title: Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.
abstract: Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.
authors.affiliation: NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Lindsay A
authors.initials: LA
authors.lastname: Baker
authors.forename: Mark
authors.initials: M
authors.lastname: Daniëls
authors.forename: Elwin A W
authors.initials: EA
authors.lastname: van der Cruijsen
authors.forename: Gert E
authors.initials: GE
authors.lastname: Folkers
authors.forename: Marc
authors.initials: M
authors.lastname: Baldus
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D015221:Potassium Channels / Q000737:chemistry*; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D048170:Streptomyces lividans / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D015221:Potassium Channels; D011510:Proteolipids; D011994:Recombinant Proteins; C412443:YIDC protein, E coli; C107790:prokaryotic potassium channel; C020485:proteoliposomes; D012293:Rifampin