title: Studying substrate binding to reconstituted secondary transporters by attenuated total reflection infrared difference spectroscopy.
abstract: The determination of protein conformational changes induced by the interaction of substrates with secondary transporters is an important step toward the elucidation of their transport mechanism. Since conformational changes in a protein alter its vibrational patterns, they can be detected with high sensitivity by infrared difference (IR(diff)) spectroscopy without the need for external probes. We describe a general procedure to obtain substrate-induced IR(diff) spectra by alternating perfusion of buffers over an attenuated total reflection (ATR) crystal containing an adhered film of a membrane protein reconstituted in lipids. As an example, we provide specific protocols to obtain melibiose and Na(+)-induced ATR-IR(diff) spectra of reconstituted melibiose permease, a sodium/melibiose co-transporter from E. coli. The presented methodology is applicable in principle to any membrane protein, provided that it can be purified and reconstituted in functional form, and appropriate substrates are available.
authors.affiliation: Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, Barcelona, Spain. victor.lorenz@gmail.com
authors.forename: Víctor A
authors.initials: VA
authors.lastname: Lórenz-Fonfría
authors.forename: Xavier
authors.initials: X
authors.lastname: León
authors.forename: Esteve
authors.initials: E
authors.lastname: Padrós
mesh_terms: D000042:Absorption; D002021:Buffers; D004926:Escherichia coli / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism; D013055:Spectrophotometry, Infrared / Q000379:methods*; D013379:Substrate Specificity; D027981:Symporters / Q000378:metabolism*
chemical_list: D002021:Buffers; D008055:Lipids; D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D012964:Sodium