Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels

Pates, Robert D.; Watts, Anthony; Uhl, Rainer; Marsh, Derek

doi:10.1016/0005-2736(85)90460-2

Cited by 26 publications

(30 citation statements)

References 19 publications

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“…For comparison, the number of lipids motionally restricted by bovine rhodopsin reconstituted in dimyristoyl phosphatidylcholine bilayers is 22 ± 2 (Ryba et al 1987). The corresponding values in native membranes are 25 ± 3 and 23 ± 2 lipids per rhodopsin for bovine and frog rod outer segment discs, respectively (Watts et al 1979; Pates et al 1985). The prediction from Equation is N b = 24 for a seven‐helix sandwich.…”

Section: Resultsmentioning

confidence: 99%

Stoichiometry of lipid interactions with transmembrane proteins—Deduced from the 3D structures

2006

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The stoichiometry of the first shell of lipids interacting with a transmembrane protein is defined operationally by the population of spin-labeled lipid chains whose motion is restricted directly by the protein. Interaction stoichiometries have been determined experimentally for a wide range of a-helical integral membrane proteins by using spin-label ESR spectroscopy. Here, we determine the spatially defined number of first-shell lipids at the hydrophobic perimeter of integral membrane proteins whose 3D structure has been determined by X-ray crystallography and lipid-protein interactions characterized by spin-labeling. Molecular modeling is used to build a single shell of lipids surrounding transmembrane structures derived from the PDB. Constrained energy optimization of the protein-lipid assemblies is performed by molecular mechanics. For relatively small proteins (up to 7-12 transmembrane helices), the geometrical first shell corresponds to that defined experimentally by perturbation of the lipid-chain dynamics. For larger, multi-subunit a-helical proteins, the lipids perturbed directly by the protein may either exceed or be less in number than those that can be accommodated at the intramembranous perimeter. In these latter cases, the motionally restricted spinlabeled lipids can be augmented by intercalation, or can correspond to a specific subpopulation at the protein interface, respectively. For monomeric b-barrel proteins, the geometrical lipid stoichiometry corresponds to that determined from lipid mobility for a 22-stranded barrel, but fewer lipids are motionally restricted than can be accommodated around an eight-stranded barrel. Deviations from the geometrical first shell, in the b-barrel case, are for the smaller protein with a highly curved barrel.

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Section: Resultsmentioning

confidence: 99%

Stoichiometry of lipid interactions with transmembrane proteins—Deduced from the 3D structures

2006

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“…NMR and EPR spin label experiments on rhodopsin in disc membranes and in artificial lipid vesicles have indicated that the protein is surrounded by a dynamic boundary layer of lipids (ca. 24) which are slightly immobilized as compared to those of the bulk phase [31–35]. The exchange rate is fast and is essentially determined by the free diffusion of the lipid molecules.…”

Section: Resultsmentioning

confidence: 99%

Evidence for the specific interaction of a lipid molecule with rhodopsin which is altered in the transition to the active state metarhodopsin II¹

1998

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Comparing the FTIR difference spectra of the rhodopsinCmetarhodopsin II transition in membranes and in dodecylmaltoside detergent, characteristic variations are observed between 1715 and 1750 cm 3I . By repeating the measurements with the rhodopsin mutant D83N/E122Q, the spectral variation between the samples in membranes versus detergent could be assigned to a difference band at 1743(+)/ 1724(3 3) cm 3I , which does not exhibit a deuteration-induced downshift. We provide evidence that this band is probably caused by the C=O stretch of only one ester group of one lipid molecule. This group interacts with the dark state of rhodopsin, whereas in metarhodopsin II, the lipid molecule behaves as if it were in the bulk lipid phase.z 1998 Federation of European Biochemical Societies.

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“…The two components of the complex spectrum were separated by computer subtraction of the immobilized component (24,25,37). The assumptions made were that for each of the specimens the complex spectrum at -40°C was representative of the immobilized component at all temperatures for that specimen and that its lineshape was independent of temperature.…”

Section: Resultsmentioning

confidence: 99%

“…Overshoot was easily observed as a distortion in the baseline. The uncertainties in estimating the end-point of the subtraction resulted in a ± 10% between the boundary layer and bulk membrane lipids (25).…”

Section: Resultsmentioning

confidence: 99%

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A Comparative Spin-Label Study of Isolated Plasma Membranes and Plasma Membranes of Whole Cells and Protoplasts from Cold-Hardened and Nonhardened Winter Rye

Windle¹

1988

Plant Physiol.

