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CaATPase from rabbit sarcoplasmic reticulum has been reconstituted into binary lipid mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1,2-dipalmitoylphosphatidylcholine-d62 (DPPC-d62) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC)/DPPC-d62. Fourier-transform infrared (FT-IR) spectroscopy has been used to monitor temperature-induced structural alterations in the individual lipid components in the presence and absence of protein. A simple two-state model is used to construct a phase diagram that is in good agreement with one constructed from differential scanning calorimetry data, for the POPE/DPPC-d62 (protein-free) system. Although these two lipids are miscible over at least most of the composition range, substantial deviations from ideal behavior are observed. An estimate of the nonideality of mixing in both the gel and liquid-crystalline phases is obtained from regular solution theory. The phase diagram for SOPC/DPPC-d62 shows gel-phase immiscibility. FT-IR studies of ternary (POPE/DPPC-d62/CaATPase) complexes indicate that both lipid components are disordered by protein at all temperatures studied. In addition, their melting events are broadened and shifted to lower temperatures compared with the appropriate binary lipid mixture. Semiquantitative estimates for the fraction of each lipid melted are obtained from the model. The effect of protein on SOPC/DPPC-d62 mixtures depends on that total lipid to protein ratio. At low protein levels, SOPC is preferentially selected by CaATPase, so that bulk lipid is enriched in DPPC-d62. At high levels of protein, both lipid components are selected. The applicability of vibrational spectroscopy for determination of the partitioning preferences of membrane proteins into regions of particular chemical structure or physical order in a complex lipid environment is demonstrated.

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CaATPase from rabbit sarcoplasmic reticulum has been isolated, purified, stripped of its native lipids, and reconstituted into binary lipid mixtures of dielaidoylphosphatidylcholine (DEPC) and dipalmitoylphosphatidylethanolamine (DPPE) or acyl-chain perdeuterated DPPE (DPPE-d62). The partitioning properties of the protein were determined from differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. Acyl-chain perdeuteration allows the separate determination of the order and melting characteristics of each lipid species with FT-IR. The binary lipid mixture has been shown to be phase separated in the gel state (Brauner, J. W., and R. Mendelsohn, 1986, Biochim. Biophys. Acta, 861:16-24). The solid phases present at low temperatures correspond to a pure DEPC phase and a mixed phase of DEPC/DPPE-d62. Insertion of protein at 37 degrees C leads to a domain of relatively protein-free DPPE-d62 and a phase containing both lipids plus CaATPase. We suggest that CaATPase selects a fixed composition (60% DEPC, 40% DPPE-d62) for its immediate environment. The composition of the lipid in the immediate vicinity of protein is largely independent of the initial DEPC/DPPE-d62 ratios in the reconstitution protocol. The relevance of these results to observations of discrete domains in native membranes is discussed.

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