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A cceleration of protein backbone NMR assignment by combinatorial labeling: A pplication to a small molecule binding study
Author(s) -
Hein Christopher,
Löhr Frank,
Schwarz Daniel,
Dötsch Volker
Publication year - 2017
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.23013
Subject(s) - chemistry , amino acid , amide , small molecule , molecule , limiting , stereochemistry , side chain , combinatorial chemistry , biochemistry , organic chemistry , mechanical engineering , engineering , polymer
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 α ‐, 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.