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.

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

“…The exact dependence between film swelling and the ionic strength of the buffer will depend, among other factors, on the nature of the lipids. For instance, a similar film swelling as obtained here for bR in purple membrane using 4 M NaCl was obtained for a membrane protein reconstituted in polar E. coli lipids using just 0.1 M NaCl 62 . If the sample occupies less than 5% of the probed volume consider re-doing the hydration step using a buffer of higher ionic strength.…”

Ion–protein interactions of a potassium ion channel studied by attenuated total reflection Fourier transform infrared spectroscopy

“…Perfusion-induced difference attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy was first applied to study the structural changes of an acetylcholine receptor upon ligand binding (Baenziger et al 1992). The method has been well reviewed by several groups (Lorenz-Fonfria et al 2012;Nyquist et al 2001;Rich and Iwaki 2007). Here it can be confirmed that the method is also applicable to a K + ion channel and is a potential tool for studying ion selectivity among alkali metal cations.…”

Structural properties determining low K+ affinity of the selectivity filter in the TWIK1 K+ channel

“…Then, the buffer was changed; the sample was washed, and the spectra were measured again. After the absorption spectra, measurements before and after buffer exchanges were completed four times; the difference spectra were calculated by subtraction, correction of membrane swelling changes, and nonspecific distortions associated with ion exchange according to Lórenz-Fonfría et al (44). The absolute absorption spectra of TWIK1 samples immersed in buffer solution (Fig.…”

“…The absolute absorption spectra of TWIK1 samples immersed in buffer solution (Fig. 1F) were obtained by subtracting the buffer contribution according to Lórenz-Fonfría et al (44). tion energy (52,53) and the RESP scheme (54) to describe the partial charges of the QM subsystem.…”