title: Transferred NOESY NMR studies of biotin mimetic peptide (FSHPQNT) bound to streptavidin: a structural model for studies of peptide-protein interactions.
abstract: Protein-protein interactions control signaling, specific adhesion, and many other biological functions. The three-dimensional structures of the interfaces and bound ligand can be approached with transferred nuclear Overhauser effect spectroscopy NMR, which can be applied to much larger proteins than conventional NMR and requires less concentrated protein. However, it is not clear how accurately the structures of protein-bound peptides can be determined by transferred nuclear Overhauser effect spectroscopy. We studied the structure of a biotin mimetic peptide (FSHPQNT) bound to streptavidin, because the X-ray structure of the complex is available to 1.74 Å resolution, and we found that conditions could be adjusted so that the off-rates were fast enough for transferred nuclear Overhauser effect spectroscopy NMR. The off-rate was determined with (19)F NMR, using a para-fluoro-phenylalanine analog of the peptide. A new criterion for a lower limit on kinetic off-rate was found, which allowed accurate structure determination at a slower off-rate. Non-specific binding of the peptide to streptavidin was not significant, because biotin blocked the peptide transferred nuclear Overhauser effect spectroscopy. Protein mediation for the long-range peptide transferred nuclear Overhauser effect spectroscopy cross-peaks was corrected by a transferred nuclear Overhauser effect spectroscopy/ROESY averaging procedure. The protein-bound structure of the peptide was determined by transferred nuclear Overhauser effect spectroscopy constrained and simulated annealing. The structure deduced from the NMR was close to the X-ray structure.
authors.affiliation: Department of Medicine, Texas A&M University Health Science Center, College of Medicine and Scott & White Memorial Hospital, Temple, TX 76508, USA. dgizachew@tamu.edu
authors.forename: Dawit
authors.initials: D
authors.lastname: Gizachew
authors.forename: Edward
authors.initials: E
authors.lastname: Dratz
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008958:Models, Molecular; D018716:Molecular Mimicry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D019809:Streptavidin / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D001710:Biotin; D019809:Streptavidin