Further experimental studies of the disulfide folding transition of ribonuclease A

Two very different mechanisms of folding have been proposed from experimental studies of disulfide formation in reduced ribonuclease A. (1) A pathway in which the rate-limiting step separates fully folded protein from all other disulfide intermediates and occurs solely in three-disulfide intermediates. (2) A multiple pathway mechanism with different rate-limiting steps for each pathway. The various rate-limiting steps involve disulfide breakage, formation, and rearrangement in intermediates with one, two, three, and four protein disulfides. To distinguish between these two mechanisms, we have carried out further studies of both unfolding and refolding. Refolding of reduced ribonuclease A requires three-disulfide intermediates to accumulate; negligible refolding occurs when only the nearly random one- and two-disulfide intermediate species are populated. Therefore, no rate-limiting steps of the type postulated in mechanism (2) occur in intermediates with one and two protein disulfides. Unfolding and disulfide reduction is an all-or-none process; no disulfide intermediates accumulate to detectable levels or precede the rate-limiting step. Mechanism (2) requires that such intermediates precede the rate-limiting step and accumulate to substantial levels. The different proposals were shown not to result from the use of different solution conditions or disulfide reagents; the two sets of data are not inconsistent. Instead, the inappropriate mechanism (2) resulted from an incorrect kinetic analysis and misinterpretation of the kinetics of disulfide formation and breakage.     

Learning about protein folding via potential functions

Over the last few years we have developed an empirical potential function that solves the protein structure recognition problem: given the sequence for an n-residue globular protein and a collection of plausible protein conformations, including the native conformation for that sequence, identify the correct, native conformation. Having determined this potential on the basis of only some 6500 native/nonnative pairs of structures for 58 proteins, we find it recognizes the native conformation for essentially all compact, soluble, globular proteins having known native conformations in comparisons with 10⁴ to 10⁶ reasonable alternative conformations apiece. In this sense, the potential encodes nearly all the essential features of globular protein conformational preference. In addition it “knows” about many additional factors in protein folding, such as the stabilization of multimeric proteins, quaternary structure, the role of disulfide bridges and ligands, proproteins vs. processed proteins, and minimal strand lengths in globular proteins. Comparisons are made with other sorts of protein folding problems, and applications in protein conformational determination and prediction are discussed. © 1994 Wiley-Liss, Inc.     

Energetics of membrane protein folding and stability

The critical role of membrane proteins in a myriad of biological and physiological functions has spawned numerous investigations over the past several decades with the long-term goal of identifying the molecular origins and energetic forces that stabilize these proteins within the membrane. Parallel structural and thermodynamics studies on several systems have provided significant insight regarding the driving forces governing folding, assembly, insertion, and translocation of membrane proteins. The present review surveys families of membrane-associated proteins including α-helical and β-barrel structures, viral surface receptors, and pore-forming toxins, citing representative proteins within each of these classes for further scrutiny in terms of structure-function relationships and global conformational stability. This overview presents seminal findings from pioneering studies on the energetics of membrane protein folding and stability to modern techniques that are exploiting the use of molecular genetics and single molecule studies. An overall consensus regarding the molecular origins of membrane protein stability is that a number of intrinsic properties resemble features of soluble proteins, yet there are distinct energetic differences arising from specific intra- and intermolecular interactions within the membrane. The combined efforts from structural, energetics, and dynamics approaches offer unique insights and improve our fundamental understanding of the driving forces dictating membrane protein folding and stability.     

Probing protein folding and stability using disulfide bonds

Disulfide bonds are required to stabilize the folded conformations of many proteins. The rates and equilibria of processes involved in disulfide bond formation and breakage can be manipulated experimentally and can be used to obtain important information about protein folding and stability. A number of experimental procedures for studying these processes, and approaches to interpreting the resulting data, are described here.     

The trials and tribulations of membrane protein folding in vitro

Membrane proteins are hard to handle and consequently the purification of functional protein in milligram quantities is a major problem. One reason for this is that once integral membrane proteins are outside their native membrane, they are prone to aggregation, are unstable and are frequently only partially functional. Knowledge of membrane protein folding mechanisms in vitro can help to understand the causes of these problems and work toward strategies to disaggregate and fold proteins correctly. Kinetic and stability studies are emerging on membrane protein folding, mainly on bacterial proteins. Mutagenesis methods have also been used to probe specific structural features or bonds in proteins. In addition, manipulation of lipid properties can be used to improve the efficiency of folding as well as the stability and function of the protein.     

