当前位置： SCI文献检索 > JOURNAL OF BIOMOLECULAR NMR期刊下所有文献 > Alpha protons as NMR probes in deuterated proteins.

Alpha protons as NMR probes in deuterated proteins.

Abstract：

:We describe a new labeling method that allows for full protonation at the backbone Hα position, maintaining protein side chains with a high level of deuteration. We refer to the method as alpha proton exchange by transamination (α-PET) since it relies on transaminase activity demonstrated here using Escherichia coli expression. We show that α-PET labeling is particularly useful in improving structural characterization of solid proteins by introduction of an additional proton reporter, while eliminating many strong dipolar couplings. The approach benefits from the high sensitivity associated with 1.3 mm samples, more abundant information including Hα resonances, and the narrow proton linewidths encountered for highly deuterated proteins. The labeling strategy solves amide proton exchange problems commonly encountered for membrane proteins when using perdeuteration and backexchange protocols, allowing access to alpha and all amide protons including those in exchange-protected regions. The incorporation of Hα protons provides new insights, as the close Hα-Hα and Hα-HN contacts present in β-sheets become accessible, improving the chance to determine the protein structure as compared with HN-HN contacts alone. Protonation of the Hα position higher than 90% is achieved for Ile, Leu, Phe, Tyr, Met, Val, Ala, Gln, Asn, Thr, Ser, Glu, Asp even though LAAO is only active at this degree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbon is labeled preferentially with a single deuteron, allowing stereospecific assignment of glycine alpha protons. In solution, we show that the high deuteration level dramatically reduces R2 relaxation rates, which is beneficial for the study of large proteins and protein dynamics. We demonstrate the method using two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing the applicability of the method to study membrane proteins.

journal_name

J Biomol NMR

journal_title

Journal of biomolecular NMR

authors

Movellan KT,Najbauer EE,Pratihar S,Salvi M,Giller K,Becker S,Andreas LB

doi

10.1007/s10858-019-00230-y

subject

Has Abstract

pub_date

2019-02-01 00:00:00

pages

81-91

issue

1-2

eissn

0925-2738

issn

1573-5001

pii

10.1007/s10858-019-00230-y

journal_volume

pub_type

杂志文章

在线工具

Alpha protons as NMR probes in deuterated proteins.