Studying substrate binding to reconstituted secondary transporters by attenuated total reflection infrared difference spectroscopy

Víctor A. Lórenz-Fonfría, Xavier León, Esteve Padrós

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8 Citations (Scopus)

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 (IRdiff) spectroscopy without the need for external probes. We describe a general procedure to obtain substrate-induced IRdiffspectra 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-IRdiffspectra 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. © 2012 Springer Science+Business Media, LLC.
Original languageEnglish
Pages (from-to)107-126
JournalMethods in Molecular Biology
Volume914
DOIs
Publication statusPublished - 1 Jan 2012

Keywords

  • Attenuated total reflection (ATR)
  • Difference spectroscopy
  • Fourier transform infrared spectroscopy (FT-IR)
  • Ligand binding
  • Melibiose permease (MelB)
  • Membrane proteins
  • Secondary transporters
  • Substrate binding

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