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.     

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.     

Outlining folding nuclei in globular proteins

Our theoretical approach for prediction of folding/unfolding nuclei in three-dimensional protein structures is based on a search for free energy saddle points on networks of protein unfolding pathways. Under some approximations, this search is performed rapidly by dynamic programming and results in prediction of Phi values, which can be compared with those found experimentally. In this study, we optimize some details of the model (specifically, hydrogen atoms are taken into account in addition to heavy atoms), and compare the theoretically obtained and experimental Phi values (which characterize involvement of residues in folding nuclei) for all 17 proteins, where Phi values are now known for many residues. We show that the model provides good Phi value predictions for proteins whose structures have been determined by X-ray analysis (the average correlation coefficient is 0.65), with a more limited success for proteins whose structures have been determined by NMR techniques only (the average correlation coefficient is 0.34), and that the transition state free energies computed from the same model are in a good anticorrelation with logarithms of experimentally measured folding rates at mid-transition (the correlation coefficient is -0.73).     

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.     

Kinetics of folding nuclei formation in proteins

When a protein folds or unfolds, it passes through many half-folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed in the vicinity of the equilibrium point. Here the "two-state" transition proceeds without any accumulation of metastable intermediates, and only the transition state ("folding nucleus") is outlined by its key influence on the folding/unfolding kinetics. This review covers recent experimental and theoretical studies of folding nuclei.     

Domains in folding of model proteins.

By means of Monte Carlo simulation, we investigated the equilibrium between folded and unfolded states of lattice model proteins. The amino acid sequences were designed to have pronounced energy minimum target conformations of different length and shape. For short fully compact (36-mer) proteins, the all-or-none transition from the unfolded state to the native state was observed. This was not always the case for longer proteins. Among 12 designed sequences with the native structure of a fully compact 48-mer, a simple all-or-none transition was observed in only three cases. For the other nine sequences, three states of behavior-the native, denatured, and intermediate states-were found. The contiguous part of the native structure (domain) was conserved in the intermediate state, whereas the remaining part was completely unfolded and structureless. These parts melted separately from each other.     

Design and folding of dimeric proteins

In a similar way in which the folding of single-domain proteins provides an important test in the study of self-organization, the folding of homodimers constitutes a basic challenge in the quest for the mechanisms that are the basis of biological recognition. Dimerization is studied by following the evolution of two identical 20-letter amino acid chains within the framework of a lattice model and using Monte Carlo simulations. It is found that when design (evolution pressure) selects few, strongly interacting (conserved) amino acids to control the process, a three-state folding scenario follows, where the monomers first fold forming the halves of the eventual dimeric interface independently of each other, and then dimerize ("lock and key" kind of association). On the other hand, if design distributes the control of the folding process on a large number of (conserved) amino acids, a two-state folding scenario ensues, where dimerization takes place at the beginning of the process, resulting in an "induced type" of association. Making use of conservation patterns of families of analogous dimers, it is possible to compare the model predictions with the behavior of real proteins. It is found that theory provides an overall account of the experimental findings.     

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)     

Post-translocational folding of secretory proteins in Gram-positive bacteria

The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells. In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form. They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin. It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates. Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions. Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress. Consequently, Gram-positive bacteria such as Bacillus subtilis encode membrane- and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall.     

Liprotides assist in folding of outer membrane proteins

Proteins and lipids can form complexes called liprotides, in which the partially denatured protein forms a shell encasing a lipid core. This effectively stabilizes a lipid micelle in an aqueous solvent and suggests that liprotides may provide a suitable vessel for membrane proteins. Accordingly we have investigated if liprotides consisting of α‐lactalbumin and oleate could aid folding of four different outer membrane proteins (OMPs) tOmpA, PagP, BamA, and OmpF. tOmpA was able to fold in the presence of the liprotide, and folding did not occur if only oleate or α‐lactalbumin were added. Although the liprotides did not fold the other three OMPs on its own, it was able to assist their folding in the presence of vesicles. Incubation with liprotides before folding into vesicles increased the folding yield of the outer membrane proteins to a level higher than using micelles of the non‐ionic surfactant DDM. Even though the liprotide was stable at both high urea concentrations and high pH, it failed to efficiently fold OmpA at high pH. Instead, optimal folding was seen at pH 8–9, suggesting that important changes in the liprotide occurred when increasing the pH. We conclude that an otherwise folding‐inactive fatty acid can be activated when presented by a liprotide and thereby work as an in vitro chaperone for outer membrane proteins.     

