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T.Heimburg and R.L.Biltonen. 1994. The thermotropic behavior of dimyristoyl phosphatidylglycerol and its interaction with cytochrome c. Biochemistry 33: 9477-9488 abstract14

Abstract: The thermotropic behavior of dimyristoylphosphatidylglycerol (DMPG) in the absence and presence of cytochrome c under low-salt conditions has been investigated using differential scanning calorimetry (DSC), 31P nuclearmagneticresonance (31P NMR),electronspinresonance(ESR),viscosity,lightscattering, and electron microscopy. In the absence of protein, the lipid undergoes a sequence of transitions over the temperature range of 7~40 C. ESR studies demonstrate increased acyl chain mobility associated with these transitions. 31P NMR indicates that the lipid, in the absence of protein, retains a lamellar structure throughout the temperature range investigated. At high lipid concentration the DSC curves exhibit a pronounced maximum in the excess heat capacity (Cp) function at about 23 C with a shoulder on the high-temperature side. As the lipid concentration is reduced to 10 mM, the Cp curves broaden, retaining a sharp maximum at about 20 C and a broader transition with a maximum at 27 C. The overall enthalpy change of 6 kcal/mol is independent of lipid concentration. Most interestingly, the lipid dispersion becomes highly viscous and optically isotropic in the main transition range (2~28 C), suggesting long-range order even at lipid concentrations as low as 10 mM. The existence of long-range order is con~1rmed by negative stain electron microscopy. The heat capacity curve in the presence of protein is broad, with a single Cp 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 specific interest is that partially saturating amounts of protein prevented the large increase in the viscosity of the dispersion in the main transition range. This result suggested that the protein prevented development of long-range order. However, under saturating conditions the viscosity of the protein-lipid complex increased with increasing temperature even beyond the transition range. This increase does not appear to be the result of formation of an extended lipid structure but is the result, according to electron microscopic evaluation, of aggregation of small protein-containing lipid vesicles. The 31P NMR spectra of the lipid in the presence of protein are isotropic, consistent with the formation of highly curved particles. Calorimetric titration studies of the binding of cytochrome c to DMPG indicate that protein binding is coupled cooperatively to changes in the state of the lipid. The binding data were analyzed in terms of a model in which the protein binds differentially to two different states of the lipid. This analysis yielded estimates of the binding affinity on the order of 106 L/mol and a stoichiometry of about 9:1, lipid to protein. The binding does not appear to involve any change in the degree of protonation, and the protein-induced change in lipid structure likely makes a significant contribution to the measured enthalpy change.