title: The nature of ligand-induced conformational change in transferrin in solution. An investigation using X-ray scattering, XAFS and site-directed mutants.
abstract: Ligand-induced conformational change in transferrins has been studied by site-directed mutagenesis of human serum half molecule (N-lobe), X-ray absorption fine structure (XAFS) spectroscopy and X-ray solution scattering. Use of recent advances in data analysis has been made for extracting model-independent molecular shapes from X-ray solution scattering data for the intact, the half molecule and its mutants. Clear evidence is provided that the transferrin molecule (intact as well as N-lobe), in its apo and holo forms, exists for the majority of the time in well-defined specific conformations representing the "fully opened" and "closed" states of the molecule, respectively. Evidence is also provided for the existence of an additional conformation, referred to here as the "intermediate" conformation for simplicity, which is trapped in the case of some of the mutants in the iron-bound form. We suggest that domain closure in the transferrin molecule is a two-step process, with the intermediate conformation representing the first stage of domain closure (approximately 20 degrees hinge-twist of domain II). Our data are not inconsistent with the ligand-free molecule sampling the closed states occasionally (< or = 10%) but are not in support of a continuous conformational search between the fully opened and closed states in the absence of iron.
authors.affiliation: CCLRC Daresbury Laboratory, Warrington, Cheshire, UK.
authors.forename: J G
authors.initials: JG
authors.lastname: Grossmann
authors.forename: J B
authors.initials: JB
authors.lastname: Crawley
authors.forename: R W
authors.initials: RW
authors.lastname: Strange
authors.forename: K J
authors.initials: KJ
authors.lastname: Patel
authors.forename: L M
authors.initials: LM
authors.lastname: Murphy
authors.forename: M
authors.initials: M
authors.lastname: Neu
authors.forename: R W
authors.initials: RW
authors.lastname: Evans
authors.forename: S S
authors.initials: SS
authors.lastname: Hasnain
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007501:Iron / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012542:Scattering, Radiation; D012996:Solutions; D013057:Spectrum Analysis; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics; D014965:X-Rays
chemical_list: D008024:Ligands; D012996:Solutions; D014168:Transferrin; D007501:Iron