Investigation of membrane protein structure using Fourier transform infrared spectroscopy.

Abstract

The structural analysis of proteins is proving to be increasingly popular among biologists attempting to understand the mechanism of action of important proteins such as enzymes, receptors, hormones, blood clotting factors and transport proteins. Each of the various techniques applied to structural studies (X-ray diffraction and circular dichroism, nuclear magnetic resonance, Raman, infrared and fluorescence spectroscopy) has provided valuable results. However, each technique also has associated problems. X-ray diffraction, to date the method of choice for structural analysis of soluble proteins, is limited in its application to the study of membrane proteins by the need for relatively large good quality crystals. At present only two membrane proteins have produced crystals which meet these requirements (Deisenhofer et al., 1985; Allen et al., 1987). Nuclear magnetic resonance spectroscopy is limited to small proteins owing to the line broadening effects associated with larger structures. Circular dichroism and Raman spectroscopy suffer from light scattering and noise problems, respectively, while fluorescence studies often require the use of perturbing probes. Infrared spectroscopy was limited for many years by the overlap of the major protein band (the amide I band) with a strong water absorption. However, the use of digital background subtraction routines and the development of Fourier transform infrared (FTIR) spectrometers has revolutionized the field of infrared spectroscopy. We now have a measurement technique which is rapid, highly accurate with respect to frequency calibration, provides excellent signal-to-noise ratio, is non-perturbing and can provide information concerning both lipid and protein components of biomembranes. The major band in the FTIR spectra of proteins is the amide I band of the protein. This absorption arises predominantly from the C=O stretch of the peptide bonds (Miyazawa, 1960) and as such is sensitive to the hydrogen bonding state of the protein. The different secondary structures present in a protein are each associated with a characteristic hydrogen bonding pattern, and so are each associated with a characteristic amide I frequency. The presence of a range of secondary structures in membrane proteins results in the production of multiple amide I absorptions. The halfwidth of each absorption is such that they cannot be resolved