Kinetic Evidence for Folding and Unfolding Intermediates in Staphylococcal Nuclease

Abstract

The complex kinetic behavior commonly observed in protein folding studies suggests that a heterogeneous population of molecules exists in solution and that a number of discrete steps are involved in the conversion of unfolded molecules to the fully native form. A central issue in protein folding is whether any of these kinetic events represent conformational steps important for efficient folding rather than side reactions caused by slow steps such as proline isomerization or misfolding of the polypeptide chain. In order to address this question, we used stopped-flow fluorescence techniques to characterize the kinetic mechanism of folding and unfolding for a Pro- variant of SNase in which all six proline residues were replaced by glycines or alanines. Compared to the wild-type protein, which exhibits a series of proline-dependent slow folding phases, the folding kinetics of Pro- SNase were much simpler, which made quantitative kinetic analysis possible. Despite the absence of prolines or other complicating factors, the folding kinetics still contain several phases and exhibit a complex denaturant dependence. The GuHCl dependence of the major observable folding phase and a distinct lag in the appearance of the native state provide clear evidence for an early folding intermediate. The fluorescence of Trp140 in the alpha-helical domain is insensitive to the formation of this early intermediate, which is consistent with a partially folded state with a stable beta-domain and a largely disordered alpha-helical region. A second intermediate is required to model the kinetics of unfolding for the Pro- variant, which shows evidence for a denaturant-induced change in the rate-limiting unfolding step. With the inclusion of these two intermediates, we are able to completely model the major phase(s) in both folding and unfolding across a wide range of denaturant concentrations using a sequential four-state folding mechanism. In order to model the minor slow phase observed for the Pro- mutant, a six-state scheme containing a parallel pathway originating from a distinct unfolded state was required. The properties of this alternate unfolded conformation are consistent with those expected due to the presence of a non-prolyl cis peptide bond. To test the kinetic model, we used simulations based on the six-state scheme and were able to completely reproduce the folding kinetics for Pro- SNase across a range of denaturant concentrations.

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

“…The lag phase rate increases from 45 s Ϫ1 at pH 5.3 to 200 s Ϫ1 at pH 8.5. These results agree closely with published data as noted in Table 1 (12,26,27).…”

“…The plot of residuals shows the reality of the lag phase, which is confirmed by similar results at other conditions and in other laboratories (see Table 1). (27). ‡ Unfold at pH 2.0, measure folding at pH 6.0, 20°C, 0.1 M sodium cacodylate, 2 mM EGTA (12).…”

Correlation between Native-State Hydrogen Exchange and Cooperative Residue Fluctuations from a Simple Model

“…Unambiguous identification of a folding pathway is possible only by direct examination of the intermediates formed during unfolding or folding processes, either using time-resolved techniques (60) or perhaps from dynamic simulations. For SNase, recent examination of a folding and unfolding mechanism by stopped-flow fluorescence techniques revealed the occurrence of a partially folded state with a stable -domain and a largely disordered R-helical region (61). Interestingly, the three highest peaks obtained here coincide with the strands 2, 3, and 5, which could lend support to a relationship between early formation and high stability.…”

Interactions between subdomains in the partially folded state of staphylococcal nuclease

“…These isolated stable subdomains clearly re£ect the modular nature of SNase. In a previous kinetic folding study of SNase, the location of the usual structure reporters in SNase, such as the Khelices or the single tryptophan at the position 140 being outside the L-subdomain prevents direct characterization of the kinetic phase involving the rapid formation of L-barrel [11]. The V66W110 and G88W110 with a single £uorescence reporter in the L-barrel are expected to be suitable for kinetic folding studies of the isolated L-subdomain.…”

“…Pulsed hydrogen exchange and NMR methods showed an early protection of hydrogen in part of the L-barrel indicating that the L-barrel is the ¢rst formed structure unit in SNase [10]. Kinetic folding and unfolding studies of a proline-free SNase also revealed a folding intermediate which is consistent with a partially folded state having a stable L-barrel and a largely disordered K-helical region [11]. These suggest that the structure of SNase can be split into two subdomains, with predominantly K-helix and L-sheet, respectively.…”