The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes

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Abstract
The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin‐5‐isothiocyanate(EITC)‐labeled l‐α‐dipalmitoylglycerophosphoethanolamine (Pam2GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator. The temperature dependence of the anisotropy decay showed that above 25°C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (η1). Extrapolation to η1= 0 Pa · s yielded a correlation time of φ= 20 ± 5 ns, t= 30°C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr= 2.0 × 109 rad2· s−1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was η2∼ 12 mPa · s. The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of η1= 1 mPa · s yielded a value of φr∼ 450 ns corresponding to Dr= 8.8 × 107 rad2· s−1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross‐sectional area of the protein part embedded in the membrane was calculated to be ∼ 9 nm2. This cross‐sectional area is large enough to include at most 14 membrane‐spanning helices. Our result also indicated that at lipid/protein molar ratios > 1.5 × 104:1 aggregation occurred in the model membranes below 30°C. However, above 30°C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.