Strategies for Protein NMR in Escherichia coli

详细信息    查看全文

• 作者：Guohua Xu ; Yansheng Ye ; Xiaoli Liu ; Shufen Cao ; Qiong Wu ; Kai Cheng ; Maili Liu ; Gary J. Pielak ; Conggang Li
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：April 1, 2014
• 年：2014
• 卷：53
• 期：12
• 页码：1971-1981
• 全文大小：735K
• 年卷期：v.53,no.12(April 1, 2014)
• ISSN：1520-4995

文摘

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-cutloop, 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 because deuteration improves in-cell spectra. Our results demonstrate that the detectability of proteins is determined by weak interactions with intercellular components and that choosing appropriate labeling strategies is critical for the success of in-cell protein NMR studies.