Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1982 Jan;37(1):265–274. doi: 10.1016/S0006-3495(82)84675-4

ESR spin-label studies of lipid-protein interactions in membranes.

D Marsh, A Watts, R D Pates, R Uhl, P F Knowles, M Esmann
PMCID: PMC1329135  PMID: 6275924

Abstract

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.

Full text

PDF
265

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boss W. F., Kelley C. J., Landsberger F. R. A novel synthesis of spin label derivatives of phosphatidylcholine. Anal Biochem. 1975 Mar;64(1):289–292. doi: 10.1016/0003-2697(75)90432-7. [DOI] [PubMed] [Google Scholar]
  2. Brotherus J. R., Jost P. C., Griffith O. H., Keana J. F., Hokin L. E. Charge selectivity at the lipid-protein interface of membranous Na,K-ATPase. Proc Natl Acad Sci U S A. 1980 Jan;77(1):272–276. doi: 10.1073/pnas.77.1.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cable M. B., Jacobus J., Powell G. L. Cardiolipin: a stereospecifically spin-labeled analogue and its specific enzymic hydrolysis. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1227–1231. doi: 10.1073/pnas.75.3.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cable M. B., Powell G. L. Spin-labeled cardiolipin: preferential segregation in the boundary layer of cytochrome c oxidase. Biochemistry. 1980 Dec 9;19(25):5679–5686. doi: 10.1021/bi00566a003. [DOI] [PubMed] [Google Scholar]
  5. Davoust J., Bienvenue A., Fellmann P., Devaux P. F. Boundary lipids and protein mobility in rhodopsin-phosphatidylcholine vesicles. Effect of lipid phase transitions. Biochim Biophys Acta. 1980 Feb 15;596(1):28–42. doi: 10.1016/0005-2736(80)90168-6. [DOI] [PubMed] [Google Scholar]
  6. Davoust J., Schoot B. M., Devaux P. F. Physical modifications of rhodopsin boundary lipids in lecithin-rhodopsin complexes: a spin-label study. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2755–2759. doi: 10.1073/pnas.76.6.2755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deese A. J., Dratz E. A., Brown M. F. Retinal rod outer segment lipids form bilayers in the presence and absence of rhodopsin: a 31P NMR study. FEBS Lett. 1981 Feb 9;124(1):93–99. doi: 10.1016/0014-5793(81)80061-0. [DOI] [PubMed] [Google Scholar]
  8. Devaux P. F., Davoust J., Rousselet A. Electron spin resonance studies of lipid-protein interactions in membranes. Biochem Soc Symp. 1981;(46):207–222. [PubMed] [Google Scholar]
  9. Esmann M., Christiansen C., Karlsson K. A., Hansson G. C., Skou J. C. Hydrodynamic properties of solubilized (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. Biochim Biophys Acta. 1980 Dec 2;603(1):1–12. doi: 10.1016/0005-2736(80)90386-7. [DOI] [PubMed] [Google Scholar]
  10. Favre E., Baroin A., Bienvenue A., Devaux P. F. Spin-label studies of lipid-protein interactions in retinal rod outer segment membranes. Fluidity of the boundary layer. Biochemistry. 1979 Apr 3;18(7):1156–1162. doi: 10.1021/bi00574a006. [DOI] [PubMed] [Google Scholar]
  11. Hoffmann W., Pink D. A., Restall C., Chapman D. Intrinsic molecules in fluid phospholipid bilayers. Fluorescence probe studies. Eur J Biochem. 1981 Mar;114(3):585–589. doi: 10.1111/j.1432-1033.1981.tb05184.x. [DOI] [PubMed] [Google Scholar]
  12. Hubbell W. L., McConnell H. M. Molecular motion in spin-labeled phospholipids and membranes. J Am Chem Soc. 1971 Jan 27;93(2):314–326. doi: 10.1021/ja00731a005. [DOI] [PubMed] [Google Scholar]
  13. Jost P. C., Capadil R. A., Vanderkooi G., Griffith O. H. Lipid-protein and lipid-lipid interactions in cytochrome oxidase model membranes. J Supramol Struct. 1973;1(4):269–280. doi: 10.1002/jss.400010404. [DOI] [PubMed] [Google Scholar]
  14. Jost P. C., Griffith O. H., Capaldi R. A., Vanderkooi G. Evidence for boundary lipid in membranes. Proc Natl Acad Sci U S A. 1973 Feb;70(2):480–484. doi: 10.1073/pnas.70.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knowles P. F., Watts A., Marsh D. Spin-label studies of lipid immobilization in dimyristoylphosphatidylcholine-substituted cytochrome oxidase. Biochemistry. 1979 Oct 16;18(21):4480–4487. doi: 10.1021/bi00588a005. [DOI] [PubMed] [Google Scholar]
  16. Marsh D., Barrantes F. J. Immobilized lipid in acetylcholine receptor-rich membranes from Torpedo marmorata. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4329–4333. doi: 10.1073/pnas.75.9.4329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marsh D., Watts A., Barrantes F. J. Phospholipid chain immobilization and steroid rotational immobilization in acetylcholine receptor-rich membranes from Torpedo marmorata. Biochim Biophys Acta. 1981 Jul 6;645(1):97–101. doi: 10.1016/0005-2736(81)90516-2. [DOI] [PubMed] [Google Scholar]
  18. Marsh D., Watts A., Maschke W., Knowles P. F. Protein--immobilized lipid in dimyristoylphosphatidylcholine-substituted cytochrome oxidase: evidence for both boundary and trapped-bilayer lipid. Biochem Biophys Res Commun. 1978 Mar 30;81(2):397–402. doi: 10.1016/0006-291x(78)91546-2. [DOI] [PubMed] [Google Scholar]
  19. Rousselet A., Devaux P. F., Wirtz K. W. Free fatty acids and esters can be immobilized by receptor rich membranes from Torpedo marmorata but not phospholipid acyl chains. Biochem Biophys Res Commun. 1979 Oct 12;90(3):871–877. doi: 10.1016/0006-291x(79)91908-9. [DOI] [PubMed] [Google Scholar]
  20. Skou J. C., Esmann M. Preparation of membrane-bound and of solubilized (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. The effect of preparative procedures on purity, specific and molar activity. Biochim Biophys Acta. 1979 Apr 12;567(2):436–444. doi: 10.1016/0005-2744(79)90129-3. [DOI] [PubMed] [Google Scholar]
  21. Uhl R., Kuras P. V., Anderson K., Abrahamson E. W. A light scattering study on the ion permeabilities of dark-adapted bovine rod outer segment disk membranes. Biochim Biophys Acta. 1980 Oct 2;601(3):462–477. doi: 10.1016/0005-2736(80)90550-7. [DOI] [PubMed] [Google Scholar]
  22. Watts A., Davoust J., Marsh D., Devaux P. F. Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results. Biochim Biophys Acta. 1981 May 20;643(3):673–676. doi: 10.1016/0005-2736(81)90365-5. [DOI] [PubMed] [Google Scholar]
  23. Watts A., Volotovski I. D., Marsh D. Rhodopsin-lipid associations in bovine rod outer segment membranes. Identification of immobilized lipid by spin-labels. Biochemistry. 1979 Oct 30;18(22):5006–5013. doi: 10.1021/bi00589a031. [DOI] [PubMed] [Google Scholar]
  24. Watts A., Volotovski I. D., Pates R., Marsh D. Spin-label studies of rhodopsin-lipid interactions. Biophys J. 1982 Jan;37(1):94–95. doi: 10.1016/S0006-3495(82)84617-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES