Methods of physical labels - a combined approach to the study of microstructure and dynamics in biological systems

Likhtenstein, G. I.; Куликов, А. В.; Kotelnikov, A. I.; Levchenko, L. A.

doi:10.1016/0165-022x(86)90047-3

Cited by 21 publications

(13 citation statements)

References 18 publications

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“…EPR methods for estimation of the depth of immersion of a nitroxide in the membrane have been reported (4)(5)(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.…”

mentioning

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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mentioning

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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“…[1] Table 3. For 8-PCSL, the smaller values of ⌬(1/P 2 ) for MnCl 2 relative to CuCl 2 , e.g., 0.30 versus 0.41 G 2 in the fluid phase, are contrary to expectations for a static dipolar relaxation mechanism (cf.…”

Section: Dependence On Ion Magnetic Momentmentioning

confidence: 98%

“…Calibrations were established for the experimental values of the saturation parameters, P and P 2 , for the integrated intensities (Eq. [1]) and spectral amplitudes (Eq.…”

Section: Spin-label T 1 Relaxation Timesmentioning

confidence: 99%

“…(1) Measurement of spin-label relaxation enhancements induced by interaction with paramagnetic ions, or their complexes, is a valuable and proven approach to studying the spatial distribution of spinlabeled functional groups of both phospholipids and proteins in membranes (1)(2)(3)(4)(5). However, to obtain reliable structural information from such spin-label EPR measurements, the mechanisms of the enhancements need to be clearly understood in the various cases.…”

Section: Introductionmentioning

confidence: 99%

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Mechanism of Relaxation Enhancement of Spin Labels in Membranes by Paramagnetic Ion Salts: Dependence on 3d and 4f Ions and on the Anions

Лившиц

Dzikovski

Marsh

2001

Journal of Magnetic Resonance

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Progressive saturation EPR measurements and EPR linewidth determinations have been performed on spin-labeled lipids in fluid phospholipid bilayer membranes to elucidate the mechanisms of relaxation enhancement by different paramagnetic ion salts. Such paramagnetic relaxation agents are widely used for structural EPR studies in biological systems, particularly with membranes. Metal ions of the 3d and 4f series were used as their chloride, sulfate, and perchlorate salts. For a given anion, the efficiency of relaxation enhancement is in the order Mn(2+) > or = Cu(2+) > Ni(2+) > Co(2+) approximately Dy(3+). A pronounced dependence of the paramagnetic relaxation enhancement on the anion is found in the order ClO(-)(4) > Cl(-) > SO(2-)(4). This is in the order of the octanol partition coefficients multiplied by spin exchange rate constants that were determined for the different paramagnetic salts in methanol. Detailed studies coupled with theoretical estimates reveal that, for the chlorides and perchlorates of Ni(2+) (and Co(2+)), the relaxation enhancements are dominated by Heisenberg spin exchange interactions with paramagnetic ions dissolved in fluid membranes. The dependence on membrane composition of the relaxation enhancement by intramembrane Heisenberg exchange indicates that the diffusion of the ions within the membrane takes place via water-filled defects. For the corresponding Cu(2+) salts, additional relaxation enhancements arise from dipolar interactions with ions within the membrane. For the case of Mn(2+) salts, static dipolar interactions with paramagnetic ions in the aqueous phase also make a further appreciable contribution to the spin-label relaxation enhancement. On this basis, different paramagnetic agents may be chosen to optimize sensitivity to different structurally correlated interactions. These results therefore will aid further spin-label EPR studies in structural biology.

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“…This is perhaps due to the experimental difficulfies as well as to the absence of an adequate theory taking into account all the peculiafifies of dynamic picture in the case of spin-labeled proteins. However, as it was shown in [4], electron-spin relaxation is quite sensitive to structure transformafions and various mofions in proteins, as well as to the dynamics in the spin system. Although the choice of an adequate model of motion is crucial for the extracfion of microdynamical parameters from the relaxation data, the analysis of relaxafion in spin-labeled proteins is usually done in terms of isotropic-mofion model, which is notable to mimic the complicated picture of motions descfibed above.…”

Section: Introductionmentioning

confidence: 94%

Investigation of the molecular motion of spin-labeled lysozyme in solution by the use of ESP spectra and electron spin relaxation

et al. 1993

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Abstract. The theory of electron spin relaxation in protein solutions taking into account the "two-motion" model of spin label mobility is developed. The relations obtained for the longitudinal and transverse relaxation rates describe the experimental results better than isotropic ("one-motion") model. The mechanism of longitudinal relaxation in solution of spin-labeled lysozyme is revealed and the correlation time of the inherent motion of spin label is evaluated. The linewidth analysis and study of ESR spectra under viscosity variation were used to obtaln the microdynamical parameters characterizing lysozyme molecular mobility. A discrepancy between the correlation time values obtained by viscosity-variation-technique and the results known from other methods is found and ascribed to the manifestation of the intermediatetime-scale mobility of protein.

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Methods of physical labels - a combined approach to the study of microstructure and dynamics in biological systems

Cited by 21 publications

References 18 publications

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

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

Mechanism of Relaxation Enhancement of Spin Labels in Membranes by Paramagnetic Ion Salts: Dependence on 3d and 4f Ions and on the Anions

Investigation of the molecular motion of spin-labeled lysozyme in solution by the use of ESP spectra and electron spin relaxation

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