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 language | English |
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Pages (from-to) | 107-126 |
Journal | Methods in Molecular Biology |
Volume | 914 |
DOIs | |
Publication status | Published - 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