Evidence from deuterium nuclear magnetic resonance for the temperature-dependent reversible self-association of erythrocyte band 3 in dimyristoylphosphatidylcholine bilayers

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Abstract
Band 3, isolated from human erythrocytes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) deuterated in the terminal methyl groups of the choline head group. By use of Triton X-100 for selective extraction and purification of band 3 and then cholate for subsequent solubilization with the lipid, a number of reconstituted complexes were produced by exhaustive detergent dialysis with protein:lipid weight ratios of between 0.32:1 and 1.25:1. Electron micrographs of negatively stained complexes showed that this method produced large vesicles of greater than 300-nm diameter. Deuterium nuclear magnetic resonance (NMR) spectra from the choline methyl deuterons in bilayer lipid above the liquid-crystal-gel phase transition temperature were shown to change systematically with increasing concentrations of band 3 in the bilayers. The measured quadrupole splittings, taken as the separation of the turning points in the recorded spectra, decreased from a value of 1.28 kHz for pure lipid to 0.98 kHz for bilayers with a protein:lipid ratio of 1.25:1 at 26.degree. C. At 35.degree. C, a more pronounced decrease in the quadrupole splittings was measured. The data from the complexes with protein:lipid ratios up to 0.7:1 (w/w) obey the mathematical treatment for a rapid two-site exchange between lipids at the protein-lipid interface and the bulk lipid phase. The temperature dependence of the measured quadrupole splitting with respect to the protein:lipid ratio indicates that the amount of lipid from the associating intramembranous surfaces of the protein. An upper limit of 70 lipids per protein monomer has been estimated at 35.degree.C, a number which suggests that the lipids having perturbed 2H NMR properties are in more than a single shell surrounding the total hydrophobic perimeter of the protein. The apparent self-association properties of the lipid. However, band 3 caused a marked broadening of the lipid phase transition which was suppressed to lower temperatures at high protein:lipid ratios. It is suggested that the temperature-dependent self-association behaviour of band 3 in membranes is in intrinsic property of the protein which is qualitatively independent of the properties of the supporting lipid.

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