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ABSTRACILipid-lipid and lipid-protein interactions in the plasma membranes of whole cells and protoplasts and an isolated plasma membrane fraction from winter rye (Secale cereale L. cv Puma) have been studied by spin labeling. Spectra were recorded between -40°C and 40°C using the freely diffusing spin-label, 16-doxyl stearic acid, as a midbilayer membrane probe. The probe was reduced by the whole cells and protoplasts and reoxidized by external potassium ferricyanide. The reoxidized probe was assumed to be localized in the plasma membrane. The spectra consisted of the superposition of a narrow and a broad component indicating that both fluid and immobilized lipids were present in the plasma membrane. The two components were separated by digital subtraction of the immobilized component. Temperature profiles of the membranes were developed using the percentage of immobilized lipid present at each temperature and the separation between the outermost hyperfine lines for the fluid lipid component. Lipid immobilization was attributed to lipid-protein interactions, lipid-cell wall interactions, and temperature-induced lipid phase transitions to the gel-state. Temperature profiles were compared for both cold-hardened and nonhardened protoplasts, plasma membranes, and plasma membrane lipids, respectively. Although cold-hardening extended the range of lipid fluidity by 5°C, it had no effect on lipid-protein interactions or activation energies of lipid mobility. Differences were found, however, between the temperature profiles for the different samples, suggesting that alterations in the plasma membrane occurred as a consequence of the isolation methods used. (29,30,33,34). The spin label, 16-doxyl stearic acid, was selected as the membrane probe because it diffuses freely throughout the membrane and because its doxyl reporter group, which is located near the hydrophobic end of the acyl chain, is positioned near the center of the membrane lipid bilayer. This places it in the region of the membrane that has the greatest sensitivity to changes in lipid fluidity and order. In the present work, we show that spectra, recorded over a wide temperature range, were composed of the superposition of an immobilized and a fluid component indicating two lipid environments for plant membranes. Temperature profiles for the specimens were developed to compare the plasma membranes from hardened and nonhardened Puma rye and to interpret spectral features in terms of the effects of plasmolysis, protoplast isolation, plasma membrane fractionation, and cold acclimation on lipid-lipid and lipid-protein interactions in the plasma membrane.The methods developed for isolating single, whole cells and protoplasts using enzymic digestion and mechanical treatment of the plant tissue (29,30) and for obtaining plasma membrane enriched fractions from tissue homogenates by phase partitioning (34) subject the plasma membrane to progressively harsher treatment. Questions have been raised of how similar the plasma membranes of protoplasts are to the plasma ...

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Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels

Cited by 26 publications

References 19 publications

Stoichiometry of lipid interactions with transmembrane proteins—Deduced from the 3D structures

Stoichiometry of lipid interactions with transmembrane proteins—Deduced from the 3D structures

Evidence for the specific interaction of a lipid molecule with rhodopsin which is altered in the transition to the active state metarhodopsin II¹

A Comparative Spin-Label Study of Isolated Plasma Membranes and Plasma Membranes of Whole Cells and Protoplasts from Cold-Hardened and Nonhardened Winter Rye

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Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels

Cited by 26 publications

References 19 publications

Stoichiometry of lipid interactions with transmembrane proteins—Deduced from the 3D structures

Stoichiometry of lipid interactions with transmembrane proteins—Deduced from the 3D structures

Evidence for the specific interaction of a lipid molecule with rhodopsin which is altered in the transition to the active state metarhodopsin II1

A Comparative Spin-Label Study of Isolated Plasma Membranes and Plasma Membranes of Whole Cells and Protoplasts from Cold-Hardened and Nonhardened Winter Rye

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Evidence for the specific interaction of a lipid molecule with rhodopsin which is altered in the transition to the active state metarhodopsin II¹