This review deals with current concepts of vibrational spectroscopy for the investigation of protein structure and function. While the focus is on infrared (IR) spectroscopy, some of the general aspects also apply to Raman spectroscopy. Special emphasis is on the amide I vibration of the polypeptide backbone that is used for secondary-structure analysis. Theoretical as well as experimental aspects are covered including transition dipole coupling. Further topics are discussed, namely the absorption of amino-acid side-chains, 1H/2H exchange to study the conformational flexibility and reaction-induced difference spectroscopy for the investigation of reaction mechanisms with a focus on interpretation tools.

Attenuated total reflection spectroscopy, where precise control of external parameters is feasible, was combined with the step-scan technique, which provides time-resolved Fourier transform-infrared spectra with microsecond time resolution. The advantages of this new approach were demonstrated by analyzing the photoreaction of the membrane protein bacteriorhodopsin (BR). By variation of temperature and pH clear-cut separation of the intermediate states L, M, N, and O, was achieved while previous infrared studies failed to separate the O intermediate in the wild-type. Therefore, we focused on a detailed description of the O−BR difference spectrum. It was proved that the major changes in secondary structure of BR are reversed in the N to O transition. Bands at 1432 and 1448 cm-1 were tentatively assigned to CH3 deformation vibrations of the retinal chromophore in the O state. At 1713 cm-1 the protonation of a carboxylic amino acid during the lifetime of O was observed. In addition to the wild-type data, the long-lived O intermediate of the E204Q and the E204T mutant have been investigated.

Active proton transfer through membrane proteins is accomplished by shifts in the acidity of internal amino acids, prosthetic groups, and water molecules.

This paper reviews state-of-the-art reaction-induced infrared difference spectroscopy of proteins. This technique enables detailed characterization of enzyme function on the level of single bonds of proteins, cofactors, or substrates. The following methods to initiate a reaction in the infrared sample are discussed: (i) light-induced difference spectroscopy, (ii) attenuated total reflection with buffer exchange, (iii) the infrared variant of stopped and continuous flow, (iv) temperature and pressure jump, (v) photolytical release of effector substances from caged compounds, (vi) equilibrium electrochemistry, and (vii) photoreduction. Illustrating applications are given including hot topics from the fields of bioenergetics, protein folding, and molecule--protein interaction.