Abstract: HX-labeling experiments in the pH-pulse mode show that protein folding can be remarkably fast. A near-native form can be reached within milliseconds. Experimental analysis of the folding process on the millisecond-to-second time scale depends upon the presence of kinetic barriers that avoid apparent two-step folding. A common barrier produces molecular intermediates; disparate barriers produce population heterogeneity that makes analysis more difficult. Results available exhibit an early, native-like two-helix intermediate in cytochrome c, an extensive, native-like, beta-sheet-plus-helix intermediate in RNase A, and a late native-like molten globular intermediate in barnase. These differences appear to reflect chance differences in the placement of the determining kinetic barriers. Requirements for observing kinetic folding intermediates are difficult to satisfy, so most intermediates are not seen, and intermediates that are seen often represent the sum of multiple preceding steps.

Abstract: The 56 amino acid B domain of protein G (GB) is a stable globular folding unit with no disulfide cross-links. The physical properties of GB offer extraordinary flexibility for evaluating the energetics of the folding reaction. The protein is monomeric and very soluble in both folded and unfolded forms. The folding reaction has been previously examined by differential scanning calorimetry (Alexander et al., 1992) and found to exhibit two-state unfolding behavior over a wide pH range with an unfolding transition near 90 degrees C (GB1) at neutral pH. Here, the kinetics of folding and unfolding two naturally occurring versions of GB have been measured using stopped-flow mixing methods and analyzed according to transition-state theory. GB contains no prolines, and the kinetics of folding and unfolding can be fit to a single, first-order rate constant over the temperature range of 5-35 degrees C. The major thermodynamic changes going from the unfolded state to the transition state are (1) a large decrease in heat capacity (delta Cp), indicating that the transition state is compact and solvent inaccessible relative to the unfolded state; (2) a large loss of entropy; and (3) a small increase in enthalpy. The most surprising feature of the folding of GB compared to that of previously studied proteins is that its folding approximates a rapid diffusion controlled process with little increase in enthalpy going from the unfolded to the transition state.

Abstract: The folding of intestinal fatty-acid binding protein has been monitored by 19F NMR after incorporation of 6-fluorotryptophan into the protein. The two resonances resulting from the two tryptophans of this protein showed different dependencies on denaturant concentration. One of the resonances was in slow chemical exchange between two resonance frequencies, native and completely unfolded. The changes for this resonance occurred over a denaturant concentration range identical to that monitored by circular dichroism or fluorescence during unfolding. The other resonance continued to show changes at concentrations of denaturant well above that needed to complete the unfolding transition as monitored by optical techniques. Site directed mutagenesis showed that tryptophan-82 was the residue responsible for the unexpected behavior. We conclude, based on complete line-shape analysis, that there are significant concentrations of one or more intermediates in equilibrium with the native and unfolded forms. The structure of the intermediate(s) is more similar to the completely unfolded form of the protein than to the native structure, since little if any secondary structure is present. Further, these structure(s) persist at high denaturant concentrations and may represent local initiating sites in the folding of this beta-sheet protein.

Abstract: The cis-trans isomerization of prolyl peptide bonds and the formation of disulfide bonds are both slow steps in protein folding. By using ribonuclease T1 as a model system, we show that these two processes can become linked in the oxidative folding of reduced proteins and that the formation of the correct disulfide bonds is facilitated in the presence of peptidyl-prolyl cis-trans isomerase. In particular, the efficiency of protein disulfide isomerase (EC 5.3.4.1) as a catalyst of disulfide bond formation in the course of oxidative folding is markedly improved when peptidyl-prolyl cis-trans isomerase is present simultaneously. Possibly, unfolded or partially folded protein chains with correct prolyl isomers are better substrates for catalysis by protein disulfide isomerase. The interdependence of the two enzymatic activities detected during in vitro folding experiments could be of importance for the de novo folding and disulfide bond formation of nascent proteins in the endoplasmic reticulum.

Abstract: The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans----cis reisomerization of the Tyr-38-Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans----cis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate rate-limiting steps by trapping partially folded structures.

Abstract: The folding of polypeptide chains in cells, following either translation or translocation through membranes, must take place under conditions of extremely high protein concentrations. In addition, folding into a correct structure must occur in the presence of other rapidly folding species, and at temperatures known to destabilize aggregation-prone folding intermediates. To facilitate folding in vivo, molecular chaperones have evolved that stabilize protein folding intermediates, thus partitioning them towards a pathway leading to the native state rather than forming inactive aggregated structures.

Abstract: The order of formation of substructures in the folding of barnase has been determined by a protein engineering procedure and corroborated and complemented by NMR experiments. Early events are the formation of the centre of the β-sheet and the C-terminus of the major α-helix. These later dock to form the major hydrophobic core. Structural studies of fragments of barnase in solution show that a peptide that spans the major α-helix is found to contain a significant fraction of its C-terminal region in the helical structures. The formation of the native secondary structure as an early event in folding and in isolated fragments is accompanied by considerable burial of hydrophobic surfaces. The experimental data support a model for protein folding in which initiation sites in secondary structure are driven by local hydrophobic interactions, and their docking via further hydrophobic interactions drives the formation of tertiary structure.

