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Infrared (IR) spectroscopy is a useful technique in the study of protein conformation and dynamics. The possibilities of the technique become apparent specially when applied to large proteins in turbid suspensions, as is often the case with membrane proteins. The present review describes the applications of IR spectroscopy to the study of membrane proteins, with an emphasis on recent work and on spectra recorded in the transmission mode, rather than using reflectance techniques. Data treatment procedures are discussed, including band analysis and difference spectroscopy methods. A technique for the analysis of protein secondary and tertiary structures that combines band analysis by curve-fitting of original spectra with protein thermal denaturation is described in detail. The assignment of IR protein bands in H2O and in D2O, one of the more difficult points in protein IR spectroscopy, is also reviewed, including some cases of unclear assignments such as loops, beta-hairpins, or 3(10)-helices. The review includes monographic studies of some membrane proteins whose structure and function have been analysed in detail by IR spectroscopy. Special emphasis has been made on the role of subunit III in cytochrome c oxidase structure, and the proton pathways across this molecule, on the topology and functional cycle of sarcoplasmic reticulum Ca(2+)-ATPase, and on the role of lipids in determining the structure of the nicotinic acetylcholine receptor. In addition, shorter descriptions of retinal proteins and references to other membrane proteins that have been studied less extensively are also included.

The effects of ceramides of natural origin on the gel-fluid and lamellar-inverted hexagonal phase transitions of phospholipids (mainly dielaidoylphosphatidylethanolamine) have been studied by differential scanning calorimetry, with additional support from infrared and 31P nuclear magnetic resonance (NMR) spectroscopy. In the lamellar phase, ceramides do not mix ideally with phospholipids, giving rise to the coexistence of domains that undergo the gel-fluid transition at different temperatures. The combination of differential scanning calorimetry and infrared spectroscopy, together with the use of deuterated lipids, allows the demonstration of independent melting temperatures for phospholipid and ceramide in the mixtures. In the lamellar-hexagonal phase transitions, ceramides (up to 15 mol %) decrease the transition temperature, without significantly modifying the transition enthalpy, thus facilitating the inverted hexagonal phase formation. 31P-NMR indicates the coexistence, within a certain range of temperatures, of lamellar and hexagonal phases, or hexagonal phase precursors. Ceramides from egg or from bovine brain are very similar in their effects on the lamellar-hexagonal transition. They are also comparable to diacylglycerides in this respect, although ceramides are less potent. These results are relevant in the interpretation of certain forms of interfacial enzyme activation and in the regulation and dynamics of the bilayer structure of cell membranes.

Fourier-transform infrared spectroscopy has been applied to the study of lipid vesicle-supported two-dimensional crystals and noncrystalline preparations of beef heart cytochrome oxidase. At room temperature, no conformational differences are seen between the noncrystalline and crystalline proteins, whose conformation is shown to consist of ca. 40% alpha-helix, 20% extended structures (including beta-sheet), 17% beta-turns, and 22% open loops plus nonstructured conformations. A novel infrared approach that combines quantitative spectral band decomposition with the study of the thermal behavior of each component has been applied. The procedure allows the independent examination of temperature-induced changes in individual structural elements (alpha-helix, beta-sheet, beta-turns, and unordered). All these reflect, upon heating the protein from 20 to 80 degrees C, a major irreversible thermal event centred at 55-60 degrees C, leading to a molecular state devoid of enzyme activity but with a defined secondary structure; in addition, when the band position, percent area (integrated intensity), and bandwidth of the various amide I components are separately plotted versus temperature, each component is seen to behave in a characteristic way. Thermal denaturation in D2O buffer shows a decrease in nonstructured conformations and an increase in beta-turns without major changes in the proportion of alpha-helix. Temperature-induced changes are not the same in amorphous and crystalline structures, the latter being in general more stable toward the thermal challenge. The above data extend and confirm previous structural studies on cytochrome oxidase using cryo-electron microscopy.