Effect of cholesterol upon the conformation of band 3 and its transmembrane fragment

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Abstract
Vesicles enriched in the anion transport protein band 3 and its transmembrane domain were prepared, and the cysteine residues were labelled with an extrinsic fluorescent probe, monobromobimane. Fluorescence energy transfer was demonstrated between intrinsic tryptophans and monobromobimane, and an average interchromophoric distance, Rav, was defined. Rav values and fluorescence emission wavelengths were used to monitor the conformation of band 3 and its transmembrane domain as a function of cholesterol content. The vesicles were treated with ovolecithin liposomes to reduce the cholesterol concentration, and there was an increase in Rav from 17.25 to 20.70 Å (1 Å = 0.1 nm) in intact band 3. A somewhat smaller increase in Rav for the transmembrane domain was observed (18.03–19.04). The tryptophan fluorescence emission wavelength was also blue shifted in the cholesterol-depleted preparations relative to the untreated samples. Combining the effects of cholesterol depletion upon Rav and the fluorescence emission maxima, it is suggested that the conformation of band 3 is influenced by the level of cholesterol in the bilayer.