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Cell‐free expression and selective isotope labelling in protein NMR
Author(s) -
Staunton David,
Schlinkert Robin,
Zanetti Giulia,
Colebrook Simon A.,
Campbell Iain D.
Publication year - 2006
Publication title -
magnetic resonance in chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.483
H-Index - 72
eISSN - 1097-458X
pISSN - 0749-1581
DOI - 10.1002/mrc.1835
Subject(s) - chemistry , heteronuclear single quantum coherence spectroscopy , heteronuclear molecule , labelling , amide , isotope , residue (chemistry) , nuclear magnetic resonance spectroscopy , biochemistry , combinatorial chemistry , stereochemistry , physics , quantum mechanics
Isotope labelling is a very powerful tool in NMR studies of proteins and has been employed in various ways for over 40 years. 15 N and 13 C incorporation, using recombinant expression systems, is now commonplace because heteronuclear experiments assist with the fundamental problems of peak resolution and assignment. The use of selective labelling for peak assignment has been restricted by the scrambling of isotope label through metabolic pathways within the expression host organism. The availability of efficient cell‐free expression systems with low levels of metabolic conversion allow the increasing use of selective isotope labelling as a tool in protein NMR. We describe two examples, one where a selective labelling scheme can identify backbone amide peaks from unassigned 1 H 15 N HSQC and HNCO spectra of a 84 residue protein, and another where a specific backbone amide in a 198 residue construct of the ninth and tenth Type III repeats from human fibronectin can be labelled and rapidly identified using a simple HSQC experiment. Copyright © 2006 John Wiley & Sons, Ltd.