Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins

Grohe, Kristof; Movellan, Kumar Tekwani; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan und Linser, Rasmus (2017): Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins. In: Journal of Biomolecular Nmr, Bd. 68, Nr. 1: S. 7-17

Abstract

We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken alpha-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using H-1 detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Chemie und Pharmazie > Department Chemie
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie
• ISSN: 0925-2738
• Sprache: Englisch