroE system (GroEL, GroES and ATP) compensates the deceleration of MBP Y283D folding. Combining kinetic folding experiments and electron microscopy of GroE particles, we demonstrate that at elevated temperatures, symmetrical GroE particles with GroES bound to both ends of the GroEL cylinder play an important role in the efficient catalysis of MBP Y283D refolding. In principle, MBP Y283D folding can be catalyzed during one encapsulation cycle. However, because the commitment to reach the native state is low after only one cycle of ATP hydrolysis, several interaction cycles are required for catalyzed folding.     

Protein-catalysed protein folding.

A number of proteins, termed chaperonins, have been identified as part of the mechanism of folding other proteins into their biologically active forms. The role of chaperonins appears to be twofold--to prevent illegitimate interactions with other proteins and to facilitate folding, possibly through an energy-dependent, catalytic function. Controlled overexpression of chaperonins may be of therapeutic value in manipulating human immune response and rescuing certain inherited human mutations.     

A computational approach to simplifying the protein folding alphabet.

What is the minimal number of residue types required to form a structured protein? This question is important for understanding protein modeling and design. Recently, an experimental finding by Baker and coworkers suggested a five-residue solution to this problem. We were motivated by their results and by the arguments of Wolynes to study reductions of protein representation based on the concept of mismatch between a reduced interaction matrix and the Miyazawa and Jernigan (MJ) matrix. We find several possible simplified schemes from the relationship of minimized mismatch versus the number of residue types (N = approximately 2-20). As a specific case, an optimal reduction with five types of residues has the same form as the simplified palette of Baker and coworkers. Statistical and kinetic features of a number of sequences are tested. Comparison of results from sequences with 20 residue types and their reduced representations indicates that the reduction by mismatch minimization is successful. For example, sequences with five types of residues have good folding ability and kinetic accessibility in model studies.     

A new principle of RNA folding based on pseudoknotting.

Tertiary interactions involving hairpin or interior loops of RNA can lead to extended quasi-continuous double helical stem regions, consisting of coaxially stacked segments of duplex RNA, bridged by single-stranded connections. This type of compact folding plays a role in various strategic regions of RNA molecules. Their role in ribosome functioning, RNA splicing and recognition of tRNA-like structures is discussed.     

A study of accessible motifs and RNA folding complexity.

mRNA molecules are folded in the cells and therefore many of their substrings may actually be inaccessible to protein and microRNA binding. The need to apply an accessibility criterion to the task of genome-wide mRNA motif discovery raises the challenge of overcoming the core O(n(3)) factor imposed by the time complexity of the currently best known algorithms for RNA secondary structure prediction. We speed up the dynamic programming algorithms that are standard for RNA folding prediction. Our new approach significantly reduces the computations without sacrificing the optimality of the results, yielding an expected time complexity of O(n(2) psi(n)), where psi(n) is shown to be constant on average under standard polymer folding models. A benchmark analysis confirms that in practice the runtime ratio between the previous approach and the new algorithm indeed grows linearly with increasing sequence size. The fast new RNA folding algorithm is utilized for genome-wide discovery of accessible cis-regulatory motifs in data sets of ribosomal densities and decay rates of S. cerevisiae genes and to the mining of exposed binding sites of tissue-specific microRNAs in A. thaliana.     

Studies on protein folding,unfolding, and fluctuations by computer simulation. II. A. Three-dimensional lattice model of lysozyme

A three-dimensional lattice model of protein designed to assimilate lysozyme is introduced. An attractive interaction is assumed to work between preassigned specific pairs of units, when they occupy the nearest-nighbor lattice points. The behavior of this lattice lysozyme is studied by a Monte Carlo simulation method. Because of the specific interunit interactions,“native state” of the lattice lysozyme is stable at low temperatures. Conformational fluctuations in the native state are observed to occur at both termini and loop regions of the main chain existing on the surface. The process of unfolding and denatured states of this model are discussed. Complete refolding from a denatured state was not observed. However, by starting from partially folded structures, the native conformation could be attained. From these observation it is concluded that, in the process of folding of proteins as simplified in a lattice model, nulceation is a rate-limiting factor. The artificial character of this model and possible improvement are discussed.     

A kinetic study of the folding of nuclease B, a possible precursor of staphylococcal nuclease A.

Nuclease B, which contains an additional flexible amino acid sequence of 19 amino acid residues bound to the NH2-terminus of nuclease A, an extracellular nuclease of Staphylococcus aureus, has been investigated in order to determine the influence of the extra residues on the refolding of the nuclease A portion from the acid denaturated state by monitoring the change in tryptophan fluorescence using a stopped-flow technique. It was found that the kinetic parameters of this refolding is similar within experimental error for nuclease A and nuclease B for the entire course (up to 40 s) studied. Therefore, the extra residues do not appear to have any detectable effect on the dynamic events involved in the refolding process. Thus, the folding of the nuclease A portion of nuclease B appears to be thermodynamically and kinetically independent of the 19 residues at the amino-terminus.     

4 anthroposcopic markers in the Northern Greece population: hand folding, arm folding, tongue rolling and tongue folding

Four anthroposcopic traits, namely hand clasping, arm folding, tongue rolling and tongue curling have been studied in a total of 7763 individuals from Thessaloniki and its surroundings, representing a sample of the population of Northern Greece. The statistical analysis of the data indicates significant sex differences only concerning tongue rolling. The frequencies obtained for the four traits under study are compared with data from the literature.