Abstract: Experimental studies of the physical interactions that stabilize protein structure are complicated by the fact that proteins do not unfold to a simple reference state. When their folded structure breaks down, protein chains do not become random coils. Instead, they enter a poorly understood ensemble of partially folded states known collectively as the denatured state. Although it has long been held that agents that promote protein unfolding act specifically on the denatured state, the idea that mutations can exert their destabilizing (or in some cases, stabilizing) effects directly on this state is not widely accepted. A large body of thermodynamic data on mutant proteins plus a limited amount of structural information describing mutational effects on denatured states indicate that 1) the denatured state plays a central role in all aspects of protein stability, including mutant effects, and 2) a quantitative understanding of how amino acid sequence encodes protein structure will probably depend on a more ...

Abstract: We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP-dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event.

Abstract: Experiments with cytochrome c (cyt c) show that an initial folding event, molecular collapse, is not an energetically downhill continuum as commonly presumed but represents a large-scale, time-consuming, cooperative barrier-crossing process. In the absence of later misfold-reorganization barriers, the early collapse barrier limits cyt c folding to a time scale of milliseconds. The collapse process itself appears to be limited by an uphill search for some coarsely determined transition state structure that can nucleate subsequent energetically downhill folding events. An earlier "burst phase" event at strongly native conditions appears to be a non-specific response of the unfolded chain to reduced denaturant concentration. The molecular collapse process may or may not require the co-formation of the amino- and carboxyl-terminal helices, which are present in an initial metastable intermediate directly following the rate-limiting collapse. After the collapse-nucleation event, folding can proceed rapidly in an apparent two-state manner, probably by way of a predetermined sequence of metastable intermediates that leads to the native protein structure (Bai et al., Science 269:192-197, 1995).

Abstract: An approach is described to monitor directly at the level of individual residues the formation of structure during protein folding. A two-dimensional heteronuclear nuclear magnetic resonance (NMR) spectrum was recorded after the rapid initiation of the refolding of a protein labeled with nitrogen-15. The intensities and line shapes of the cross peaks in the spectrum reflected the kinetic time course of the folding events that occurred during the spectral accumulation. The method was used to demonstrate the cooperative nature of the acquisition of the native main chain fold of apo bovine α-lactalbumin. The general approach, however, should be applicable to the investigation of a wide range of chemical reactions.

Abstract: All possible protein folding intermediates exist in equilibrium with the native protein at native as well as non-native conditions, with occupation determined by their free energy level. The study of these forms can illuminate the fundamental principles of protein structure and folding. Hydrogen exchange methods can be used to detect and characterize these partially unfolded forms at native conditions and as a function of mild denaturant and temperature. This information illuminates the requirements that govern the ability of kinetic and equilibrium methods to study folding intermediates.

Abstract: NMR has emerged as an important tool for studies of protein folding because of the unique structural insights it can provide into many aspects of the folding process. Applications include measurements of kinetic folding events and structural characterization of folding intermediates, partly folded states, and unfolded states. Kinetic information on a time scale of milliseconds or longer can be obtained by real-time NMR experiments and by quench-flow hydrogen-exchange pulse labeling. Although NMR cannot provide direct information on the very rapid processes occurring during the earliest stages of protein folding, studies of isolated peptide fragments provide insights into likely protein folding initiation events. Multidimensional NMR techniques are providing new information on the structure and dynamics of protein folding intermediates and both partly folded and unfolded states.

Abstract: We have probed the nature of the individual kinetic steps in the folding of the Tetrahymena ribozyme by studying the folding kinetics of mutant ribozymes. After rapid formation of the first structural subdomain, a slow step precedes stable formation of the second subdomain. The two central helices of the second subdomain form in an interdependent manner, and this structural subunit therefore also constitutes a kinetic folding unit. The slow folding step includes formation of tertiary interactions in a triple-helical scaffold that orients the two subdomains of the RNA. The rapid and early formation of short range secondary structure, the hierarchical formation of kinetic folding units corresponding to structural subdomains, and the formation of tertiary interactions between subdomains late during the folding process appear to be common features of the folding mechanism for both RNA and proteins.

Abstract: To determine when secondary structure forms as two chains coalesce to form an alpha-helical dimer, the folding rates of variants of the coiled coil region of GCN4 were compared. Residues at non-perturbing positions along the exterior length of the helices were substituted one at a time with alanine and glycine to vary helix propensity and therefore dimer stability. For all variants, the bimolecular folding rate remains largely unchanged; the unfolding rate changes to largely account for the change in stability. Thus, contrary to most folding models, widespread helix is not yet formed at the rate-limiting step in the folding pathway. The high-energy transition state is a collapsed form that contains little if any secondary structure, as suggested for the globular protein cytochrome c (Sosnick et al., Proteins 24: 413-426, 1996).

Abstract: Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new acceptor sites was analysed by pulse-labelling under two sets of conditions that are known to reduce the rate of folding: (i) addition of dithiothreitol to the growth medium and (ii) introduction of deletions in the propeptide. A variety of effects was observed, depending on the position of the new acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation reactions can compete in vivo and that glycosylation does not necessarily precede folding. The approach described may be generally applicable for the analysis of protein folding in vivo.

