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

Abstract

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...

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).…”

“…The absence of retinal precludes the DSC transition at pH 6.5 in pure water (Cladera et al, 1992a). Nevertheless, without retinal but in the presence of salts (buffer) at both alkaline and at neutral pH a DSC transition appears for both intact and cleaved BR, the enthalpy of which is 30% ( 5% of that of the corresponding reconstituted samples (Cladera et al, 1992a;Khan et al, 1992). Assuming this 30% enthalpy value for the non-regenerated BR, and bearing in mind that retinal reconstitution for our AB‚CDEFG samples was about 60%, the average ∆H values obtained in our DSC experiments would be about 290 and 87 kJ/mol for regenerated and retinal-free AB‚CDEFG, respectively, at pH 7.5, whereas the corresponding ∆H values would be about 259 and 78 kJ/mol at pH 9.5.…”

“…Furthermore we have demonstrated the importance of bound cations in the stability of BR in a previous study into the deionized and different cation-regenerated membranes (Cladera et al, 1988). Retinal does seem to play a major role in BR stability since the bleached or even the Schiff-base reduced proteins do not undergo the main DSC denaturation transition in water at pH 6.5 (Cladera et al, 1992a). Nevertheless, in the presence of salts at pH 7.5 bleached BR shows a small and wide DSC transition with a T m at about 82°C and a ∆H value of about 110 kJ/mol, similar to that found for this sample at pH 7.0 (T m ) 85°C, ∆H ) 96 kJ/mol) by Kahn et al (1992).…”

“…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, 1988Cladera et al, , 1992a Khan et al, 1992;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.…”

Structural Characterization of the C2 Domains of Classical Isozymes of Protein Kinase C and Novel Protein Kinase Cε by using Infrared Spectroscopy

“…The bands located at 1663 cm −1 and 1688 cm −1 arise from β-turns [3,32]. Additionally, there is a band at about 1612 cm −1 , which has been assigned to side chain absorption [18,21,22], and so, its contribution is not included in the calculation of the secondary structure of PKC-C2. The secondary structure of PKCα-C2 domain (Table 1) is 60% β-sheet (taking into account the 1635 and 1675 cm −1 bands), 12% α-helix/dihedral turns (similar to α-helix), 15% β-turns and 12% open loops and non-structured conformation.…”