Differential scanning calorimetry demonstrates that the bleached form of the purple membrane does not possess any measurable thermal transition in water, up to 105"C, whereas in 0.1 M phosphate pH 7.5 it shows a transition at about 8 2 T , with an enthalpy of 110 kJ/mol. In the latter medium, the native membrane shows the main transition at 97"C, with an enthalpy of 390 kJ/mol. The reduced form of the purple membrane shows two small transitions in water, as well as in 0.1 M phosphate, which do not seem to be related to the main thermal transition of the native membrane. Fouriertransform infrared spectra in D 2 0 show that the two modified samples, as well as the native one, undergo similar secondary structural changes upon thermal denaturation. These changes appear to extend through a wide temperature range for both modified forms, particularly for the bleached one. The results suggest that the main thermal transition in the purple membrane is due to a cooperative conformational change involving the disruption of the network of electrostatic and hydrogen-bonding interactions which originate from the protonated Schiff base. In the two modified membranes, these conformational changes appear to proceed smoothly through a rather low or non-cooperative process. The thermal behaviour of the bleached membrane in water resembles that of the molten globule state described for several globular proteins.Bacteriorhodopsin is a retinylidene protein found in the purple membrane patches of Halobacterium halobium cells. It translocates protons from the inside to the outside of the cell upon light absorption, thus forming a proton gradient across the celular membrane. The protein is arranged in seven transmembrane a-helices, and possess a retinal molecule attached to a lysine residue through a protonated Schiff base [I -31. The bacteriorhodopsin molecules are distributed through the bilayer in trimers, which in turn are ordered in a two-dimensional hexagonal array. Thermal denaturation studies of bacteriorhodopsin have shown the high stability of the purple complex. At neutral pH, the melting temperature (t,,,) is found near 95°C for the major transition of the native complex, which is found to be an irreversible process [4]. Extreme pH values [5], deionization [6], delipidation [7], or incorporation of bacteriorhodopsin in vesicles [8] lowers the t,, but in all cases the enthalpy of denaturation is about four times lower than those found for globular proteins [9]. This lower denaturation enthalpy seems to be the general situation for all the intrinsic membrane proteins so far investigated, a fact related to the lack of protein/water interactions in their denatured state [lo].The thermal transition is accompanied by decoloration due to the hydrolysis of the retinal Schiff base. On the other hand, it is known that the retinal molecule occupies a central position in the seven a-helical rods [l 11 and thus it can act as a tightening or clamping element. These features prompted us to investigate the influence of the retinal molecule...

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