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

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

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NMR processing:

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NMR assignment:

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Side-chains:

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Structure from NMR restraints:

Ab initio:

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Rosetta-NMR (Robetta)

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GeNMR

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Torsion angles from chemical shifts:

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Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

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Proshift

PPM

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Protein solubility:

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04-10-2017, 12:51 AM

nmrlearner

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

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 hydrogenâ??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 Î±-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 1.3Â*mm rotor. Tracking of the HN peak intensities for 2Â*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 1H 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.

Source: Journal of Biomolecular NMR

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