Lateral Diffusion, Protein Mobility, and Phase Transitions in Escherichia coli Membranes. A Spin Label Study

The coefficient of lateral diffusion in Escherichia coli membranes is determined as D_diff = 3.25 × 10–8 cm2/sec at 40° using a spin label technique developed previously. This value compares well with the rate of lateral diffusion in dipalmitoyllecithin (DPL) membranes at 50°. The rate of rotational and translational diffusion of membrane proteins may be estimated from the hopping frequency (v ≈ 107 Hz) of the lipid molecules. For a protein with a radius R = 25 Å we obtain a rotational relaxation time τ_r of about τ_r = 75 μsec and a coefficient of lateral diffusion D_diffP ~ 3 × 10–10 cm2/sec. The electron spin resonance (esr) spectra of spin labels incorporated in DPL model membranes undergo characteristic changes in the temperature range of the lipid phase transition. The transition temperature, T_t, determined from the changes in spectral intensity and the order parameter S depends on the distance between the paramagnetic center and the membrane surface. If the NO group is deeply buried within the hydrocarbon phase the obtained values of T_t agree well with dilatometric and spectroscopic measurements (90° light scattering, 8-anilino-1-naphthalenesulfonate (ANS) fluorescence). Lower values of T_t are, however, observed if the NO group is near the semipolar region of the membrane. The same spectral changes have been observed in intact membranes of an E. coli fatty acid auxotroph (containing predominantly trans-Δ9-octadecenoic acid, cis-Δ9-octadeccnoic acid, and trans-Δ9-hexadecenoic acid), indicating a lipid phase transition in these membranes. The transition temperatures T_t obtained with stearic acid labels carrying the NO group near the methyl end of the chain agree well with the previously reported breaks in the temperature dependence of some transport systems of the respective E. coli mutant (P. Overath et al., Nature (London), New Biol., 234, 264 (1971)). This shows that these breaks are caused by phase transitions of the membrane lipids. The occurrence of a lipid phase transition in E. coli membranes and the approximate equality of the coefficient of lateral diffusion in these membranes with the value of D_diff in DPL model membranes strongly support the presence of (continuous) lipid layers in the E. coli membranes. “Polar” spin labels (Tempo, digitoxigenin) and stearic acid labels with the NO groups near the carboxyl end indicate a “pretransition” some 6–8° below the main transition. This shows that the result of spin label studies with membranes may depend critically on the position of the paramagnetic center within the membrane.