title: Protein (19)F NMR in Escherichia coli.
abstract: Although overexpression and (15)N enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, (15)N enrichment alone is insufficient for detecting most globular proteins. Here, we explain this dichotomy and overcome the problem while extending the capability of in-cell NMR by using (19)F-labeled proteins. Resonances from small (approximately 10 kDa) globular proteins containing the amino acid analogue 3-fluoro-tyrosine can be observed in cells, but for larger proteins the (19)F resonances are broadened beyond detection. Incorporating the amino acid analogue trifluoromethyl-L-phenylalanine allows larger proteins (up to 100 kDa) to be observed in cells. We also show that site-specific structural and dynamic information about both globular and disordered proteins can be obtained inside cells by using (19)F NMR.
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Conggang
authors.initials: C
authors.lastname: Li
authors.forename: Gui-Fang
authors.initials: GF
authors.lastname: Wang
authors.forename: Yaqiang
authors.initials: Y
authors.lastname: Wang
authors.forename: Rachel
authors.initials: R
authors.lastname: Creager-Allen
authors.forename: Evan A
authors.initials: EA
authors.lastname: Lutz
authors.forename: Heidi
authors.initials: H
authors.lastname: Scronce
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Slade
authors.forename: Rebecca A S
authors.initials: RA
authors.lastname: Ruf
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Pielak
mesh_terms: D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D054879:Halogenation; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011480:Protease Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; D002918:Chymotrypsin