EFFECTS OF MEMBRANE THICKNESS ON THE MOLECULAR-DYNAMICS AND ENZYMATIC-ACTIVITY OF RECONSTITUTED CA-ATPASE

We have studied the effect of phospholipid chain length on the activit y and molecular dynamics of reconstituted Ca-ATPase from skeletal sarc oplasmic reticulum (SR), using time-resolved phosphorescence anisotrop y (TPA) and electron paramagnetic resonance (EPR). We used reconstitut ed Ca-ATPase in exogenous phosphatidylcholines with monounsaturated ch ains 14-24 carbons long, to determine their effects on the physical pr operties of the Ca-ATPase and to correlate these physical changes with changes in the ATPase activity. In agreement with previous studies, w e found that the enzymatic activity was maximal with a chain length of 18 and decreased substantially with longer or shorter chains. Our TPA results show that chain lengths longer or shorter than the optimal 18 result in a significantly decreased mobility of the Ca-ATPase, indica ted by higher residual anisotropy and suggesting extensive protein agg regation. Saturation-transfer EPR data obtained with a spin label boun d to a different site also indicates substantial immobilization of the enzyme, supporting the TPA results. There is good agreement between t he fractional inhibition of the Ca-ATPase activity and the fraction of the enzyme in large aggregates. Solubilization in the nonionic deterg ent C(12)E(8) demonstrated that inhibition of enzyme activity is rever sible. In contrast to the large effects on protein mobility, these cha nges in chain length had little or no effect on hydrocarbon chain mobi lity as detected by conventional EPR at different depths in the membra ne. We conclude that the Ca-ATPase has an optimum lipid bilayer thickn ess, presumably matching the thickness of the hydrophobic transmembran e surface of the enzyme, and that deviation from this optimum thicknes s produces a hydrophobic mismatch that induces protein aggregation and hence Ca-ATPase inhibition. This is consistent with our proposal that protein dynamics and protein-protein interactions are of primary impo rtance to the Ca-ATPase mechanism.