FTIR Analysis of Proteins and Protein-Membrane Interactions.

Fourier transform infrared (FTIR) spectroscopy has become one of the major techniques of structural characterization of proteins, peptides, and protein-membrane interactions. 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 intermolecular interactions. The 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 13C, 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, the structural features of membrane pore forming proteins and peptides, and much more. The purpose of this chapter 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, is also presented.