title: Determining binding sites in protein-nucleic acid complexes by cross-saturation.
abstract: Cross-saturation experiments have been shown to give accurate information regarding the interacting surfaces in protein-protein and protein-RNA complexes. The rate of magnetization transfer depends on a number of factors including geometry, spin topology, and effective correlation times. To assess the influence of these variables on such experiments, and to determine the range of applicability of the technique, we have simulated the time-course of magnetization transfer across the interface in a variety of protein-nucleic acid complexes (434 Cro, SRY, MetJ and U1A), each having a different binding geometry. The simulations have been carried out primarily to provide information about the experimentally accessible targets for selective saturation, such as the anomeric protons and the imino protons of the nucleic acid. Saturation of either of these groups of signals leads to partial excitation throughout the nucleic acid molecule, and the resulting transfer of saturation to the labelled protein moiety can be readily detected by the reduction in intensity of particular peaks in the HSQC spectrum of the protein. The simulations show that information can be obtained about the residues in contact with the nucleic acid without any need for deuteration. Experimental cross-saturation data have been obtained from the Mbp1-DNA complex and interpreted in conjunction with the simulations to map out the binding surface in detail.
authors.affiliation: Division of Molecular Structure, National Institute for Medical Research, London, UK. alane@nimr.mrc.ac.uk
authors.forename: A N
authors.initials: AN
authors.lastname: Lane
authors.forename: G
authors.initials: G
authors.lastname: Kelly
authors.forename: A
authors.initials: A
authors.lastname: Ramos
authors.forename: T A
authors.initials: TA
authors.lastname: Frenkiel
mesh_terms: D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009687:Nuclear Proteins; D009696:Nucleic Acids / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D016601:RNA-Binding Proteins; D012097:Repressor Proteins / Q000737:chemistry; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C083209:MBP1 protein, S cerevisiae; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009696:Nucleic Acids; D011506:Proteins; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; C075339:U1A protein; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C060486:methionine repressor protein, Bacteria; C016649:phage repressor proteins; D012313:RNA; D004247:DNA