Lateral and rotational diffusion of bacteriorhodopsin in lipid bilayers: experimental test of the Saffman-Delbrück equations.

Abstract

Lateral diffusion of bacteriorhodopsin and a lipid analogue has been measured in dimyristoylphosphatidylcholine bilayers as a function of temperature, phospholipid/protein (mol/mol; L/P) ratio, and aqueous phase viscosity. The protein lateral diffusion coefficients measured above the temperature at which the lipid gel-liquid/crystalline phase transition occurs (Tc) are combined with previously determined rotational diffusion coefficients to provide a test of the Saffman-Delbrfick equations [Saffman, P. G. & Delbrfick, M. (1975) Procm NatL Acade Sci USA 72,[3111][3112][3113]. Insertion of the diffusion coefficients into these equations enables the protein diameter to be calculated. The value of 4.3 ± 0.5 nm so obtained is in reasonable agreement with the known structure of bacteriorhodopsin. A 12-fold increase in the viscosity of the aqueous phase reduces protein lateral diffusion coefficients by 50%, which is also consistent with the Saffman-Delbruick equations. Both protein and lipid lateral diffusion coefficients decrease with decreasing L/P ratio above the T,. It is argued that, at a high L/P ratio, this effect is probably due to changes in membrane viscosity while, at a low L/P ratio, "crowding" effects (steric restrictions) and protein aggregation become important. When comparing diffusion measurements made in different systems, it is important to take the effect of the L/P ratio into account. When this is done, other published measurements offreely diffusing membrane proteins are in good agreement with the present results and the predictions of the Saffman-Delbruick equations. Below the Ta, the presence of protein enhances diffusion rates. The overall effect is to smooth out the large change in diffusion coefficient that occurs at the T,.It is probable that diffusion plays a crucial role in a number of membrane functions [e.g., receptor-mediated processes (1), electron transfer (2), and photoreception (3)]. Much has been learned about this field by the use of optical techniques developed for measuring diffusion of membrane components (for review, see refs. 4-6). Fluorescence microphotolysis (7) is a versatile means for measuring translational and rotational diffusion in single cells, isolated membranes, artificial membranes, and solution (8-13). Transient dichroism of intrinsic chromophores or triplet probes has provided data on the rotational diffusion of a variety of membrane proteins (14). In the case of triplet probes, rotational diffusion has also been measured by using phosphorescence (15,16), delayed fluorescence (17), and fluorescence-depletion signals (18).A fruitful approach for analyzing parameters that potentially might restrict and regulate mobility in cellular membranes is the study of artificial bilayer membranes. Membranes made from a single lipid species (19, 20) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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