Protein-lipid interactions. High-field deuterium and phosphorus nuclear magnetic resonance spectroscopic investigation of the cytochrome oxidase-phospholipid interaction and the effects of cholate

Abstract

Deuterium quadrupole-echo Fourier transform nuclear magnetic resonance spectra (at 34 MHz) and phosphorus-3 1 Fourier transform nuclear magnetic resonance spectra (at 60.7 MHz) have been obtained of 1-(6,6-dideuteriopalmitoyl)-2-oleyl-sn-glycero-3-phosphocholine dispersions in excess water in the absence of, and complexed with, the membrane enzyme cytochrome oxidase (cytochrome c:02 oxidoreductase, EC 1.9.3.1). Thereby, we have investigated the effects of the detergent sodium cholate, and of temperature, on protein-lipid interactions in this system. Our results strongly suggest that residual detergent in these protein-lipid complexes causes a significant disordering of hydrocarbon chain and head group organization as determined by deuterium quadrupole splittings (AvQ) and phosphorus chemical shielding anisotropies (Aa). At low (-2 wt %) cholate levels, C6 AuQ and 31P ha values in the protein-lipid complexes (containing T h e nature of the interactions between proteins and lipids in biological membranes is a topic of considerable current t From the Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (D.M.R. and E.O.), and the Department of Chemistry and the Laboratory for Bioenergetics, State University of New York at Albany, Albany, New York 12222 (J.C.H. and T.E.K.). Received April I I , 1979; revised manuscript received Augusl 15, 1979. This work was supported by the National Institutes of Health (Grant HL-19481 to E.O. and Grants GM-16767 and HL12576 to T.E.K.) and in part by the Alfred P. Sloan Foundation (E.O.). *Correspondence should be addressed to this author. He is an Alfred P. Sloan Research Fellow, 1978-1980. *Supported in part by a National Institutes of Health, Cell and Molecular Biology Training grant (Grant GM-07283). -70 wt % protein) are only about 8% smaller than in pure phospholipid bilayers at the same temperature, between 20 and 35 “C, suggesting rather similar structural organization. There are, however, significant line-width increases, especially on approaching T, in both deuterium and phosphorus spectra, indicating increased correlation times, in the region of the membrane surface. These results obtained on protein-lipid complexes containing an unsaturated phospholipid are in agreement with those of a previous study utilizing a disaturated phospholipid [Kang, S . Y., Gutowsky, H. S . , Hsung, J. C., Jacobs, R., King, T. E., Rice, D., & Oldfield, E. (1979) Biochemistry 18, 3257-32671 but differ significantly from those of a similar study employing 1-(5,5-dideuteriopalmitoyl)-2-oleyl-sn-glycero-3-phosphocholine [Seelig, A,, & Seelig, J . (1978) Hoppe-Seyler’s 2. Physiol. Chem. 359, 1747-17561. interest due to the frequent observation that a variety of membrane enzymes appear to require lipids in order to express their full biological activity [e.g., Yu et al. (1975), Gennis & Jonas (1977), Tanford & Reynolds (1976), Bennett et al. (1978), and Vik & Capaldi (1977)l. The most frequent model for the nature of the protein-lipid interaction is that the lipids are somehow immobilized (Jost et al., 1973a,b, 1977; Marsh et al., 1978; Warren et al., 1974, 1975; Longmuir et al., 1977) by the rigid protein “wall” (Moore et al., 1978), a cholesterol-like effect (Hong & Hubbell, 1972; Kleemann & McConnell, 1976), and that as a result the lipid hydrocarbon chain organization becomes far more restricted, leading to increased molecular order parameters (Dahlquist et al., 1977; MarEelja, 1976; Jost & Griffith, 1978; Scott & Cherng, 1978). 0006-2960/79/0418-5885S01 .OO/O , 1