Conformational stability and folding mechanisms of dimeric proteins

The folding of multisubunit proteins is of tremendous biological significance since the large majority of proteins exist as protein-protein complexes. Extensive experimental and computational studies have provided fundamental insights into the principles of folding of small monomeric proteins. Recently, important advances have been made in extending folding studies to multisubunit proteins, in particular homodimeric proteins. This review summarizes the equilibrium and kinetic theory and models underlying the quantitative analysis of dimeric protein folding using chemical denaturation, as well as the experimental results that have been obtained. Although various principles identified for monomer folding also apply to the folding of dimeric proteins, the effects of subunit association can manifest in complex ways, and are frequently overlooked. Changes in molecularity typically give rise to very different overall folding behaviour than is observed for monomeric proteins. The results obtained for dimers have provided key insights pertinent to understanding biological assembly and regulation of multisubunit proteins. These advances have set the stage for future advances in folding involving protein-protein interactions for natural multisubunit proteins and unnatural assemblies involved in disease.     

A novel procedure for identification of the folding/unfolding patterns of dimeric proteins

The unfolding of proteins has been widely used for investigating the thermodynamic properties of monomeric proteins but has been used infrequently for dimeric (or oligomeric) proteins, because of the inherent cooperation of denaturation and dissociation of the dimers (oligomers). Here, we introduce a thermodynamic parameter K_obs to discriminate the diverse folding patterns of dimeric proteins. K_obs remains constant as the protein concentration increases for the true one-step curve of unfolding pattern (A), increases and reaches a plateau for one-step curves with monomeric intermediate pattern (B), and increases steadily with no plateau for one-step curves with dimeric intermediate pattern (C).     

Cross-linking of dimeric CitS and GltS transport proteins

CitS of Klebsiella pneumoniae and GltS of Escherichia coli are Na+-dependent secondary transporters from different families that are believed to share the same fold and quaternary structure. A 10 kDa protein tag (Biotin Acceptor Domain [BAD]) was fused to the N-terminus of both proteins (CitS-BAD1 and GltS-BAD1, respectively) and inserted in the central cytoplasmic loop that connects the two halves of the proteins (CitS-BAD260 and GltS-BAD206). Both CitS constructs and GltS-BAD206 were produced and shown to be active transporters, but GltS-BAD1 could not be detected in the membrane. Distance relationships in the complexes were studied by cross-linking studies. Both CitS constructs were shown to be in the dimeric state after purification in detergent by cross-linking with glutaraldehyde. The concentration of glutaraldehyde resulting in 50% cross-linking was significantly higher for CitS-BAD1 than for CitS and CitS-BAD260. Remarkably, GltS and GltS-BAD260 were not cross-linked by glutaraldehyde because of the lack of productive reactive sites. Cross-linking of GltS was observed when the N-terminal 46 residues of CitS with or without BAD at the N-terminus were added to the N-terminus of GltS. The stretch of 46 residues contains the first transmembrane segment of CitS that is missing in the GltS structure. The data support an orientation of the monomers in the dimer with the N-termini close to the dimer interface and the central cytoplasmic loops far away at the ends of the long axis of the dimer structure in a view perpendicular to the membrane.     

Cross-linking of dimeric CitS and GltS transport proteins

Abstract

CitS of Klebsiella pneumoniae and GltS of Escherichia coli are Na⁺-dependent secondary transporters from different families that are believed to share the same fold and quaternary structure. A 10 kDa protein tag (Biotin Acceptor Domain [BAD]) was fused to the N-terminus of both proteins (CitS-BAD1 and GltS-BAD1, respectively) and inserted in the central cytoplasmic loop that connects the two halves of the proteins (CitS-BAD260 and GltS-BAD206). Both CitS constructs and GltS-BAD206 were produced and shown to be active transporters, but GltS-BAD1 could not be detected in the membrane. Distance relationships in the complexes were studied by cross-linking studies. Both CitS constructs were shown to be in the dimeric state after purification in detergent by cross-linking with glutaraldehyde. The concentration of glutaraldehyde resulting in 50% cross-linking was significantly higher for CitS-BAD1 than for CitS and CitS-BAD260. Remarkably, GltS and GltS-BAD260 were not cross-linked by glutaraldehyde because of the lack of productive reactive sites. Cross-linking of GltS was observed when the N-terminal 46 residues of CitS with or without BAD at the N-terminus were added to the N-terminus of GltS. The stretch of 46 residues contains the first transmembrane segment of CitS that is missing in the GltS structure. The data support an orientation of the monomers in the dimer with the N-termini close to the dimer interface and the central cytoplasmic loops far away at the ends of the long axis of the dimer structure in a view perpendicular to the membrane.     