Abstract: Molecular chaperones are proteins that promote the correct folding, assembly, and transport of other protein mo1ecules.l The most widely studied of the molecular chaperones are the chaperonins; these form a subgroup found in prokaryotic cells, in mitochondria, and in pla~tids.*-~ They are oligomeric proteins of high molecular weight, which work in conjunction with a smaller, also oligomeric, coprotein. One such chaperonin is cpn60 from Escherichia coli, the product of the gro-EL gene locus. This has a subunit mass of approximately 60,000 daltons, and the whole protein exists as an assembly of 14 subunits in a “double-doughnut” structure, each ring comprising 7 subunits. The coprotein is produced by thegro-ES locus and is termed cpnlO by virtue of its 10,000-dalton subunit molecular weight. CpnlO exists as a “singledoughnut” structure of 7 subunits.6 In conjunction, these proteins have been shown to aid the folding and/or assembly of bacteriophages, multimeric and monomeric protein molecule^.^^* They function by binding to unfolded or partially folded protein molecules and, by transducing the energy of ATP hydrolysis, increasing the yield of the natively assembled form.9 The mechanistic detail of this process remains undefined. Here we report on preliminary kinetic experiments that explore the interactions of cpn60 with (a) the folding intermediates of Bacillus stearothemophilus lactate dehydrogenase (LDH), (b) with ATP and an unreactive analogue (AMP-PNP), and (c) with the coprotein cpnl0. From these results with LDH we suggest a mechanism that explains the ability of chaperonins to improve the efficiency of protein folding in general.

Abstract: This paper reviews the background and outlines the utility of a 3D → 1D transformation of peptide conformation. Although this transformation leads to a linearized notation of protein secondary and tertiary structures that may be used for an objective description and classification of protein folding*, nevertheless, the method is intended to be descriptive and is not meant to be predictive.

Abstract: The native state of a protein molecule in aqueous solutions represents one of the lowest states of Gibbs energy [Anfinsen, C.B. (1973) Science 181, 223-230]. Much progress has been made about the rules of protein folding [King, J. (1989) Chem. Eng. News 67, 32-54] and the dominant forces in protein folding [Dill, K.A. (1990) Biochemistry 29, 7133-7155]. However, the quantitative contributions of different Gibbs energy terms to protein stability remains a controversial issue [Moult, J., & Unger, R. (1991) Biochemistry 30, 3816-3824]. A molecular thermodynamic model has been proposed for the Gibbs energy of folding a residue in aqueous homopolypeptides from a random-coiled state to either the alpha-helix state or the beta-sheet state [Chen, C.-C., Zhu, Y., King, J.A., & Evans, L.B. (1992) Biopolymers 32, 1375-1392]. In this work, we present a generalization of the molecular thermodynamic model for the Gibbs energy of folding natural and synthetic heteropolypeptides from random-coiled conformations into alpha-helical conformations. The generalized model incorporates the intrinsic folding potential due to residue-solvent interactions, the cooperative folding effect due to residue-residue interactions, and the location and length of alpha-helices. The utility of the model was demonstrated by examining the stability of alpha-helical conformations of a number of natural polypeptides including C-peptide (residues 1-13) and S-peptide (residues 1-20) of RNase A (bovine pancreatic ribonuclease A), the P alpha fragment in BPTI (bovine pancreatic trypsin inhibitor), and synthetic polypeptides (the copolymers of different amino acid residues) including alanine-based peptides (16 or 17 residues long) in water. The computed Gibbs energies correspond well with the experimental data on helicity. The results also accounted for the effects of amino acid substitution and temperature on the stability of alpha-helical conformations of the test polypeptides.

Abstract: Publisher Summary This chapter describes the use of stopped-flow circular dichroism and 19 F NMR as probes of secondary and tertiary structure formation, respectively, during the folding of rat intestinal fatty-acid binding protein (IFABP). IFABP is a small predominantly β-structure protein that contains two tryptophans but neither cysteine nor proline, thereby avoiding the folding complications associated with those residues. The equilibrium and kinetic folding properties of this protein have been examined, primarily by monitoring fluorescence and circular dichroism changes during the transition. Although no significant concentration of intermediates was detected at equilibrium, at least one transient intermediate was shown to be present during both unfolding and refolding of the protein. The problems associated with determining the mechanism of protein folding have proven to be difficult to overcome. As such, any techniques that can follow changes in secondary structure or at specific sites during folding are useful. Both stopped-flow CD and 19 F NMR have proven important to the study of the folding of IFABP and may be helpful in the study of other proteins as well.

Abstract: The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogen-deuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 back-bone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the beta-sheet and the alpha-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slow-folding species are less prominent.

Abstract: We study the kinetics of folding of a heteropolymer containing a specific sequence of hydrophobic, hydrophilic, and neutral residues. The heteropolymer, representing the alpha carbon sequence of a model protein, folds into a β‐barrel structure below a characteristic folding transition temperature. Several measures are introduced to probe the dynamics of approach to a folded state with particular emphasis on the kinetics of formation of nonbonded contacts starting from a high temperature random configuration. The measures of compactness are used to argue that even below the folding temperature, nonbonded contacts are formed at a very slow rate. This result is further corroborated by analyzing the dynamics of relaxation of the nonbonded interactions which are responsible for the formation of folded structures at low temperatures. We also show that below the folding transition temperature and for short times, the heteropolymer undergoes large structural fluctuations. Examination of the time dependence of the...