THERMOTROPIC BEHAVIOR OF DIMYRISTOYLPHOSPHATIDYLGLYCEROL AND ITS INTERACTION WITH CYTOCHROME-C

The thermotropic behavior of dimyristoylphosphatidylglycerol (DMPG) in the absence and presence of cytochrome c under low-salt conditions ha s been investigated using differential scanning calorimetry (DSC), P-3 1 nuclear magnetic resonance (P-31 NMR), electron spin resonance (ESR) , viscosity, light scattering, and electron microscopy. In the absence of protein, the lipid undergoes a sequence of transitions over the te mperature range of 7-40 degrees C. ESR studies demonstrate increased a cyl chain mobility associated with these transitions. P-31 NMR indicat es that the lipid, in the absence of protein, retains a lamellar struc ture throughout the temperature range investigated. At high lipid conc entration the DSC curves exhibit a pronounced maximum in the excess he at capacity (C-p) function at about 23 degrees C with a shoulder on th e high-temperature side. As the lipid concentration is reduced to 10 m M, the C-p curves broaden, retaining a sharp maximum at about 20 degre es C and a broader transition with a maximum at 27 degrees C. The over all enthalpy change of 6 kcal/mol is independent of lipid concentratio n. Most interestingly, the lipid dispersion becomes highly viscous and optically isotropic in the main transition range (20-28 degrees C), s uggesting long-range order even at lipid concentrations as low as 10 m M. The existence of long-range order is confirmed by negative stain el ectron microscopy. The heat capacity curve in the presence of protein is broad, with a single C-p maximum and an overall enthalpy change of 1.7 kcal/mol. Similarly, the temperature dependence of the ESR spectra shows none of the detail observed in the absence of the protein. Of s pecific interest is that partially saturating amounts of protein preve nted the large increase in the viscosity of the dispersion in the main transition range. This result suggested that the protein prevented de velopment of long-range order. However, under saturating conditions th e viscosity of the protein-lipid complex increased with increasing tem perature even beyond the transition range. This increase does not appe ar to be the result of formation of an extended lipid structure but is the result, according to electron microscopic evaluation, of aggregat ion of small protein-containing lipid vesicles. The P-31 NMR spectra o f the lipid in the presence of protein are isotropic, consistent with the formation of highly curved particles. Calorimetric titration studi es of the binding of cytochrome c to DMPG indicate that protein bindin g is coupled cooperatively to changes in the state of the lipid. The b inding data were analyzed in terms of a model in which the protein bin ds differentially to two different states of the lipid. This analysis yielded estimates of the binding affinity on the order of 10(6) L/mol and a stoichiometry of about 9:1, lipid to protein. The binding does n ot appear to involve any change in the degree of protonation, and the protein-induced change in lipid structure likely makes a significant c ontribution to the measured enthalpy change.