Strategies for Protein NMR in Escherichia coli.
 

Strategies for Protein NMR in Escherichia coli.

Related Articles Strategies for Protein NMR in Escherichia coli.

Biochemistry. 2014 Mar 5;

Authors: Xu G, Ye Y, Liu X, Cao S, Wu Q, Cheng K, Liu M, Pielak GJ, Li C

Abstract
In-cell NMR spectroscopy provides insight into protein conformation, dynamics and function at atomic resolution in living cells. Systematic evaluation of isotopic labeling strategies is necessary to observe the target protein in the sea of other molecules in the cell. Here, we investigate the detectability, sensitivity and resolution of in-cell NMR spectra of the globular proteins, GB1, ubiquitin, calmodulin and bcl-xl, resulting from uniform 15N enrichment (with and without deuteration), selective 15N-leu enrichment, 13C methyl enrichment of isoleucine, leucine, valine and alanine, fractional 13C enrichment and 19F labeling. Most of the target proteins can be observed by 19F labeling and 13C enrichment with direct detection because selectively labeling suppresses background signals, and deuteration improves in-cell spectra. Our results demonstrate the detectability of proteins is determined by weak interactions with intercellular components and that choosing appropriate labeling strategies is critical for the success in in-cell protein NMR studies.


PMID: 24597855 [PubMed - as supplied by publisher]



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