A kinetically significant intermediate in the folding of barnase

Abstract

O ne fundamental question about the pathway of protein folding is whether or not there are folding intermediates; that is, whether a pathway is two-state or multistate. The next question is whether intermediates are on-pathway, that is, on the route between the denatured state, D, and the native state, N. Unequivocal proof of mechanism by kinetics is often neither simple nor straightforward because much of kinetic evidence is by its very nature indirect and hence ambiguous. But the kineticist has two powerful tools for defining mechanisms. The first is the principle of microscopic reversibility. It is a necessary consequence of two-state kinetics that the ratio of forward and reverse rate constants is identical to the independently measured equilibrium constant. A corollary is that a two-state reaction must have the same transition state in its forward and reverse reactions. These tests apply both to true reversible first-order reactions and to pseudofirst order reactions that have highenergy intermediates. The second is direct observation of intermediates. In general, kinetics is at its most powerful in disproving particular mechanisms because it can show that only a restricted set of mechanisms is consistent with observed data. For example, the kineticist can formulate the simplest reaction scheme consistent with the kinetics. But, it is nearly always possible to fit to the data more complex mechanisms that have additional intermediates. Thus, reactions that are apparently two state always have the possibility of containing high-energy intermediates that are not amenable to direct detection and are kinetically silent. Conversely, it is possible to demonstrate unequivocally by using the consequences of microscopic reversibility that a reaction scheme has to have a minimum number of intermediates and hence eliminate all mechanisms with a smaller number of steps. It is more difficult to prove whether the intermediates are on-pathway or are side-reactions that are nonproductive.My laboratory has over the period of a decade developed the 110-residue protein barnase as a paradigm for analyzing the stability of proteins and the pathway of folding via an intermediate (1-3). We have demonstrated that an intermediate occurs during the folding of barnase by rigorously applying all three tests (1,4,5). Parallel studies with chymotrypsin inhibitor 2 (CI2) have shown that it folds cleanly by two-state kinetics (6), and those experiments act as controls for those on barnase. Despite the apparently incontrovertible evidence and other data that are inconsistent with a two-state folding pathway for barnase, Bai and coworkers claimed first (7)

Identification and characterization of protein folding intermediates

“…The first evidence for a folding intermediate came from a roll-over effect in the chevron plot [20]. More recently, analysis of pulse labeling hydrogen exchange protection factors has shown that the intermediate is an on-pathway species to the native state [54]. The structural features of the barnase folding intermediate has been addressed both experimentally (e.g.…”

Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

“…Although some small proteins are thought to fold by a 2-state mechanism in which the unfolded state transitions in a highly cooperative manner to the folded conformer [20] (Figure 1a), it is becoming increasingly clear that for many proteins, folding involves the formation of one or more transiently formed intermediates [1,[6][7][8][9][10]21] (Figure 1b). Sufficiently stable intermediates can be detected kinetically using stop-flow or continuous-flow techniques and proven to be onpathway by kinetic modeling [8,9,22] be populated to a level that is detectable at equilibrium by thermodynamic experiments, and in amenable cases be proven to be on-pathway by relaxation dispersion NMR, a technique which will be described in detail in later sections.…”

“…NOESY experiments exploit the phenomenon of cross-relaxation between hydrogen atoms that are close in space. Cross-peaks are generally visible between atoms that are separated by less than 6Å, and their intensity is proportional to 1/r 6 Chapter 2…”

“…Levinthal"s solution to the paradox: protein folding is not a random process, but instead proceeds by some directed mechanism [5]. Indeed, it appears that many of the proteins whose folding has been studied to date follow directed pathways involving the transient accumulation of specific intermediate states with defined structure [1,[6][7][8][9][10]. In principle, structural studies of folding intermediates would provide a wealth of information about the folding process [1,10] and potentially also shed light as to how proteins misfold [11].…”

Folding of the Pit1 homeodomain near the speed limit

“…All of the folding limbs for the fast phase converge at ∼3 × 10 5 s −1 . The slow phases exhibited rollover, characteristic of multistate kinetics (12)(13)(14).The dependence of the logarithms of the relaxation rate constants λ on [Urea] were fitted simultaneously assuming that each phase was two-state and each individual rate constant (k) had the standard linear dependence on [Urea], k ¼ k 0 expðm½urea ∕RTÞ, (Fig. 3).…”