Coupling of conformational folding and disulfide-bond reactions in oxidative folding of proteins

The oxidative folding of proteins consists of conformational folding and disulfide-bond reactions. These two processes are coupled significantly in folding-coupled regeneration steps, in which a single chemical reaction (the "forward" reaction) converts a conformationally unstable precursor species into a conformationally stable, disulfide-protected successor species. Two limiting-case mechanisms for folding-coupled regeneration steps are described. In the folded-precursor mechanism, the precursor species is preferentially folded at the moment of the forward reaction. The (transient) native structure increases the effective concentrations of the reactive thiol and disulfide groups, thus favoring the forward reaction. By contrast, in the quasi-stochastic mechanism, the forward reaction occurs quasi-stochastically in an unfolded precursor; i.e., reactive groups encounter each other with a probability determined primarily by loop entropy, albeit modified by conformational biases in the unfolded state. The resulting successor species is initially unfolded, and its folding competes with backward chemical reactions to the unfolded precursors. The folded-precursor and quasi-stochastic mechanisms may be distinguished experimentally by the dependence of their kinetics on factors affecting the rates of thiol--disulfide exchange and conformational (un)folding. Experimental data and structural and biochemical arguments suggest that the quasi-stochastic mechanism is more plausible than the folded-precursor mechanism for most proteins.     

Size and folding in globular proteins

We have modeled protein folding by packing a unified length of regular structural elements (alpha-helices and beta-sheets) into a 'cube'. In a globular protein with m alpha-helices and n beta-strands, this unified length is expressed in units of heptapeptides in alpha-helices, and in units of tripeptides in beta-strands. Calculations using published data show that a 4-helix bundle (m = 4, n = 0) has at least 2 x 2 x 2 helical heptapeptides; the 16-strand beta-barrel of porin (m = 0, n = 16) is at most 4 x 4 x 4 tripeptides in beta-strands. Compact, recurring protein modules with mixed helices and beta-strands are the ones that actually acquire a geometrically quasi-spherical, or cubic, shape.     

The folding of dimeric cytoplasmic malate dehydrogenase. Equilibrium and kinetic studies.

Porcine heart cytoplasmic malate dehydrogenase (s-MDH) is a dimeric protein (2 x 35 kDa). We have studied equilibrium unfolding and refolding of s-MDH using activity assay, fluorescence, far-UV and near-UV circular dichroism (CD) spectroscopy, hydrophobic probe-1-anilino-8-napthalene sulfonic acid binding, dynamic light scattering, and chromatographic (HPLC) techniques. The unfolding and refolding transitions are reversible and show the presence of two equilibrium intermediate states. The first one is a compact monomer (MC) formed immediately after subunit dissociation and the second one is an expanded monomer (ME), which is little less compact than the native monomer and has most of the characteristic features of a 'molten globule' state. The equilibrium transition is fitted in the model: 2U <--> 2M(E) <--> 2M(C) <--> D. The time course of kinetics of self- refolding of s-MDH revealed two parallel folding pathways [Rudolph, R., Fuchs, I. & Jaenicke, R. (1986) Biochemistry 25, 1662-1669]. The major pathway (70%) is 2U-->2M*-->2M-->D, the rate limiting step being the isomerization of the monomers (K1 = 1.7 x 10(-3) s(-1)). The minor pathway (30%) involves an association step leading to the incorrectly folding dimers, prior to the very slow D*-->D folding step. In this study, we have characterized the folding-assembly pathway of dimeric s-MDH. Our kinetic and equilibrium experiments indicate that the folding of s-MDH involves the formation of two folding intermediates. However, whether the equilibrium intermediates are equivalent to the kinetic ones is beyond the scope of this study.     

The folding and evolution of multidomain proteins

Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent. Furthermore, recent studies indicate that multidomain proteins have evolved mechanisms to minimize the problems of interdomain misfolding.     

Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates

Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III.     

