Structural Characterization of Membrane Proteins and Peptides by FTIR and ATR-FTIR Spectroscopy

Abstract

Fourier transform infrared (FTIR) spectroscopy is widely used in structural characterization of proteins or peptides. While the method does not have the capability of providing the precise, atomic-resolution molecular structure, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or upon intermolecular interactions. Sensitivity of vibrational frequencies to atomic masses has led to development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as (13)C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein. The same isotope effect is used to identify site-specific conformational changes in proteins by site-directed or segmental isotope labeling. Negligible light scattering in the infrared region provides an opportunity to study intermolecular interactions between large protein complexes, interactions of proteins and peptides with lipid vesicles, or protein-nucleic acid interactions without light scattering problems often encountered in ultraviolet spectroscopy. Attenuated total reflection FTIR (ATR-FTIR) is a surface-sensitive version of infrared spectroscopy that has proved useful in studying membrane proteins and lipids, protein-membrane interactions, mechanisms of interfacial enzymes, and molecular architecture of membrane pore or channel forming proteins and peptides. The purpose of this article was to provide a practical guide to analyze protein structure and protein-membrane interactions by FTIR and ATR-FTIR techniques, including procedures of sample preparation, measurements, and data analysis. Basic background information on FTIR spectroscopy, as well as some relatively new developments in structural and functional characterization of proteins and peptides in lipid membranes, are also presented.