Spin-Probe—spin-Label Investigations of Model Membranes

Hyde, James S.; Popp, Carol A.; Schreier, Shirley

doi:10.1016/b978-0-12-225402-4.50060-7

Cited by 5 publications

(3 citation statements)

References 7 publications

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“…3. Judging from previous work, this is not unexpected since polar moieties like the nitroxide attached near the terminal position of the chain result in a "bending back" of that position to the aqueous surface (15,22). Thus n-doxyl PCs with nitroxides near the chain terminus should be used with caution.…”

Section: Discussionmentioning

confidence: 99%

“…For two paramagnetic reagents with similar sizes, the ratio of the exchange rates with a given nitroxide is independent of steric factors and would depend on distance through the concentration gradient only. The interaction of fast-relaxing paramagnetic reagents such as 02 and NiAA with nitroxides in fluid media is dominated by Heisenberg exchange and produces changes in the spin-lattice relaxation time (T1) proportional to We,, (15). The experimental quantity AP1/2 is related to the exchange rate according to AP1/2°c WeX/T*2, RESULTS A structural model of bR based on electron diffraction (16) and earlier EPR results (1) is shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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“…Current literature data on cancer detection with EPR suggests that 16-doxyl stearic acid (16-DS) should be used for the study of conformational changes of HSA, due to its structural congruence with HSA and the position of the nitroxide group at the terminal part of the FA chain, also claiming that 16-DS is highly specific for HSA. − However, many studies have shown that when FAs, labeled with the doxyl group located at different positions on the carbon chain, are bound to albumin, the doxyl group of the 16-DS is located in a more polar and less fixed environment than that of the 5-doxyl stearic acid (5-DS). , There is nothing special in the binding of 16-DS to HSA; quite to the contrary, it may have some disadvantages over using other SLFAs, e.g., 5-DS. Namely, studies of incorporation of 16-DS to artificial and natural membranes clearly indicated that 16-DS may “bend-back” to the aqueous surface, so that the polar nitroxide group on the C-16 atom is not located inside the membrane. − This suggests that the same bending effect may occur during the binding of 16-DS to HSA subdomains so that the polar label on C-16 is out. Indeed, the study of the accessibility of different SLFAs bound to bovine serum albumin (BSA) has shown that 16-DS is more accessible to water-soluble ferricyanide than 5-DS, suggesting that it protrudes from the protein’s hydrophobic pocket …”

Section: Introductionmentioning

confidence: 99%

Binding of Doxyl Stearic Spin Labels to Human Serum Albumin: An EPR Study

et al. 2014

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The binding of spin-labeled fatty acids (SLFAs) to the human serum albumin (HSA) examined by electron paramagnetic resonance (EPR) spectroscopy was studied to evaluate the potential of the HSA/SLFA/EPR technique as a biomarking tool for cancer. A comparative study was performed on two spin labels with nitroxide groups attached at opposite ends of the fatty acid (FA) chain, 5-doxyl stearic (5-DS) and 16-doxyl stearic (16-DS) acid. The effects of incubation time, different [SLFA]/[HSA] molar ratios, ethanol, and temperature showed that the position of the nitroxide group produces certain differences in binding between the two SLFAs. Spectra for different [SLFA]/[HSA] molar ratios were decomposed into two spectral components, which correspond to the weakly and strongly bound SLFAs. The reduction of SLFA with ascorbate showed the existence of a two component process, fast and slow, confirming the decomposition results. Warfarin has no effect on the binding of the two SLFAs, whereas ibuprofen significantly decreases the binding of 5-DS and has no effect on 16-DS. Together, the results of this study indicate that both SLFAs, 5-DS and 16-DS, should be used for the study of HSA conformational changes in blood induced by various medical conditions.

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Autobiography of James S. Hyde

Hyde

2017

Appl Magn Reson

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The papers, book chapters, reviews, and patents by James S. Hyde in the bibliography of this document have been separated into EPR and MRI sections, and within each section by topics. Within each topic, publications are listed chronologically. A brief summary is provided for each patent listed. A few publications and patents that do not fit this schema have been omitted. This list of publications is preceded by a scientific autobiography that focuses on selected topics that are judged to have been of most scientific importance. References to many of the publications and patents in the bibliography are made in the autobiography.

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Spin-Probe—spin-Label Investigations of Model Membranes

Cited by 5 publications

References 7 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.

Binding of Doxyl Stearic Spin Labels to Human Serum Albumin: An EPR Study

Autobiography of James S. Hyde

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