Retinal proteins as model systems for membrane protein folding

Experimental folding studies of membrane proteins are more challenging than water-soluble proteins because of the higher hydrophobicity content of membrane embedded sequences and the need to provide a hydrophobic milieu for the transmembrane regions. The first challenge is their denaturation: due to the thermodynamic instability of polar groups in the membrane, secondary structures in membrane proteins are more difficult to disrupt than in soluble proteins. The second challenge is to refold from the denatured states. Successful refolding of membrane proteins has almost always been from very subtly denatured states. Therefore, it can be useful to analyze membrane protein folding using computational methods, and we will provide results obtained with simulated unfolding of membrane protein structures using the Floppy Inclusions and Rigid Substructure Topography (FIRST) method. Computational methods have the advantage that they allow a direct comparison between diverse membrane proteins. We will review here both, experimental and FIRST studies of the retinal binding proteins bacteriorhodopsin and mammalian rhodopsin, and discuss the extension of the findings to deriving hypotheses on the mechanisms of folding of membrane proteins in general. This article is part of a Special Issue entitled: Retinal Proteins—You can teach an old dog new tricks.     

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.     

Reducing the computational complexity of protein folding via fragment folding and assembly

Understanding, and ultimately predicting, how a 1-D protein chain reaches its native 3-D fold has been one of the most challenging problems during the last few decades. Data increasingly indicate that protein folding is a hierarchical process. Hence, the question arises as to whether we can use the hierarchical concept to reduce the practically intractable computational times. For such a scheme to work, the first step is to cut the protein sequence into fragments that form local minima on the polypeptide chain. The conformations of such fragments in solution are likely to be similar to those when the fragments are embedded in the native fold, although alternate conformations may be favored during the mutual stabilization in the combinatorial assembly process. Two elements are needed for such cutting: (1) a library of (clustered) fragments derived from known protein structures and (2) an assignment algorithm that selects optimal combinations to "cover" the protein sequence. The next two steps in hierarchical folding schemes, not addressed here, are the combinatorial assembly of the fragments and finally, optimization of the obtained conformations. Here, we address the first step in a hierarchical protein-folding scheme. The input is a target protein sequence and a library of fragments created by clustering building blocks that were generated by cutting all protein structures. The output is a set of cutout fragments. We briefly outline a graph theoretic algorithm that automatically assigns building blocks to the target sequence, and we describe a sample of the results we have obtained.     

Zinc-dependent dimerization of the folding catalyst, protein disulfide isomerase

Protein disulfide isomerase (PDI), an essential folding catalyst and chaperone of the endoplasmic reticulum (ER), has four structural domains (a-b-b'-a'-) of approximately equal size. Each domain has sequence or structural homology with thioredoxin. Sedimentation equilibrium and velocity experiments show that PDI is an elongated monomer (axial ratio 5.7), suggesting that the four thioredoxin domains are extended. In the presence of physiological levels (<1 mM) of Zn(2+) and other thiophilic divalent cations such as Cd(2+) and Hg(2+), PDI forms a stable dimer that aggregates into much larger oligomeric forms with time. The dimer is also elongated (axial ratio 7.1). Oligomerization involves the interaction of Zn(2+) with the cysteines of PDI. PDI has active sites in the N-terminal (a) and C-terminal (a')thioredoxin domains, each with two cysteines (CGHC). Two other cysteines are found in one of the internal domains (b'). Cysteine to serine mutations show that Zn(2+)-dependent dimerization occurs predominantly by bridging an active site cysteine from either one of the active sites with one of the cysteines in the internal domain (b'). The dimer incorporates two atoms of Zn(2+) and exhibits 50% of the isomerase activity of PDI. At longer times and higher PDI concentrations, the dimer forms oligomers and aggregates of high molecular weight (>600 kDa). Because of a very high concentration of PDI in the ER, its interaction with divalent ions could play a role in regulating the effective concentration of these metal ions, protecting against metal toxicity, or affecting the activity of other (ER) proteins that use Zn(2+) as a cofactor.     

New insights into structural determinants of prion protein folding and stability

Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.     

New insights into structural determinants of prion protein folding and stability

Abstract

Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.     

Polytopic membrane protein folding and assembly in vitro and in vivo (Review)

The insertion and folding of proteins in biological membranes during protein synthesis in vivo is fundamental to membrane biogenesis. At present, however, certain molecular aspects of this process can only be understood by complementary studies in vitro. We bring together in vitro and in vivo results, highlighting how the studies inform each other and increase our knowledge of the folding and assembly of polytopic membrane proteins. A notable recent advance is the high-resolution crystal structure of the protein machinery responsible for membrane protein insertion into the endoplasmic reticulum. This provides an opportunity to combine in vitro and in vivo studies at a more sophisticated level and address mechanistic aspects of polytopic protein insertion and folding. Quality control is another important aspect of membrane biogenesis, and we give an overview of the current understanding of this process, focusing on cystic fibrosis as a well-studied paradigm. Mutations in the associated membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), can cause the quality control mechanisms to prevent the mutant protein reaching its normal site of action, the cell surface. In vitro studies of CFTR shed light on the possible origins of other clinically relevant folding mutants and highlight the potential synergy between in vitro and in vivo approaches.     

