EVIDENCE FOR PHOSPHOLIPID MICRODOMAIN FORMATION IN LIQUID-CRYSTALLINELIPOSOMES RECONSTITUTED WITH ESCHERICHIA-COLI LACTOSE PERMEASE

The well-characterized integral membrane protein lactose (lac) permeas e from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyre ne-labeled phospholipid analogs into yl-sn-glycero-3-sn-glycero-3-phos pho-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, th e presence of lac permease (at a protein:phospholipid stoichiometry P/ L of 1:4.000) increased the rate of excimer formation by -(pyren-1-yl) ]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefol d. 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 bila yer fluidity due to permease is unlikely, thus implying that the augme nted 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 reconsti tuted into POPG vesicles at P/L 1:4000. The importance of the length o f 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 m onoclonal antibody. Interestingly, excimer formation by -(pyren-1-yl)] hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfol d in the presence of lac permease. Results obtained with the correspon ding pyrenyl phosphatidylglycerols and -methanols were qualitatively s imilar to those above, thus indicating that lipid headgroup-protein in teractions are not involved. Inclusion of hosphoethanolamino-N-(5-fluo resceinthio-carbamoyl) (DPPF, X = 0.005) into reconstituted lactose pe rmease vesicles containing PPDPC caused a nearly 90% decrease in excim er fluorescence, whereas in control vesicles lacking the reconstituted protein only 40% quenching was evident. The addition of 1,2-dipalmitd yl-sn-glycero-3-phospho-rac'-glycerol (DPPG) decreased I-E/I-M for PPD PC, revealing the driving force for the lateral segregation of this pr obe to become attenuated. More specifically, for protein-free bilayers at X(DPPG) = 0.10 the rate of lateral diffusion of PPDPC in POPG is d iminished, as evidenced by the 24% decrement in I-E/I-M under these co nditions the increase in I-E/I-M due to lac permease was strongly redu ced, by similar to 84%. The present data are interpreted in terms of t he 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 1 2 hydrophobic, membrane-spanning alpha-helical segments of lactose per mease range between 28.5 and 37.5 Angstrom and thus exceed the hydroph obic thickness of POPG of similar to 25.8 Angstrom. Therefore, to redu ce the free energy of the assembly, longer lipids such as PPDPC and DP PF are accumulated in the immediate vicinity of lactose permease in fl uid, liquid crystalline POPG bilayers.