title: Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study.
abstract: Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial15N-,13Cα-, and13C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves15N, 1-13C, 2-13C, fully15N/13C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide1H and15N resonances need to be obtained, even in cases where sensitivity is the limiting factor.
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt, 60438, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Hein
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt, 60438, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Löhr
authors.affiliation: Merck KGaA, Discovery Pharmacology, Global Research and Development, Darmstadt, 64293, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Schwarz
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt, 60438, Germany.
authors.forename: Volker
authors.identifier: http://orcid.org/0000-0001-5720-212X
authors.initials: V
authors.lastname: Dötsch
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D002474:Cell-Free System; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D000081406:Peptidyl-Prolyl Isomerase F; D008024:Ligands; D009587:Nitrogen Isotopes; D021983:Cyclophilins
keywords: BEST-TROSY; cell-free expression; cyclophilin D; ligand screening; selective isotope labeling