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Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Published on: November 29, 2013
Hydrogen-exchange stability analysis of Bergerac-Src homology 3 variants allows the characterization of a folding
Ana-Rosa Viguera1, Luis Serrano
1Unidad de Biofisica-Consejo Superior de Investigaciones Cientificas-Universidad del Pais Vasco, P.O. Box 644, 48080 Bilbao, Spain. gbbviria@lg.ehu.es
Abstract:
Amide hydrogendeuterium exchange rates have been determined for two mutants of alpha-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 high-energy barrier crossings. Comparison of DeltaG(ex) and m(ex) 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 beta-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.
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