Abstract
We have developed a quantitative and relatively model-independent measure of lipid fluidity using EPR and have applied this method to compare the temperature dependence of lipid hydrocarbon chain fluidity, overall protein rotational mobility, and the calcium-dependent enzymatic activity of the Ca-ATPase in sarcoplasmic reticulum. We define membrane lipid fluidity to be T/eta, where eta is the viscosity of a long chain hydrocarbon reference solvent in which a fatty acid spin label gives the same EPR spectrum (quantitated by the order parameter S) as observed for the same probe in the membrane. This measure is independent of the reference solvent used as long as the spectral line shapes in the membrane and the solvent match precisely, indicating that the same type of anisotropic probe motion occurs in the two systems. We argue that this empirical measurement of fluidity, defined in analogy to the macroscopic fluidity (T/eta) of a bulk solvent, should be more directly related to protein rotational mobility (and thus to protein function) than are more conventional measures of fluidity, such as the rate or amplitude of rotational motion of the lipid hydrocarbon chains themselves. This new definition thus offers a fluidity measure that is more directly relevant to the protein's behavior. The direct relationship between this measure of membrane fluidity and protein rotational mobility is supported by measurements in sarcoplasmic reticulum. The overall rotational motion of the spin-labeled Ca-ATPase protein was measured by saturation-transfer EPR. The Arrhenius activation energy for protein rotational mobility (11-12 kcal/mol/degree) agrees well with the activation energy for lipid fluidity, if defined as in this study, but not if more conventional definitions of lipid fluidity are used. This agreement, which extends over the entire temperature range from 0 to 40 degrees C, suggests that protein mobility depends directly on lipid fluidity in this system, as predicted from hydrodynamic theory. The same activation energy is observed for the calcium-dependent ATPase activity under physiological conditions, suggesting that protein rotational mobility (dependent on lipid fluidity) is involved in the rate-limiting step of active calcium transport.
Highlights
We have developed a quantitativeandrelativelySince the general acceptance that biological membranes are model-independent measure of lipid fluidityusing EPR fluid structures, there has been an active discussion of the and have applied this method to compare the temper- possible rolethat changes in membrane fluidity could play in ature dependence of lipid hydrocarbon chain fluidity, triggering or modulating membrane functions (reviewed by overall protein rotational mobility, and the calcium- Katesand Manson, 1984; Shinitzky, 1984; Deuticke and dependent enzymatic activity of the Ca-ATPase in sar- Haest, 1987)
An apparent exception to this principle was obtained productive associations between Ca-ATPase polypeptide with cholesterol, which decreases lipid fluidity in SR vesicles chains, resulting in a rate constant for the process of protein without affecting enzymatic activity (Warren etal., 1975; association that is slower than the diffusion-limited process by several orders of magnitude (Berg and von Hippel, 1985), reconciling the time scale of protein rotation
When either the mean molecular weight of the spin-labeled CaATPase oligomer or the fluidity of the hydrocarbon chain environment is altered, we find that therelationship between membrane fluidity and protein rotationaml obility agrees with that predicted theoretically (Saffman and Delbriick, 1975)
Summary
Since the general acceptance that biological membranes are model-independent measure of lipid fluidityusing EPR fluid structures, there has been an active discussion of the and have applied this method to compare the temper- possible rolethat changes in membrane fluidity could play in ature dependence of lipid hydrocarbon chain fluidity, triggering or modulating membrane functions (reviewed by overall protein rotational mobility, and the calcium- Katesand Manson, 1984; Shinitzky, 1984; Deuticke and dependent enzymatic activity of the Ca-ATPase in sar- Haest, 1987). We argue that functioning of membrane-bound enzymes (Bennett et al, this empiricaml easurement of fluidity, defined in anal- 1980; Caffrey and Feigenson, 1981; Johannsson et al, 1981a, ogy to themacroscopic fluidity (!l‘/q) of a bulksolvent, 1981b; Navarro et al, 1984; East et al, 1984),it is commonly should be more directly related to protein rotational proposed that an important general mechanism for the regumobility (and to protein function) than aremore lation of membrane function is the modulation of both specific conventional measures of fluidity, such as the rate or interactions at the lipid-protein interface (Fong and Mcamplitude of rotational motionof the lipid hydrocarbonNamee, 1987;Bigelow and Thomas, 1987) and membrane chains themselves This new definition offers a fluidity, presumably through changes in fatty acid unsaturafluidity measure that is more directly relevant to the tion andcholesterol content
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