title: Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins.
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 α-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 using1H 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.
authors.affiliation: Department Chemie und Pharmazie, Ludwig-Maximilians-Universität München, 81377, Munich, Germany.|Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
authors.forename: Kristof
authors.initials: K
authors.lastname: Grohe
authors.affiliation: Department Chemie und Pharmazie, Ludwig-Maximilians-Universität München, 81377, Munich, Germany.|Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
authors.forename: Kumar Tekwani
authors.initials: KT
authors.lastname: Movellan
authors.affiliation: Department Chemie und Pharmazie, Ludwig-Maximilians-Universität München, 81377, Munich, Germany.|Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
authors.forename: Suresh Kumar
authors.initials: SK
authors.lastname: Vasa
authors.affiliation: Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Giller
authors.affiliation: Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Department Chemie und Pharmazie, Ludwig-Maximilians-Universität München, 81377, Munich, Germany. rasmus.linser@lmu.de.|Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany. rasmus.linser@lmu.de.
authors.forename: Rasmus
authors.identifier: http://orcid.org/0000-0001-8983-2935
authors.initials: R
authors.lastname: Linser
mesh_terms: D000577:Amides / Q000737:chemistry; D000818:Animals; D002645:Chickens; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006860:Hydrogen Bonding; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D013049:Spectrin / Q000737:chemistry*; D014867:Water / Q000737:chemistry; D018909:src Homology Domains
chemical_list: D000577:Amides; D014867:Water; D013049:Spectrin
keywords: H-bond determination; MAS; Protein structure; Proton detection; Solid-state NMR; Water accessibility