Dynamical and temperature-dependent effects of lipid-protein interactions. Application of deuterium nuclear magnetic resonance and electron paramagnetic resonance spectroscopy to the same reconstitutions of cytochrome c oxidase

Abstract

2H NMR and EPR spectra have been obtained as a function of temperature and protein concentration from the same samples of beef heart cytochrome c oxidase reconstituted into 1-(16,16,16-trideuteriopalmitoyl)-2-palmitoleoyl-sn-glycero-3-phosphocholine. At all temperatures, the EPR spectra show the characteristic "bound" and "free" components, while the 2H NMR spectra show only a narrow distribution of orientational order parameters. At temperatures near the phase transition of the pure lipid, the dependence of the 2H NMR average orientational order on protein concentration fits a two-stage model in which the phospholipid molecular exchange rapidly between two states tentatively identified as sites either on or off the protein surface. From this model, the 2H NMR spectra yield a value of 0.18 mg of phospholipid per mg of protein as necessary to cover the surface of cytochrome c oxidase, which is the same value as derived from the EPR spectra at -20 degrees C. Both the 2H NMR and EPR spectra vary markedly with temperature. At temperatures well above the phase transition of the pure lipid, the average orientational parameters derived from the 2H NMR spectra are independent of protein concentration and are the same as for the lipid alone. Qualitatively, the EPR spectra show large apparent decreases in the average orientational order with increasing temperature. Analysis of 2H NMR relaxation rates indicates an additional motion in the presence of protein with a correlation time of 10(-6)-10(-7) s. If this new motion is associated with exchange between the two states, a minimum value of 10(6)-10(7) s-1 for the exchange rate is obtained, assuming that the lipids on the protein surface are much more motionally restricted than the rest of the lipid. Such an exchange rate is compatible with the observed differences in 2H NMR and EPR spectra. These results are consistent with short-lived, energetically weak interactions between cytochrome c oxidase and the phospholipids used in this study.

Transverse nuclear spin relaxation in phospholipid bilayer membranes

“…Measured values of T1 lead to T , I s while a typical value of the Larmor frequency in *H NMR, fo = 35 MHz, gives N 5 X lo4 s. Since the total second moment of 2H spins on acyl chains is M2 N 1.1 x 10' s -~, a lower limit for 7M is given by T~ 2 3 X 10" s and a reasonable estimate of T~ corresponds to rM N s (Seelig & Seelig, 1980). The relatively shorter T2 values in the liquid-crystalline phase are compatible with values of 72 in the range 10-7-10-8 s. Indeed, with this interpretation of T2, the reduction of T2 always observed upon addition of proteins to phospholipid bilayer membranes may be interpreted as indicating a significant slowing down of the acyl chains at the lipid-protein interface (Paddy et al, 1981;Bienvenue et al, 1982).…”

“…The generalization of these equations to include more than one type of motion is obvious [see, for example, Paddy et al (1981), eq 131.…”

Ordered and disordered phospholipid domains coexist in membranes containing the calcium pump protein of sarcoplasmic reticulum.

“…However, there is not universal agreement with this view. The major objection to this simple model has been that deuterium nuclear magnetic resonance (21H NMR) probes display a spectrum characteristic of a more disordered environment in protein-lipid recombinant membranes than in pure lipid bilayers (6,7). A possible resolution of the apparent disparity between ESR and 2H NMR has been suggested in terms of both the different time resolutions of these two techniques and a proposed disordered protein surface (6,8).…”

Physicochemical Determinations of Sterols by Deuterium and Carbon-13 Nuclear Magnetic Resonance

“…The idea proposed by Paddy et al (1981) is the following: One can describe the dynamics of such a system by a hierarchy in time of N motions (fulfilling the time-scale conditions previously mentioned that correlation time τ ί < 4.10 ~6 s), each motion being characterized by its correlation time, nature, activation energy, and order parameter. To simplify the analysis, it is assumed that motions are independent, have separated time scales, and possess axial symmetry.…”