Hydrogen-deuterium exchange of streptavidin and its complex with biotin studied by 2D-attenuated total reflection Fourier transform infrared spectroscopy

Hydrogen-deuterium exchange for streptavidin and its complex with biotin is studied by means of attenuated total reflection (ATR) Fourier transform in frared (FTIR) spectroscopy. To analyze the spectral changes upon deuteratio n, difference spectra and two-dimensional correlation spectra are calculate d. We find that the exchange rate varies with the secondary structure in wh ich the exchanging amide protons are incorporated. The most slowly exchangi ng protons, with a characteristic time constant on the order of hours, are part of the beta-sheet secondary structure. The separation in time of excha nge of the beta-sheet from other structural elements allows the amide II an d II' frequencies of the beta-sheet (1530 and 1445 cm(-1)) to be identified . A second component which exchanges more rapidly than the beta-sheet is ch aracterized by its amide I frequencies 1680, 1640, and 1465 cm(-1). This co mponent is attributed to the exchange of amide groups in secondary structur es other than the central beta-barrel. Binding of the ligand results in a s lower exchange rate of the rapid component. These changes are interpreted i n terms of structural differences observed by previous X-ray studies of one loop in the protein involved in the binding of the ligand. The presence of the ligand is found to inhibit the exchange of similar to 10 amide protons during the time of the experiment (10 h). The protected amide groups are m ost likely part of the beta-sheet structure, and their retarded exchange is tentatively interpreted in terms of a reduced flexibility of the tetrameri c protein upon binding of the ligand.