Microdomain dynamics in folding proteins

The basic formulas for the incorporation into the diffusion–collision model of the stabilities of intermediate states on the folding pathway are derived and discussed. A hypothetical two-step folding pathway is calculated in detail. A model for the production of incorrectly folded intermediates is suggested and some numerical estimates made. Implications and future directions in the evolution of the model are discussed. Three appendices deal with some mathematical aspects of the model.     

Mechanism of acid-induced folding of proteins

We have previously shown [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577] that beta-lactamase, cytochrome c, and apomyoglobin are maximally unfolded at pH 2 under conditions of low ionic strength, but a further decrease in pH, by increasing the concentration of HCl, refolds the proteins to the A state with properties similar to those of a molten globule state. To understand the mechanism of acid-induced refolding of protein structure, we studied the effects of various strong acids and their neutral salts on the acid-unfolded states of ferricytochrome c and apomyoglobin. The conformational transition of cytochrome c was monitored at 20 degrees C by using changes in the far-UV CD and in the Soret absorption at 394 nm, and that of apomyoglobin was monitored by changes in the far-UV CD. Various strong acids (i.e., sulfuric acid, perchloric acid, nitric acid, trichloroacetic acid, and trifluoroacetic acid) refolded the acid-unfolded cytochrome c and apomyoglobin to the A states as was the case with HCl. For both proteins neutral salts of these acids caused similar conformational transitions, confirming that the anions are responsible for bringing about the transition. The order of effectiveness of anions was shown to be ferricyanide greater than ferrocyanide greater than sulfate greater than thiocyanate greater than perchlorate greater than iodide greater than nitrate greater than trifluoroacetate greater than bromide greater than chloride.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prion proteins: folding and aggregation properties

The partial unfolding or alternative folding of a class of polypeptides is at the origin of fascinating events in living cells. In their non-native conformation, these constitutive polypeptides called prions are at the origin of a protein-based structural heredity. These polypeptides are closely associated to a class of fatal neurodegenerative illnesses in mammals and to the emergence and propagation of phenotypic traits in baker's yeasts. The structural transition from the correctly folded, native form of a prion protein to a persistent misfolded form that ultimately may cause cell death or the transmission of phenotypic traits is not yet fully understood. The mechanistic models accounting for this structure-based mode of inheritance and the extent of partial unfolding of prions or their alternative folding and the subsequent aggregation process are developed and discussed. Finally, the potential regulation of prion propagation by molecular chaperones is presented.     

Comparing folding codes for proteins and polymers

Proteins fold to unique compact native structures. Perhaps other polymers could be designed to fold in similar ways. The chemical nature of the monomer “alphabet” determines the “energy matrix” of monomer interactions—which defines the folding code, the relationship between sequence and structure. We study two properties of energy matrices using two-dimensional lattice models: uniqueness, the number of sequences that fold to only one structure, and encodability, the number of folds that are unique lowest-energy structures of certain monomer sequences. For the simplest model folding code, involving binary sequences of H (hydrophobic) and P (polar) monomers, only a small fraction of sequences fold uniquely, and not all structures can be encoded. Adding strong repulsive interactions results in a folding code with more sequences folding uniquely and more designable folds. Some theories suggest that the quality of a folding code depends only on the number of letters in the monomer alphabet, but we find that the energy matrix itself can be at least as important as the size of the alphabet. Certain multi-letter codes, including some with 20 letters, may be less physical or protein-like than codes with smaller numbers of letters because they neglect correlations among inter-residue interactions, treat only maximally compact conformations, or add arbitrary energies to the energy matrix.     

Structural dynamics, stability and folding of proteins

The present concepts of protein folding in vitro are reviewed. According to these concepts, amino acid sequence of protein, which has appeared a result of evolutionary selection, determines the native structure of protein, the pathway of protein folding, and the existence of free energy barrier between native and denatured states of protein. The latter means that protein macromolecule can exist in either native or denatured state. And all macromolecules in the native state are identical but for structural fluctuations due to Brownian motion of their atoms. Identity of all molecules in native state is of primary importance for their correct functioning. The dependence of protein stability, which is measured as the difference between free energy of protein in native and denatured states, on temperature and denaturant concentration is discussed. The modern approaches characterizing transition state and nucleation are regarded. The role of intermediate and misfolded states in amorphous aggregate and amyloid fibril formation is discussed.