Hairpin configuration of H-2Kk in liposomes formed by detergent dialysis.

Abstract

H-2Kk is a transmembrane glycoprotein having the N-terminal region of the heavy chain exposed at the cell surface and the C-terminal region exposed at the cytoplasmic face in its native configuration in the plasma membrane. The configuration of H-2Kk in liposomes formed by detergent dialysis was investigated by using fluorescently labeled H-2 and Co2+ ions to quench fluorescence. H-2Kk was incorporated into sealed lipid vesicles when deoxycholate was removed by dialysis from a mixture of protein and lipid. Including 20 mM carboxyfluorescein (CF) in the mixture prior to dialysis resulted in CF trapped inside the vesicles at concentrations where self-quenching occurred. Vesicles with CF trapped inside were shown to be osmotically active and impermeable to Na+ and Co2+ ions. In order to examine the configuration of H-2Kk in these liposomes, the heavy chain was covalently labeled by using the sulfhydryl reactive fluorescent reagents fluorescein-5-ylmaleimide (NFM) or 5-[[2-[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS). In both cases, approximately equal amounts of fluorescent label were incorporated into the N- and C-terminal regions of the protein. Incorporation of the labeled H-2 into liposomes and examination of the effect of Co2+ on the fluorescence showed that all of the label was accessible to quenching by Co2+ and thus exposed on the outside of the liposome. The results demonstrate that the H-2Kk is incorporated into these liposomes in a hairpin configuration, not in the transmembrane configuration found in native membranes.(ABSTRACT TRUNCATED AT 250 WORDS)