Exclusion of a Transmembrane-Type Peptide from Ordered-Lipid Domains (Rafts) Detected by Fluorescence Quenching: Extension of Quenching Analysis to Account for the Effects of Domain Size and Domain Boundaries

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Abstract
Sphingolipid/cholesterol-rich rafts are membrane domains thought to exist in the liquid-ordered state. To understand the rules governing the association of proteins with rafts, the behavior of a model membrane-inserted hydrophobic polypeptide (LW peptide, acetyl-K2W2L8AL8W2K2-amide) was examined. The distribution of LW peptide between coexisting ordered and disordered lipid domains was probed by measuring the amount of LW Trp fluorescence quenched by a nitroxide-labeled phospholipid that concentrated in disordered lipid domains. Strong quenching of the Trp fluorescence (relative to quenching in model membranes lacking domains) showed that LW peptide was concentrated in quencher-rich disordered domains and was largely excluded from ordered domains. Exclusion of LW peptide from the ordered domains was observed both in the absence and in the presence of 25−33 mol % cholesterol, indicating that the peptide is relatively excluded both from gel-state domains (which form in the absence of cholesterol) and from liquid-ordered-state domains (which form at high cholesterol concentrations). Because exclusion was also observed when ordered domains contained sphingomyelin in place of DPPC, or ergosterol in place of cholesterol, it appeared that this behavior was not strongly dependent on lipid structure. In both the absence and the presence of 25 mol % cholesterol, exclusion was also not strongly dependent upon the fraction of the bilayer in the form of ordered domains. To evaluate LW peptide behavior in more detail, an analysis of the effects of domain size and edges upon quenching was formulated. This analysis showed that quenching can be affected both by domain size and by whether a fluorescent molecule localized at domain edges. Its application to the quenching of LW peptide indicated that the peptide did not preferentially reside at the boundaries between ordered and disordered domains.