pH dependence of bacteriorhodopsin thermal unfolding

Abstract

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.

Scanning Calorimetry and Fourier-Transform Infrared Studies into the Thermal Stability of Cleaved Bacteriorhodopsin Systems

“…These values compare well with those already published (Jackson & Sturtevant, 1978;Brouillette et al, 1987;Cladera et al, 1992a;Kahn et al, 1992;. The absence of retinal precludes the DSC transition at pH 6.5 in pure water (Cladera et al, 1992a).…”

“…All samples cleaved with chymotrypsin were regenerated with all-trans-retinal after cleavage. The results for native BR compare well with others already published in the literature (Jackson & Sturtevant, 1978;Brouillette et al, 1987;Cladera et al, 1992a;, i.e., a pre-transition at about 73 or 80°C and the main denaturation transition, centered at about 81 or 95°C, at pH 9.5 and 7.5, respectively. The AB‚CDEFG sample shows a single transition endotherm at about 88-89°C, pH 7.5, and 72-76°C, pH 9.5, depending upon the scan rate, without any detectable pre-transition (Table 1); here the cleavage of the 71-72 peptide bond is practically 100%, so the single endotherm corresponds to the denaturation of the cleaved bacteriorhodopsin.…”

A pathway for the thermal destabilization of bacteriorhodopsin

“…In order to facilitate comparison with previous studies [9,13], all the above experiments were repeated in a 50 mM bicarbonate, 100 mM NaC1, pH 9.5 buffer. IR spectra reveal that the end-point of the thermal denaturation process at pH 9.5 is undistinguishable from the equivalent phenomenon at pH 7.5, but the denaturation process appears to be completed after 5 rain at 92°C when the pH is 9.5 (data not shown).…”

The role of retinal in the thermal stability of the purple membrane

“…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].…”