Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease

The well-characterized integral membrane protein lactose (lac) permease from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyrene-labeled phospholipid analogs into 1-palmitoyl-2-oleoyl-sn-glycero-3-sn-glycero-3-phospho-rac'-glycerol (POPG) liposomes. Effects of lac permease on bilayer lipid dynamics were investigated by measuring the excimer-to-monomer fluorescence intensity ratio I-E/I-M. Compared to control vesicles, the presence of lac permease (at a protein:phospholipid stoichiometry P/L of 1:4.000) increased the rate of excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefold. Decreasing P/L from approximately 1:4.000 to 1:7.600 decreased the I-E/I-M for PPDPC from 0.16 to 0.05, respectively. An increase in bilayer fluidity due to permease is unlikely, thus implying that the augmented I-E/I-M should arise from partial lateral segregation of PPDPC in the vesicles. This notion is supported by the further 38% increase in I-E/I-M observed for the pyrene-labeled Cys-148 lac permease reconstituted into POPG vesicles at P/L 1:4000. The importance of the length of the lipid-protein boundary is implicated by the reduction in I-E/I-M resulting from the aggregation of the lac permease in vesicles by a monoclonal antibody. Interestingly, excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfold in the presence of lac permease. Results obtained with the corresponding pyrenyl phosphatidylglycerols and -methanols were qualitatively similar to those above, thus indicating that lipid headgroup-protein interactions are not involved. Inclusion of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresceinthio-carbamoyl) (DPPF, X = 0.005) into reconstituted lactose permease vesicles containing PPDPC caused a nearly 90% decrease in excimer fluorescence, whereas in control vesicles lacking the reconstituted protein only 40% quenching was evident. The addition of 1,2-dipalmitdyl-sn-glycero-3-phospho-rac'-glycerol (DPPG) decreased I-E/I-M for PPDPC, revealing the driving force for the lateral segregation of this probe to become attenuated. More specifically, for protein-free bilayers at X(DPPG) = 0.10 the rate of lateral diffusion of PPDPC in POPG is diminished, as evidenced by the 24% decrement in I-E/I-M under these conditions the increase in I-E/I-M due to lac permease was strongly reduced, by similar to 84%. The present data are interpreted in terms of the hydrophobic mismatch theory, which predicts that integral membrane proteins will draw lipids of similar hydrophobic thickness into their vicinity. In brief, the approximate lengths of most of the predicted 12 hydrophobic, membrane-spanning alpha-helical segments of lactose permease range between 28.5 and 37.5 Angstrom and thus exceed the hydrophobic thickness of POPG of similar to 25.8 Angstrom. Therefore, to reduce the free energy of the assembly, longer lipids such as PPDPC and DPPF are accumulated in the immediate vicinity of lactose permease in fluid, liquid crystalline POPG bilayers.