Nonlocal Interactions Stabilize Long Range Loops in the Initial Folding Intermediates of Reduced Bovine Pancreatic Trypsin Inhibitor

Ittah, Varda; Haas, Elisha

doi:10.1021/bi00013a042

Cited by 89 publications

(83 citation statements)

References 79 publications

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“…2) (Ittah and Haas 1995). We hypothesized that the earliest steps in the folding transition are the closure of few long loop (25-35 residues) segments by NLIs between clusters with mostly non-polar residues at their ends (Ittah and Haas 1995;Haas 2005).…”

Section: The Loop Hypothesismentioning

confidence: 99%

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

et al. 2013

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The extremely fast and efficient folding transition (in seconds) of globular proteins led to the search for some unifying principles embedded in the physics of the folding polypeptides. Most of the proposed mechanisms highlight the role of local interactions that stabilize secondary structure elements or a folding nucleus as the starting point of the folding pathways, i.e., a "bottom-up" mechanism. Nonlocal interactions were assumed either to stabilize the nucleus or lead to the later steps of coalescence of the secondary structure elements. An alternative mechanism was proposed, an "up-down" mechanism in which it was assumed that folding starts with the formation of very few non-local interactions which form closed long loops at the initiation of folding. The possible biological advantage of this mechanism, the "loop hypothesis", is that the hydrophobic collapse is associated with ordered compactization which reduces the chance for degradation and misfolding. In the present review the experiments, simulations and theoretical consideration that either directly or indirectly support this mechanism are summarized. It is argued that experiments monitoring the time-dependent development of the formation of specifically targeted early-formed sub-domain structural elements, either long loops or secondary structure elements, are necessary. This can be achieved by the timeresolved FRET-based "double kinetics" method in combination with mutational studies. Yet, attempts to improve the time resolution of the folding initiation should be extended down to the sub-microsecond time regime in order to design experiments that would resolve the classes of proteins which first fold by local or non-local interactions.

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Section: The Loop Hypothesismentioning

confidence: 99%

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

et al. 2013

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“…In this respect, it will be instructive to investigate how our predictions might be modulated by the application of other tractable explicitchain models that incorporate nonnative interactions (40,60), interaction heterogeneity (12,61,62), and/or side-chain effects (13) and ascertain whether a similar trend exists in all-atom simulations (63). Experiments revealed that some proteins do fold by first forming nonlocal contacts (64,65). It will be extremely illuminating if differences in contact pattern between TPs and the non-TP population in the unfolded basin can be quantified by future single-molecule experiments.…”

Section: Initial Stages Of Transition Paths Have Statistically Atypicmentioning

confidence: 99%

Transition paths, diffusive processes, and preequilibria of protein folding

Zhang

Chan

2012

Proc. Natl. Acad. Sci. U.S.A.

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Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties. P rotein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1,2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7)(8)(9)(10)(11)(12)(13)(14)(15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16-25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the ...

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“…A quite different view of the dimensions of reduced BPTI has emerged from studies in which energy transfer measurements were used to analyze the distributions of distances between fluorescent probes attached to different sites along the polypeptide~Amir & Haas, 1988;Amir et al, 1992;Ittah & Haas, 1995!. The results of these studies indicate the presence of distinct subpopulations of molecules with different distributions of intramolecular distances. The average distances calculated for the more compact subpopulation are nearly as small as those predicted for the native protein.…”

Section: The Nature and Role Of Nonrandom Structure In Fully Reduced mentioning

confidence: 99%

Early events in the disulfide‐coupled folding of BPTI

Bułaj

Goldenberg

1999

Protein Science

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Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor~BPTI! have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein~between Cys14 and 38!, but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the @14-38# intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfideformation steps were carried out. The results of these studies indicate that the formation of @14-38# from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 8C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming @14-38# or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of @14-38# did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.Keywords: @14-38# intermediate; bovine pancreatic trypsin inhibitor; disulfide bonds; protein folding; unfolded proteins For more than two decades, bovine pancreatic trypsin inhibitor BPTI! has served as an important model for both theoretical and experimental studies of protein folding~Levitt & Warshel, 1975;Creighton, 1978;Goldenberg et al., 1989;Weissman & Kim, 1991;Goldenberg, 1992;Daggett & Levitt, 1993;Darby et al., 1995!. Pioneering work by Creighton~1978! demonstrated that the folded structure of BPTI depends on its three disulfide bonds and that these disulfides could be used as experimental probes of conformation during oxidative refolding of the reduced protein. These experiments revealed that only a fraction of the possible disulfidebonded intermediates accumulate to significant levels during refolding, thus providing some of the first experimental evidence to support the view that proteins fold via specific pathways. In subsequent studies, a variety of techniques, including NMR spectroscopy and mutational analysis, have been used to analyze the conformations of the intermediates and the interactions that stab...

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Nonlocal Interactions Stabilize Long Range Loops in the Initial Folding Intermediates of Reduced Bovine Pancreatic Trypsin Inhibitor

Cited by 89 publications

References 79 publications

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

Transition paths, diffusive processes, and preequilibria of protein folding

Early events in the disulfide‐coupled folding of BPTI

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