title: Utilization of methyl proton resonances in cross-saturation measurement for determining the interfaces of large protein-protein complexes.
abstract: Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220-223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of (1)H(2)O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach.
authors.affiliation: Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Aomi 2-41-6, Koto-ku, Tokyo, 135-0064, Japan. hid@jbirc.aist.go.jp
authors.forename: Hideo
authors.initials: H
authors.lastname: Takahashi
authors.forename: Mayumi
authors.initials: M
authors.lastname: Miyazawa
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ina
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Fukunishi
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Mizukoshi
authors.forename: Haruki
authors.initials: H
authors.lastname: Nakamura
authors.forename: Ichio
authors.initials: I
authors.lastname: Shimada
mesh_terms: D003198:Computer Simulation; D030562:Databases, Protein; D041961:Deuterium Exchange Measurement; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D046912:Multiprotein Complexes; D011522:Protons; D013205:Staphylococcal Protein A