J. Mol. Biol. (1983) l{~i, 211-2i7 Lipid Bilayer Thickness Varies Linearly with Acyl Chain Length in Fluid Phosphatidylcholine Vesicles The thickness of the lipid bilayer in vesicles made of pure phosphatidylcholines, with acyl chain lengths ranging from l0 to 24 carbons, has been determined by analysis of continuous X-ray scattering data from vesicle pellets at temperatures above the lipid phase transition temperature. Bilayer thickness was found to vary linearly with the number of carbons per acyl chain. The lines for saturated and monounsaturated acyl chains were slightly displaced but had similar slopes. For the saturated species di-12 : 0, di-14 : 0, di-16 : 0, and did8 : 0 phosphatidylcholine the surface areas per molecule were all 65-7 to 66"5 A2, while the monounsaturated species and di-10 : 0 phosphatidylcholine all occupied 67"7 to 70"1 A2 per molecule. It is generally agreed that a lipid bilayer is the basic structural motif of most biological membranes (Stoeckenius & Engelman, 1969) and that interactions with lipids modulate the function of at least some membrane proteins in important ways. Since a bilayer arrangement of phospholipids is characterized by a distinct central hydrophobic region bounded by two polar interracial regions, we expect the thickness of the hydrophobic region to influence such properties as ion permeability, capacitance, and structure and function of transmembrane proteins. In particular, optimal lipid bilayer thickness is required for maximal activity of at least two integral membrane proteins, the Ca2+-ATPase from sarcoplasmic reticulum (Caffrey & Feigenson, 1981 ; Johannsson et al., 1981 ; Moore et al., 1981) and the ion channel gramicidin (Haydon & Hladky, 1972). We have also explored the effect of lipid bilayer thickness on the planar organization of the transmembrane protein bacteriorhodopsin (Lewis & Engleman, 1983). In order to interpret the results of such experiments, it is necessary to characterize the equilibrium thickness of pure lipid bilayers under similar experimental conditions. The simplest approach to measurement of the thickness of the hydrophobic region is to determine the distance between the phosphate groups across the bilayer, d, and then to subtract the thickness of the appropriate portion of the polar region. Therefore, we have used X-ray scattering to determine d in vesicles made with pure phosphatidylcholines with acyl chain lengths from 10 to 24 carbon atoms, each at a temperature above the phase transition temperature, t m, of the phosphatidylcholine species. By using vesicles consisting of one or a few bilayers, strong sampling of the bilayer transform is avoided, and the resulting continuous X-ray scattering permits direct determination of d (Engelman, 1970,1971 ; Wilkins et al., 1971). This method avoids possible errors in the measurement of water content or in the assumptions of partial specific volumes, which are necessary to derive the bilayer thickness from the interlamellar distance in multilamellar samples. 211 0022-2836/83/1402t 1-07 $03.00/0 © 1983 AcademicPress Inc. (London) Ltd.