Oxygen diffusion–concentration in phospholipidic model membranes. An electron spin resonance–saturation study

Vachon, André; Lecomte, Conrad; Berleur, François; Roman, V. K.; Fatome, M.; Braquet, P.

doi:10.1039/f19878300177

Cited by 9 publications

(4 citation statements)

References 1 publication

(1 reference statement)

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“…In addition, the relaxation times estimated from CW saturation methods are effective relaxation times since they are affected by nitrogen nuclear relaxation rates (Hyde & Sarna, 1978;Subczynski & Hyde, 1981). However, changes in Pl/2 due to the presence of an exchange reagent reflect parallel changes in 7le due to collisions (Hyde & Sarna, 1978;Subczynski & Hyde, 1981;Vachon et al, 1987).…”

Section: Theorymentioning

confidence: 99%

Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines

Altenbach¹,

Flitsch²,

Khorana³

et al. 1989

Biochemistry

287

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Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.

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Section: Theorymentioning

confidence: 99%

Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines

Altenbach¹,

Flitsch²,

Khorana³

et al. 1989

Biochemistry

287

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“…Another useful probe of topography for membrane proteins is molecular oxygen (Subczynski and Hyde, 1981;Vachon et al, 1987;Altenbach et al, 1989). Like CROX, it is fast relaxing and causes efficient spin lattice relaxation upon collision with a nitroxide.…”

Section: Conformation Of Membrane-bound Melittinmentioning

confidence: 99%

Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation recovery and continuous wave power saturation electron paramagnetic resonance

Altenbach¹,

Froncisz²,

Hyde³

et al. 1989

Biophysical Journal

131

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Melittin spin-labeled specifically with a nitroxide at positions 7, 21, 23, or the amino terminus was bound to phospholipid membranes, and the exposure of the spin label to the aqueous phase was investigated by measurement of Heisenberg exchange with chromium oxalate in the solution. The exchange frequency was determined by saturation recovery electron paramagnetic resonance (EPR) using a loop-gap resonator. This method allows use of very low concentrations (less than 1 mM) of chromium oxalate compared with conventional measurements of EPR line broadening (typically 50 mM), thus avoiding problems associated with high metal ion concentration. Differences in exchange frequency between the various positions were also estimated by continuous wave power saturation methods. In either approach, the spin label at lysine 7 was found to be the most exposed to chromium oxalate whereas that at lysine 23 was found to be the least exposed. This is consistent with a model for the membrane bound peptide in which an amphiphilic helix lies with its axis parallel to the bilayer surface and the hydrophobic moment points toward the bilayer interior.

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“…Polar molecules preferentially partition into the aqueous phase, and nonpolar molecules preferentially partition into the membrane phase. The bilayer interior, however, is nonuniform, with gradients of fluidity (11,12) and polarity (13) (14) Wex = 4trpgdDm(x)Cm(x), [21 where p is the exchange probability, g is a steric factor, d is the collision diameter, Dm(x) is the position-dependent relative diffusion coefficient, and Cm(x) is the position-dependent concentration given by Eq. 1.…”

Section: Methodsmentioning

confidence: 99%

A collision gradient method to determine the immersiondepth of nitroxides in lipid bilayers: application to spin-labeled mutants ofbacteriorhodopsin.

Altenbach¹,

Greenhalgh²,

Khorana³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

433

636

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Ten mutants of bacteriorhodopsin, each containing a single cysteine residue regularly spaced along helix D and facing the lipid bilayer, were derivatized with a nitroxide spin label. Collision rates of the nitroxide with apolar oxygen increased with distance from the membrane/solution interface. Collision rates with polar metal ion complexes decreased over the same distance. Although the collision rates depend on steric constraints imposed by the local protein structure and on the depth in the membrane, the ratio of the collision rate of oxygen to those of a polar metal ion complex is independent of structural features of the protein. The logarithm of the ratio is a linear function of depth within the membrane. Calibration of this ratio parameter with spin-labeled phospholipids allows localization of the individual nitroxides, and hence the bacteriorhodopsin molecule, relative to the plane of the phosphate groups of the bilayer. The spacing between residues is consistent with the pitch ofan a-helix. These results provide a general strategy for determining the immersion depth of nitroxides in bilayers.Site-directed spin labeling has become a powerful tool for determination of membrane protein structures and their disposition with respect to the bilayer (1-3). In previous studies, information on the region where transmembrane helices intersect with the membrane/solution interface has been obtained by analysis of a consecutive series of mutants that traverse the interface (1). If it were possible to determine the vertical distance of a spin-labeled side chain from the plane of the phosphates of the lipid headgroups, analysis of only one or two spin-labeled mutants would be sufficient to determine the position ofa transmembrane domain relative to the membrane.EPR methods for estimation of the depth of immersion of a nitroxide in the membrane have been reported (4-6). These methods are based upon the dipolar interactions of the nitroxide with paramagnetic reagents constrained to the aqueous phase and require knowledge of the spacial distribution of the paramagnetic reagents in solution. This distribution may be readily deduced for a pure bilayer with a nitroxide on a lipid chain, but not for a nitroxide attached to a protein in a bilayer. This is because the protein has an unknown excluded volume for the paramagnetic reagent in the aqueous phase.In this report, we make use of site-directed spin labeling to introduce nitroxides along the entire length of helix D of bacteriorhodopsin (bR). The collision frequencies of the nitroxides with paramagnetic reagents are dependent upon position, and this effect is shown to provide an approach for localization of nitroxides in the membrane interior. MATERIALS AND METHODSEgg yolk phosphatidylcholine (PC) and 1-palmitoyl-2-(ndoxylpalmitoyl) PC spin-labeled isomers with n = 5, 7, 10, 12, and 16 were obtained from Avanti Polar Lipids. Ethylenediamine-N,N'-diacetic acid (EDDA), nickel(II) acetylacetonate (NiAA), and Ni(OH)2 were obtained from Aldrich. bR mutants were prepare...

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Oxygen diffusion–concentration in phospholipidic model membranes. An electron spin resonance–saturation study

Cited by 9 publications

References 1 publication

Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines

Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines

Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation recovery and continuous wave power saturation electron paramagnetic resonance

A collision gradient method to determine the immersiondepth of nitroxides in lipid bilayers: application to spin-labeled mutants ofbacteriorhodopsin.

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