Abstract: Little is known about the kinetic process in which stable intermediates in protein folding are formed: whether their folding is highly cooperative (two-state) or weakly cooperative is controversial. We report here that the folding and unfolding kinetics of the pH 4-stable intermediate (I1) of apomyoglobin are measurable, in the millisecond time range, when monitored by stopped-flow measurements of tryptophan fluorescence. The kinetics confirm that folding of I1 is strongly cooperative, but there is a burst phase (missing amplitude) in unfolding. If the faster steps in unfolding of I1 can be measured directly by suitable fast-reaction methods, they will give information about the nature of the folding transition.

Abstract: Understanding the folding mechanisms of large, highly structured RNAs is important for understanding how these molecules carry out their function. Although models for the three-dimensional architecture of several large RNAs have been constructed, the process by which these structures are formed is only now beginning to be explored. The kinetic folding pathway of the Tetrahymena ribozyme involves multiple intermediates and both Mg2+-dependent and Mg2+-independent steps. To determine whether this general mechanism is representative of folding of other large RNAs, a study of RNase P RNA folding was undertaken. We show, using a kinetic oligonucleotide hybridization assay, that there is at least one slow step on the folding pathway of RNase P RNA, resulting in conformational changes in the P7 helix region on the minute timescale. Although this folding event requires the presence of Mg2+, the slow step itself does not involve Mg2+ binding. The P7 and P2 helix regions exhibit distinctly different folding behavior and ion dependence, implying that RNase P folding is likely to be a complex process. Furthermore, there are distinct similarities in the folding of RNase P RNA from both Bacillus subtilis and Escherichia coli, indicating that the folding pathway may also be conserved along with the final structure. The slow folding kinetics, Mg2+-independence of the rate, and existence of intermediates are basic features of the folding mechanism of the Tetrahymena group I intron that are also found in RNase P RNA, suggesting these may be general features of the folding of large RNAs.

Abstract: Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable.

Abstract: A valuable approach to understanding the forces that maintain protein structure is to analyze the thermodynamic effects of mutations on protein folding. The folding process is most often described using an energetic model that assumes a two-state transition between the native and denatured states. However, some results obtained using this approach for mutants of the protein staphylococcal nuclease have contradicted expectations from our current understanding of protein energetics. The application of differential scanning calorimetry to a set of mutant nuclease proteins allowed us to measure directly the effects of mutations on the enthalpy and heat capacity changes of unfolding, as well as on the cooperativity. We found that most of these effects can be understood with a three-state model of folding including a distinct intermediate, but not with the two-state model. Use of a three-state instead of a two-state model leads to large differences in conclusions about the stability effects of some mutations, s...

Abstract: The Arc repressor is a small, homodimeric protein. Studies of mutant proteins show that the side chains that form the hydrophobic core are the most important determinants of structure. A variety of hydrogen bonds and salt bridges also contribute to stabilization of the native structure, but these can often be replaced by hydrophobic interactions. The transition state for folding/unfolding is dimeric and contains a large amount of buried hydrophobic surface, but the beta-sheet of native Arc is not formed. Moreover, relatively little side chain information appears to be used in the transition state, suggesting that tight packing of the hydrophobic core and optimization of hydrogen-bond geometry are events that occur later in folding.

Abstract: New classes of small proteins have recently beenfound that refold rapidly with two-state kinetics from a substantially unfolded conformation (“U”) and without the accumulation of a folding intermediate. Barnase, on the other hand, is representative of a class of proteins that display multistate kinetics and refold from a partly structured conformation, a folding intermediate (I). The accumulation of I on the folding pathway of barnase is highly dependent on the experimental conditions: a transition from multistate to two-state folding behavior can be induced simply by changing the reaction conditions away from physiological, i.e., elevated temperatures, high concentration of denaturant, or low pH. We argue that the change in folding behavior results from the denatured state changing under different conditions. The denatured state seems compact and partly structured at conditions that favor folding but is disorganized at denaturing conditions. At physiological pH and temperature, the denatured state (Dphy...

Abstract: Future research on protein folding must confront two serious dilemmas. (1) It may never be possible to observe at high resolution the very important structures that form in the first few milliseconds of the refolding reaction. (2) The energy functions used to predict structure from sequence will always be approximations of the true energy function. One strategy to resolve both dilemmas is to view protein folding from a different perspective, one that no longer emphasizes time and unique trajectories through conformation space. Instead, free energy replaces time as the reaction coordinate, and ensembles of equilibrium states of partially folded proteins are analyzed in place of trajectories of one protein chain through conformation space, either in vitro or in silico. Initial characterization of the folding of staphylococcal nuclease within this alternative conceptual framework has led to an equilibrium folding pathway with several surprising features. In addition to the finding of two bundles of four hydrophobic segments containing both native and non-native interactions, a gradient in relative stability of different substructures has been identified, with the most stable interactions located toward the amino terminus and the least stable toward the carboxy terminus. Hydrophobic bundles with up-down topology and stability gradients may be two examples of numerous tactics used by proteins to facilitate rapid folding and minimize aggregation. As NMR methods for structural analysis of partially folded proteins are refined, higher resolution descriptions of the structure and dynamics of the polypeptide chain outside the native state may provide many insights into the processes and energetics underlying the self-assembly of folded structure.