Calorimetric and fluorescence depolarization studies on the lipid phase transition of bacteriorhodopsin-dimyristoylphosphatidylcholine vesicles

Abstract

The thermotropic lipid phase transition of dimyristoylphosphatidylcholine vesicles reconstituted with bacteriorhodopsin was investigated as a function of the lipid to protein ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. Two attractive features of this system are that the lipid phase transition induces lipid-protein segregation and that the state of aggregation of the protein is known. Above the lipid phase transition and above molar lipid to protein ratios of about 100, bacteriorhodopsin is monomeric. Well below the phase transition, bacteriorhodopsin is aggregated in a hexagonal protein lattice. With increasing amounts of incorporated bacteriorhodopsin, the calorimetric transition broadens, and a second component develops at a temperature which is lower than that of the unperturbed transition. The latter transition was assigned to the disaggregation of the bacteriorhodpsin lattice which occurs 6-7 degrees C below the phase transition of the protein-free lipids according to previous measurements. The van't Hoff enthalpy of the phase transition, as determined from the fluorescence depolarization of diphenylhexatriene, is in surprisingly good agreement with that obtained from differential scanning calorimetry over a wide range of lipid to protein ratios. The differential scanning calorimetry data can be simulated on the basis of a model which takes the protein segregation and crystallization specifically into account. The essential feature of this model is that calorimetrically detectable lipid melting occurs in the temperature region of the protein crystallization, since for the disaggregation of the close packed bacteriorhodopsin lattice additional lipids of an intermediate chain conformation are required.

15N T2′ relaxation times of bacteriorhodopsin transmembrane amide nitrogens

“…In agreement with Heyn et al, 43 the DMPC gel to liquid crystalline phase transition was found to be broadened by the presence of BR and a typical axially symmetric spectrum was only observed above 30°C at a 1 : 150 ratio. The 2 H methylene quadrupolar splittings measured on choline-deuterated DMPC (DMPC-d4) were identical with those determined previously on unoriented samples.…”

Miscibility of phosphatidylcholine binary mixtures in unilamellar vesicles: Phase equilibria

“…The effect of acyl chain-length (Shimsbick & McConnell, 1973;Chapman et al, 1974;Lentz et al, 1976;Mabrey & Sturtevant, 1976;Jacobs et al, 1977;Kremer et al, 1977;Lee, 1978;Marcelja & Wolf, 1979;Scott & Cheng, 1979;Freire & Snyder, 1980;Chen & Sturtevant, 1981), polar head group (Blume & Ackerman, 1974;Chapman et al, 1974;Wu & McConnell, 1975;Lentz & Litman, 1978), or perturber molecules such as anesthetics (Jain & Wu, 1977;Mountcastle et al, 1978;Heyn et al, 1981) upon the width of the phase transition, have been reported. The theory on the cooperativity of lipid phase transition in bilayer systems is inconsistent with the idea that the thermotropic phase transition of lipid bilayers follows thermodynamically first-order kinetics.…”

Characterization of cooperative conformational transitions by Fourier transform infrared spectroscopy: application to phospholipid binary mixtures

“…Efforts to quantitatively describe the extent of transition using observable experimental parameters generally have assumed the following relation between the degree of transition, a , and the observable parameter, r where rA and rg are the values of the experimental parameter in states A and B, respectively (24). The disadvantage of this method is that it assumes a linear dependence of r on a .…”