Hydrogen-exchange stability analysis of Bergerac -Src homology 3 variants allows the characterization of a folding intermediate in equilibrium

Abstract

Amide hydrogen͞deuterium exchange rates have been determined for two mutants of ␣-spectrin Src homology 3 domain (WT), containing an elongated stable (SHH) and unstable (SHA) distal loop. SHA, similarly to WT, follows a two-state transition, whereas SHH apparently folds via a three-state mechanism. Native-state amide hydrogen exchange is effective in ascribing energetic readjustments observed in kinetic experiments to species stabilized within the denatured base and distinguishing those from highenergy barrier crossings. Comparison of ⌬G ex and mex parameters for amide protons of these mutants demonstrates the existence of an intermediate and allows the identification of protons protected in this state. The consolidation of a form containing a prefolded long ␤-hairpin induces the switch to a three-state mechanism in an otherwise two-state folder. It can be inferred that the unbalanced high stability of individual elements of secondary structure in a polypeptide could ultimately complicate its folding mechanism.kinetics ͉ ␤-hairpin T he Src homology 3 (SH3) domain from ␣-spectrin is a wellcharacterized model system for protein folding and stability (1). It was shown early that this domain folds by a two-state transition (2) and that its isolated secondary structure elements are quite unstructured (3). We have investigated (4) the consequences of fusing the SH3 domain with a short sequence KITVNGKTYE (BHH) that tends to fold as a stable ␤-hairpin (5). In the SHHBergerac mutant (SHH for short) the two-residue distal ␤-turn (N47-D48) of ␣-spectrin SH3 has been substituted by BHH. SHH is more stable and folds significantly faster than the short WT ␣-spectrin SH3 at moderate urea concentrations, but unlike WT, its folding rate levels off under strong folding conditions (Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org), thus deviating from the simplest two-state folding mechanism. Mutation of two residues in the inserted BHH region of SHH (Ile-2Ј and Val-4Ј to Ala, SHA-Bergerac) destabilizes the protein and switches it back to a two-state folding process (Fig. 6). When fused to WT, the inserted BHH and BHH I2ЈA V4ЈA sequences adopt a fully folded ␤-hairpin structure protruding from the protein (4), but the corresponding isolated peptides are, respectively, Ϸ30% folded (BHH) and fully unfolded (BHH I2ЈA V4ЈA).Understanding the reasons behind rate constant deviations could be important in improving the methods available for in vitro protein stabilization, as well as in revealing limiting steps related to switches from two to higher order folding mechanisms. Deviations in chevron plots do not have an unequivocal interpretation. Rate constants reflect free energy differences from reactants to the highest energy level in the reaction coordinate or transition state ( ‡). The deviations often occurring at lower denaturant concentrations in refolding experiments are usually assigned to changes within the denatured ensemble, i.e., a new energy level or intermediate state (I) is stabilized...

Simulation and Experiment Conspire to Reveal Cryptic Intermediates and a Slide from the Nucleation-condensation to Framework Mechanism of Folding

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Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

“…Eventually, at high enough concentrations of denaturant, all the residues in the protein will exchange via a global unfolding mechanism, and thus all will show the same denaturantdependence of exchange. [31][32][33][34] Accordingly, we performed NHX measurements on CTPR2 and CTPR3. Because p u depends on the concentration of denaturant, Eq.…”

Engineering Stabilising β-Sheet Interactions into a Conformationally Flexible Region of the Folding Transition State of Ubiquitin

“…Two examples of the introduction of a b-hairpin peptide extension, derived from a sequence that folds in isolation, have been shown to extend the folding nucleus and accelerate folding by templating the formation of key native-like contacts. [16][17][18][19] However, the consequence of engineering stable elements of secondary structure as folding nucleation sites reveals deviations from the two-state model with roll-over effects in the refolding kinetic data at low concentrations of denaturant consistent with the population of a collapsed compact state involving non-native interactions.…”