2-DIMENSIONAL FT-IR SPECTROSCOPY - A POWERFUL METHOD TO STUDY THE SECONDARY STRUCTURE OF PROTEINS USING H-D EXCHANGE

Two-dimensional infrared spectroscopy has been used for the first time to study the conformation of proteins by hydrogen-deuterium exchange. In order to generate the two-dimensional synchronous and asynchronous maps, hydrogen-deuterium exchange of the amide protons of proteins de posited on attenuated total reflection crystals has been used as an ex ternal perturbation, Offing to the fact that the amide protons associa ted with each conformation are not exchanged at the same rate, the dif ferent conformational contributions of the amide bands could be separa ted. The use of different sampling time domains turned out to be very helpful in order to separate more efficiently the fast kinetics from t he slower ones. The results obtained on myoglobin show that this metho d is particularly useful to unravel the different components under the poorly resolved amide I, II, and II' bands of proteins. The analysis of the synchronous and asynchronous maps of myoglobin demonstrates tha t the amide I band of this protein is composed of at least four compon ents that could be assigned to alpha-helic al, intermolecular beta-she et, beta-turn, and random coil conformations.