Rl. Cornea et Dd. Thomas, EFFECTS OF MEMBRANE THICKNESS ON THE MOLECULAR-DYNAMICS AND ENZYMATIC-ACTIVITY OF RECONSTITUTED CA-ATPASE, Biochemistry, 33(10), 1994, pp. 2912-2920
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.