包含体蛋白质的复性研究进展

包含体的形成是异源蛋白质在大肠杆菌中高效表达的必然结果,也是目前产生重组蛋白质最有效的方法之一。不可溶、无生物活性的包含体必须经过变性、复性才能获得天然结构,完整特定的生物学功能。聚集是造成重组蛋白质复性产率低下的主要因素,因此理解蛋白质聚集机制,减少和防止聚集的发生是建立高效、高产率复性方法的关键。分子伴侣、低分子量添加物等在复性过程中的应用及新的复性方法的建立都大大提高了重组蛋白质复性产率。     

On prediction of folding nuclei in globular proteins

The approach described in this paper on the prediction of folding nuclei in globular proteins with known three dimensional structures is based on a search of the lowest saddle points through the barrier separating the unfolded state from the native structure on the free-energy landscape of protein chain. This search is performed by a dynamic programming method. Comparison of theoretical results with experimental data on the folding nuclei of two dozen of proteins shows that our model provides good phi value predictions for proteins whose structures have been determined by X-ray analysis, with a less limited success for proteins whose structures have been determined by NMR techniques only. Consideration of a full ensemble of transition states results in more successful prediction than consideration of only the transition states with the minimal free energy. In conclusion we have predicted the localization of folding nuclei for three dimensional protein structures for which kinetics of folding is studied now but the localization of folding nuclei is still unknown.     

Hydrophobicity and structural classes in proteins.

The bulk hydrophobic character for the 20 natural amino acid residues, has been obtained from a database of 60 protein structures, grouped in the four structural classes alpha alpha, beta beta, alpha + beta and alpha/beta. The hydrophobicity coefficients thus obtained are compared with Ponnuswamy's original values using scales normalized to average = 0.0 and standard deviation = 1.0. Even though most of the amino acid residues do not change their hydropathic character in the different structural classes, their behaviour suggests the convenience that averaging methods should only consider proteins of the same structural class and that this information should be included in the secondary structure methods.     

Chaperonins--keeping a lid on folding proteins

Two classes of chaperonins are known in all groups of organisms to participate in the folding of newly synthesized proteins. Whereas bacterial type I chaperonins use a reversibly binding cofactor to temporarily sequester folding substrate proteins within the cylindrical chaperonin cavity, type II chaperonins in archaea and the eukaryotic cytosol appear to have evolved a built-in lid for this purpose. Not entirely surprisingly, this has consequences for the folding modes of the two types of chaperonins.     

Characterization of the folding degree of proteins

MOTIVATION: The characterization of the folding degree of chains is central to the elucidation of structure--function relationships in proteins. Here we present a new index for characterizing the folding degree of a (protein) chain. This index shows a range of features that are desirable for the study of the relation between structure and function in proteins. RESULTS: A novel index characterizing the folding degree of (protein) chains is developed based on the spectral moments of a matrix representing the dihedral angles (phi, omega and epsilon) of the protein main chain. The proposed index is normalized to the chain size, is not correlated to the gyration radius of the backbone chain and is able to distinguish between structures for which the sum of the main-chain dihedral angles is identical. The index is well correlated to the percentages of helix and strand in proteins, shows a linear dependence with temperature changes, and is able to differentiate among protein families. AVAILABILITY: On request from the author.     

In vitro folding of alpha-helical membrane proteins

For large-scale production, as required in structural biology, membrane proteins can be expressed in an insoluble form as inclusion bodies and be refolded in vitro. This requires refolding conditions where the native form is thermodynamically stable and where nonproductive pathways leading to aggregation are avoided. Examples of successful refolding are reviewed and general guidelines to establish refolding protocols of membrane proteins are presented.