Spin-label

3 Spin Labels. - Spin labels have traditionally been a tool of ESR spectroscopy, where the ESR resonance of the free radical label (usually a nitroxide-containing species, such as TEMPO) is observed directly. However, spin labels have increasingly become a tool of NMR as T1 relaxation enhancing agents. 

Aime et have used the coordinatively-unsaturated Gd chelate, Gd-D03A, as a relaxation label which binds semi-selectively to solvent-exposed carboxylates on the surface of a protein. In this complex, Gd(III) is 7-coordinate, leaving two sites available for oxygen donors from the protein. In experimental studies, paramagnetic Ti relaxation enhancements were measured for the amide protons by inversion-recovery HSQC and used to provide distance 

Metal ions that are bound to proteins as paramagnetic labels are often conjugated by flexible linkages, in which case the location of the metal ion with respect to the protein is not well defined. To handle this problem, Iwahara et have developed a computational strategy which more accurately back-calculates the paramagnetic relaxation enhancement based on a multiple-structure representation of the paramagnetic label in simulated annealing calculations. 

2 Protein-lipid interaction. Ellena et have studied the localization of perfluorocarbons in lipid membranes, using for this purpose Gd and O2 as relaxation-enhancing agents of the resonance. 

Hilty et alP have used spin labels to study protein-lipid interactions in mixed micelles containing dihexanoylphosphatidylcholine and the E. coli outer-membrane protein X (OmpX). As paramagnetic relaxation probes, they used several different nitroxide spin labels attached to the lipid as well as Gd-DOTA, which remains in the aqueous phase. Spectral perturbations were monitored in TROSY and ID H NMR spectra. 

Applications of spin labels to problems in structural biology have continued to grow over the four decades since McConnell s original proposal. We mention here only two examples, which provided early support for the method. 

Morrisett, et al.17 studied phase transitions in E. coli membranes using three different spin labels, including 5-doxyl stearate (5-DS) (7). 

They observed abrupt changes in the slope of Arrhenius plots for reactions catalyzed by NADH oxidase and p-lactate oxidase that correlate well with phase transitions detected by the ESR spectra of the nitroxide spin labels bound covalently to the enzymes (Table 5.4). 

Another example comes from the work of Johnson, et a/.18 These workers studied spin labels dissolved in lipid bilayer dispersions of dipalmitoylphos-phatidylcholine and cholesterol (9 1 by weight) in the hope that anisotropic rotational diffusion of the spin label would mimic the motion of the bilayer components. In addition to 5-DS, which is sensitive to rotational motion about the NO bond, they used the steroidal nitroxide 8, which tends to rotate about an axis perpendicular to the N-O bond. ESR measurements were carried out at both 9 and 35 GHz and at temperatures ranging from 30 to 30 °C. Rather different results were obtained with the two spin labels, largely as a result of the different axes of rotation. Because the rotation rates were very slow, ESR spectra appeared as powder patterns rather than isotropic spectra and special methods were needed to extract the motional data. 

Atkins, Electron Spin Resonance, Specialist Periodical Report, Royal Society of Chemistry, London, 1973,1,47 1974,2, 52 1976,3, 35 1977,4,1. 

The Determination of the Topology of the Membrane-NPY Interface 5.3.3.1 Spin Labels 

Spin labels contain unpaired electrons that by highly efficient electron-nuclear spin dipolar coupling lead to accelerated transverse or longitudinal relaxation. The effect is rather far-reaching (at least up to 10 A), and its general use is described in Chapt. 15. 

The doxylstearates may be prepared as concentrated (e. g. 0.3 M) methanolic stock solutions. They should be added such that their final concentration in the micelle solution is about one spin label per micelle. When possible, the spin label should be added to the micelle solution before the peptide to ensure that it integrates into the micelles properly and does not bind specifically with the peptide. 

In principle, spin label effects can be observed in both homonuclear and heteronuclear spectra. When no isotope-enriched peptide is available, integration of TOCSY spectra in the presence and in the absence of spin label gives information of spatial proximity of 

The so-called snorkelling effect of Arg or Lys side chains [35] has been observed frequently and may lead to erroneous interpretation of the data. 

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Figure Bl.15.14. Comparison of 95.1 GHz (A) and 9.71 GHz (B) EPR spectra for a frozen solution of a nitroxide spin label attached to insulin measured at 170 K.

The structure of spin-labeled acetylenes includes 7r-electron-rich pyrazole and... 

Sodium dodecyl sulfate micelle 71,72,77,79 Spin label 139 Starch 100, 104 —, crosslinked 106 —, graft polymers 105, 107, 125, 127 Styrene 160—162 Styrene-divinylbenzene resins 167 Styrenesulfonic acid, copolymers 74—76 Surface area 147... 

Interaction of spin labels with transition metals. S. S. Eaton and G. R. Eaton, Coord. Chem. Rev., 1978,26, 207-262 (218). 

The values for the lipid molecules compare well (althoughJgiey are still somewhat larger) with the experimental value of 1.5x10 cm /s as measured with the use of a nitroxide spin label. We note that the discrepancy of one order of magnitude, as found in the previous simulation with simplified head groups, is no longer observed. Hence we may safely conclude that the diffusion coefficient of the lipid molecules is determined by hydrodynamic interactions of the head groups with the aqueous layer rather than by the interactions within the lipid layer. The diffusion coefficient of water is about three times smaller than the value of the pure model water thus the water in the bilayer diffuses about three times slower than in the bulk. 

In the second case, the 180° pulse is applied only to nucleus A, causing an exchange of spin labels of the A spin states to occur, so the direction of rotation of the X magnetization vectors is reversed during the second... 

An expansion of the method can be found in second-site SLAPSTIC, where, in favorable cases, a second binding site close to the binding site of interest can be used to infer a spin label via a second ligand [116]. 

Berliner, L.J. (1976) Spin-labelling Theory and Applications Academic Press, New York. 

Ligeza A, Tikhonov AN, Hyde JS, Subczynski WK. 1998. Oxygen permeability of thylakoid membranes Electron paramagnetic resonance spin labeling study. Biochim Biophys Acta 1365 453. 

However, the equilibrium of the indicator adsorbed at an interface may also be affected by a lower dielectric constant as compared to bulk water. Therefore, it is better to use instead pH, the interfacial and bulk pK values in Eq. (50). The concept of the use at pH indicators for the evaluation of Ajy is also basis of other methods, like spin-labeled EPR, optical and electrochemical probes [19,70]. The results of the determination of the Aj by means of these methods may be loaded with an error of up to 50mV [19]. For some the potentials determined by these methods, Ajy values are in a good agreement with the electrokinetic (zeta) potentials found using microelectrophoresis [73]. It is proof that, for small systems, there is lack of methods for finding the complete value of A>. 

Fig. 1.11 Structure of spin-labelled adenosine (l-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylate (5-aminoadenosine) -amide).

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