Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study.
Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study.
Biopolymers. 2016 Dec 30;:
Authors: Hein C, Löhr F, Schwarz D, Dötsch V
Abstract
Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial (15) N-, (13) C(?) -, and (13) C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves (15) N, 1-(13) C, 2-(13) C, fully (15) N/(13) C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide (1) H and (15) N resonances need to be obtained, even in cases where sensitivity is the limiting factor. This article is protected by copyright. All rights reserved.
PMID: 28035667 [PubMed - as supplied by publisher]
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