SCANNING CALORIMETRY AND FOURIER-TRANSFORM INFRARED STUDIES INTO THE THERMAL-STABILITY OF CLEAVED BACTERIORHODOPSIN SYSTEMS

Differential scanning calorimetry and Fourier-transform infrared spect roscopy have been used to characterize the thermal stability of bacter iorhodopsin (BR) cleaved within different loops connecting the helical rods. The results are compared to these of the native protein. We sho w that the denaturation temperature and enthalpy of BR cleaved at pept ide bond 71-72 or 155-156 are lower than those of the intact protein, and that these values become even lower for the BR cleaved at both pep tide bonds, The effect of cleavage on the denaturation temperature and enthalpy values seems to be additive as has been previously suggested [Khan, T. W., Sturtevant, J. M., & Engelman, D. M. (1992) Biochemistr y 31, 8829], The thermal denaturation of all the samples was irreversi ble and scan-rate dependent. When cleaved at the 71-72 bond BR follows quantitatively the predictions of the two-state kinetic model at pH 9 .5, with an activation energy of 374 kJ/mol, similar to that of native BR. Calorimetry experiments with different populations of intact and cleaved BR provide diner evidence for some intermolecular cooperativit y upon denaturation, The denatured samples maintain a large proportion of alpha helices and beta structure, a fact which seems to be related to their low denaturation enthalpy as compared to that of water-solub le, globular proteins.