Effect of temperature, pH, and metal ion binding on the secondary structure of bacteriorhodopsin: FT-IR study of the melting and premelting transition temperatures

We have measured the temperature dependence of the FT-IR spectra of bacteri orhodopsin (bR) as a function of the pH and of the divalent cation regenera tion with Ca2+ and Mg2+. It has been found that although the irreversible m elting transition shows a strong dependence on the pH of the native bR, the premelting reversible transition at 78-80 degreesC shows very little varia tion over the pH range studied. It is further shown that the acid blue bR s hows a red-shifted amide I spectrum at physiological temperature, which sho ws a more typical alpha -helical frequency component at 1652 cm(-1) and cou ld be the reason for the observed reduction of its melting temperature and lack of an observed premelting transition. Furthermore, the thermal transit ions for Ca2+- and Mg2+-regenerated bR (Ca-bR and Mg-bR, respectively) each show a premelting transition at the same 78-80 degreesC temperature as the native purple membrane, but the irreversible melting transition has a slig ht dependence on the cation identity. The pH dependence of the Ca2+-regener ated bR is studied, and neither transition varies over the pH range studied . These results are discussed in terms of the cation contribution to the se condary structural stability in bR.