Thermodynamic characterization of lipid interactions and conformational stability of membrane proteins

The interaction of two membrane proteins, cytochrome b₅ and cytochrome c oxidase with phosphatidylcholine lipid bilayers has been investigated using high-sensitivity scanning calorimetry and fluorescence spectroscopy. In both cases, the incorporation of each of these integral membrane proteins into large single lamellar phospholipid vesicles causes a reduction in the enthalpy associated with the lipid phase transition, a lowering of the transition temperature and a broadening of the lipid melting profile. The dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome b₅ prevents 14 ± lipids from undergoing the gel to liquid crystalline transition and cytochrome c oxidase prevents 99 ± 5 lipid molecules from undergoing the phase transition. Steady state fluorescence spectroscopy of the reconstituted membrane proteins indicates that integral membrane protein acts as a barrier to the lateral mobility of the lipid as probed by pyrene decanoic acid, and increases the apparent order of the phospholipid above the phase transition temperature as determined by diphenylhexatriene fluorescence anistropy measurements.

The thermal denaturation of cytochrome c oxidase was investigated using high sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. The thermal denaturation of cytochrome oxidase is characterized by an enthalpy change of 550 ± 50 Kcal/mole enzyme complex. This irreversible transition occurs at 63°C with the membrane reconstituted enzyme as opposed to 56°C with the detergent-solubil1 zed enzyme, indicating the lipid bilayer has a stabilizing influence on the enzyme complex. The denaturation of the enzyme complex is not a two state process but is composed of four melting steps. Differential solubility thermal gel analysis has permitted the quantitative identification of the contribution of individual subunits of the enzyme complex to the melting steps of the complex heat capacity profile. This gel analysis technique provides an ideal complement to the study of complex membrane protein systems by high sensitivity differential scanning calorimetry.