Statistical mechanics of protein folding by exhaustive enumeration

It is hard to construct theories for the folding of globular proteins because they are large and complicated molecules having enormous numbers of nonnative conformations and having native states that are complicated to describe. Statistical mechanical theories of protein folding are constructed around major simplifying assumptions about the energy as a function of conformation and/or simplifications of the representation of the polypeptide chain, such as one point per residue on a cubic lattice. It is not clear how the results of these theories are affected by their various simplifications. Here we take a very different simplification approach where the chain is accurately represented and the energy of each conformation is calculated by a not unreasonable empirical function. However, the set of amino acid sequences and allowed conformations is so restricted that it becomes computationally feasible to examine them all. Hence we are able to calculate melting curves for thermal denaturation as well as the detailed kinetic pathway of refolding. Such calculations are based on a novel representation of the conformations as points in an abstract 12-dimensional Euclidean conformation space. Fast folding sequences have relatively high melting temperatures, native structures with relatively low energies, small kinetic barriers between local minima, and relatively many conformations in the global energy minimum's watershed. In contrast to other folding theories, these models show no necessary relationship between fast folding and an overall funnel shape to the energy surface, or a large energy gap between the native and the lowest nonnative structure, or the depth of the native energy minimum compared to the roughness of the energy landscape. Proteins 32:425–437, 1998. © 1998 Wiley-Liss, Inc.     

Correspondence between anomalous m- and DeltaCp-values in protein folding

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant LA30 has a curved unfolding limb, which is most consistent with TSE mobility. The kinetic m-values (derivatives of the rate constants with respect to denaturant concentration) in themselves depend on denaturant concentration. To obtain complementary information about putative mobile TSEs, we have carried out a thermodynamic analysis of the three proteins, based on data for refolding and unfolding over the range 10 degrees C to 70 degrees C. The data at all temperatures can be fitted to two-state model systems. Importantly, for all three proteins the activation heat capacities are, within error, identical to the heat capacities measured in independent experiments under equilibrium conditions. Although the equilibrium heat capacities are essentially invariant with regard to denaturant concentration, the activation heat capacities, similar to the structurally equivalent kinetic m-values, show marked denaturant dependence. Furthermore, the values of beta++ at different denaturant concentrations measured by m-values and by heat capacity values are very similar. These observations are consistent with significant transition state movements within the framework of two-state folding. The basis for TSE movement appears to be enthalpic rather than entropic, suggesting that the binding energy of denaturant-protein interactions is a major determinant of the response of energy landscape contours to changing environments.     

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.     

Comparison of intramolecular and intermolecular reactions in protein folding

The intrinsic component of the standard free energy change for the formation of a disulfide bond in a protein molecule is compared to that for an analogous chemical reaction. The former reaction, which represents theintramolecular formation of a disulfide bond in a protein molecule from a cysteine group containing a mixed disulfide bond with glutathione, and a free cysteine residue, is a unimolecular reaction. In contrast, its chemical analogue is a bimolecular reaction, and corresponds to theintermolecular disulfide interchange between a mixed disulfide-bonded compound between a cysteine residue and glutathione, and a free cysteine molecule. The difference in the intrinsic free energy of the above two reactions is estimated by two different approaches. First, a theoretical estimate of the magnitude of the difference in free energy of the two reactions (for a standard state of 1 M) is obtained using a gas-phase statistical thermodynamic approach, which indicates that the intramolecular reaction is energetically favored over its intermolecular counterpart by as much as 15.6 kcal/mole. For comparison, an experimentally derived value is also obtained, using experimental data from a study by Konishi et al. of the regeneration of the protein ribonuclease A (RNase A) from its reduced form by reduced and oxidized glutathiones. The intrinsic component of the free energy change of the intramolecular reaction, as it occurs in the protein molecule, is obtained from such experimental data by accounting explicitly for the free energy change (assumed to be solely an entropy change) pertaining to the conformational changes (ring closure) that the protein molecule undergoes in the course of the reaction. On the basis of the value derived from such an experimental approach, the intramolecular reaction is also energetically more favorable as compared to its intermolecular analogue, but only by a difference of 2.3 kcal/mole (for a standard state of 1 M). The large apparent discrepancy between the two values estimated from the theoretical and experimental approaches is rationalized by the postulation of several additional factors not inherent in the gas-phase theoretical estimate, such as dehydration and intramolecular hydrogen-bonding effects, which can largely compensate for the otherwise favorable energetics of the intramolecular reaction.     

Charge-charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa

Gaining a better understanding of the denatured state ensemble of proteins is important for understanding protein stability and the mechanism of protein folding. We studied the folding kinetics of ribonuclease Sa (RNase Sa) and a charge-reversal variant (D17R). The refolding kinetics are similar, but the unfolding rate constant is 10-fold greater for the variant. This suggests that charge-charge interactions in the denatured state and the transition state ensembles are more favorable in the variant than in RNase Sa, and shows that charge-charge interactions can influence the kinetics and mechanism of protein folding.