Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems ² Ana I. Azuaga, ‡ Francesc Sepulcre, § Esteve Padro ´s, § and Pedro L. Mateo* ,‡ Departmento de Quı ´mica Fı ´sica, Facultad de Ciencias, e Instituto de Biotecnologı ´a, UniVersidad de Granada, 18071 Granada, Spain, and Unitat de Biofı ´sica, Departament de Bioquı ´mica i de Biologia Molecular, Facultat de Medicina, UniVersitat Auto ` noma de Barcelona, 08193 Bellaterra, Barcelona, Spain ReceiVed July 24, 1996 X ABSTRACT: Differential scanning calorimetry and Fourier-transform infrared spectroscopy have been used to characterize the thermal stability of bacteriorhodopsin (BR) cleaved within different loops connecting the helical rods. The results are compared to those of the native protein. We show that the denaturation temperature and enthalpy of BR cleaved at peptide 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 peptide 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) Biochemistry 31, 8829]. The thermal denaturation of all the samples was irreversible 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 direct evidence for some intermolecular cooperativity upon denaturation. The denatured samples maintain a large proportion of R helices and  structure, a fact which seems to be related to their low denaturation enthalpy as compared to that of water-soluble, globular proteins. Thermodynamic-data analysis and energetic characteriza- tion of the thermal stability of membrane proteins (Sanchez- Ruiz & Mateo, 1987; Ruiz-Sanz et al., 1992) as well as that of several globular proteins (Sanchez-Ruiz et al., 1988; Conejero-Lara et al., 1991) cannot be undertaken because of their non-equilibrium, irreversible denaturation (Sanchez- Ruiz, 1992). This irreversibility is usually due to non- equilibrium processes taking place on protein unfolding (Klibanov & Ahern, 1987). Even in this case differential scanning calorimetry (DSC) 1 can provide the denaturation enthalpy (ΔH) and temperature (T m ) values of the transition for a given scan rate. Thus T m can be used as an operative parameter, at least in comparative, relative terms, to char- acterize the thermal stability of a protein which unfolds under non-equilibrium conditions. Fourier-transform infrared spec- troscopy (FTIR) is a very suitable technique to complement protein DSC studies, particularly in the case of membrane proteins, since it provides structural information concerning the native and denatured states and can also be used to observe the denaturation process itself (Surewicz & Mantsch, 1988; Surewicz et al., 1993; Arrondo et al., 1993; Jackson & Mantsch, 1995). Bacteriorhodopsin (BR), the only protein present in the purple membrane of Halobacterium salinarium, is one of the best known and well-studied intrinsic membrane proteins, both from a structural and a functional point of view (Henderson et al., 1990; Rothschild, 1992; Lanyi, 1993; Grigorieff et al., 1996). Previous denaturation studies carried out by DSC and spectroscopic techniques have thrown light on the importance of several structural features on BR’s stability, such as the presence of the retinal moiety, the favorable packing and interactions of the seven helices within the bilayer, the presence of cations in the medium, and the possible role of the extramembranous loops connecting the helices (Jackson & Sturtevant, 1978; Brouillette et al., 1987; Cladera et al., 1988, 1992a; Khan et al., 1992; Sanchez-Ruiz & Galisteo, 1993; Taneva et al., 1995). The question still remains, however, as to what extent these stability factors, and certain others proposed in the literature (Khan et al., 1992), are quantitatively comparable with each other, and whether they are interconnected to any degree or can be considered in principle as being simply additive factors. We have made DSC and FTIR studies into the thermal stability of bacteriorhodopsin cleaved within different loops (individual peptide bonds 71-72 and 155-156, and both) at neutral and alkaline pH values and different scan rates and compared these results to those of the native protein. The denaturation of some of the samples followed the two- state kinetic model. When different BR molecule popula- tions were present in the sample a clear cooperative intermolecular interaction appeared on denaturation. The ² This work was supported by DGICYT Grants PB93-1163 and PB92-0622 from the Ministerio de Educacio ´n y Ciencia (Spain), and 1995SGR00481 from the DGR (Generalitat de Catalunya). A.I.A. held a predoctoral fellowship from the Junta de Andalucı ´a. * Author to whom correspondence should be addressed. Tel: +34- (9)58-243333/1. FAX: +34-(9)58-272879/274258. ‡ Universidad de Granada. § Universitat Auto `noma de Barcelona. X Abstract published in AdVance ACS Abstracts, November 15, 1996. 1 Abbreviations: DSC, differential scanning calorimetry; FTIR, Fourier-transform infrared spectroscopy; BR, bacteriorhodopsin; ABCDEFG, uncleaved bacteriorhodopsin; AB‚CDEFG, chymotrypsin- cleaved bacteriorhodopsin in the 71-72 peptide bond; ABCDE‚FG, NaBH-cleaved bacteriorhodopsin in the 155-156 peptide bond; AB‚CDE‚FG, double-cleaved bacteriorhodopsin in the 71-72 and 155- 156 peptide bonds; FWHH, full width at half height; k, resolution enhancement factor. 16328 Biochemistry 1996, 35, 16328-16335 S0006-2960(96)01845-4 CCC: $12.00 © 1996 American Chemical Society