title: Strategies for protein NMR in Escherichia coli.
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-cutloop, resulting from uniform (15)N enrichment (with and without deuteration), selective (15)N-Leu enrichment, (13)C-methyl enrichment of isoleucine, leucine, valine, and alanine, fractional (13)C enrichment, and (19)F labeling. Most of the target proteins can be observed by (19)F labeling and (13)C 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.
authors.affiliation: Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Center for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences , Wuhan 430071, P. R. China.
authors.forename: Guohua
authors.initials: G
authors.lastname: Xu
authors.forename: Yansheng
authors.initials: Y
authors.lastname: Ye
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Liu
authors.forename: Shufen
authors.initials: S
authors.lastname: Cao
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Maili
authors.initials: M
authors.lastname: Liu
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Pielak
authors.forename: Conggang
authors.initials: C
authors.lastname: Li
mesh_terms: D002147:Calmodulin / Q000737:chemistry / Q000502:physiology; D002478:Cells, Cultured; D004926:Escherichia coli / Q000737:chemistry / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D025801:Ubiquitin / Q000737:chemistry / Q000502:physiology
chemical_list: D002147:Calmodulin; D029968:Escherichia coli Proteins; D025801:Ubiquitin