Distance between skeletal protein 4.1 and the erythrocyte membrane bilayer measured by resonance energy transfer

To assess the molecular architecture of the human erythrocyte skeletal protein 4.1:bilayer interface, the distance between a donor sulfhydryl-specific fluorescent probe attached to a region near the glycophorin-binding domain of protein 4.1 and an acceptor lipophilic probe in the exposed leaflet of inside-out vesicles (IOVs) was measured by fluorescence resonance energy transfer. To prevent aggregation and loss of function, protein 4.1 was labeled in situ on the surface of IOVs, purified, and rebound onto fresh IOVs. The labeled protein 4.1 was similar to the native protein in its gel electrophoretic pattern and its binding affinity to stripped-IOVs (Kd 35 +/- 4 nM). Energy transfer was assessed using two donor-acceptor pairs, 5-[2-[(iodoacetyl)amino]ethyl] aminonaphthalene-1-sulfonic acid and 3,3'-ditetradecyloxacarbocyanine perchlorate, or 5-iodoacetamidofluorescein and tetramethylrhodamine phosphatidylethanolamine. Using both donor fluorescence intensity and lifetime quenching measurements, an average distance of 75 +/- 5 A between the probe on the protein and the surface of IOVs was found. In parallel fluorescence resonance energy transfer studies with protein 4.1 and liposomes with a phospholipid composition similar to the inner leaflet of the red cell membrane, a closer distance was found (49 +/- 5 A). Two control experiments validated energy transfer: (a) the spectrum of a mixture of IOVs separately labeled with donor and acceptor was different from the spectrum of the doubly labeled IOVs at identical donor and acceptor concentrations; and (b) no energy transfer was observed following detergent disruption of the geometric relationship between donor and acceptor. Taken together, these observations suggest that membrane-bound protein 4.1 is elongated and that the labeled site is located at a position deep in the 30-kDa N-terminal glycophorin-binding domain of the protein. The data are also consistent with the view that the cytoplasmic tail of glycophorin is interposed between protein 4.1 and the lipids. These experiments represent the first measurement of a distance between a skeletal protein and the lipid bilayer.