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 deposited on attenuated total reflection crystals has been used as an external perturbation. Owing to the fact that the amide protons associated with each conformation are not exchanged at the same rate, the different conformational contributions of the amide bands could be separated. The use of different sampling time domains turned out to be very helpful in order to separate more efficiently the fast kinetics from the slower ones. The results obtained on myoglobin show that this method 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 that the amide I band of this protein is composed of at least four components that could be assigned to alpha -helical, intermolecular beta -sheet, beta -turn, and random coil conformations.
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