Nitroxide spin label

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 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. 

If the g- and hyperfine anisotropies are known from analysis of a solid-state spectrum, the line-width parameters (1, and yt can be used to compute the rotational correlation time, tr, a useful measure of freedom of motion. Line widths in ESR spectra of nitroxide spin labels, for example, have been used to probe the motional freedom of biological macromolecules.14 Since tr is related to the molecular hydrodynamic volume, Ft, and the solution viscosity, r, by a relationship introduced by Debye 15... 

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). 

Figure 5.11 Effect of a slowing of the rate of rotational motion on the simulated ESR spectrum of a typical nitroxide spin label. Broadening becomes even more pronounced and non-uniform as the rate is further decreased.

The e.s.r. spectra of y-irradiated diethyl phosphate and its salts have been studied111 and the structures of the phosphonates(89)112 and some nucleoside phosphates113 have been studied from the e.s.r. spectra of the nitroxide spin-labelled compounds. An e.s.r. study of electron transfer in dinucleoside phosphate anions indicates preferen-... 

NITRENE NITROGENASE NITROGEN FIXATION Nitroxide spin-labeled phospholipids, PHOSPHOLIPID FLIP-FLOP NOISE... 

Neutral Nitroxide spin label — Reduced spin label... 

Miscellaneous bRC. - The bRC of R. sphaeroides contains one accessible cysteine at the H subunit (His H-156). Poluektov et al.m have bound a specific nitroxide spin label to this Cys and used temperature-dependent multifrequency (9 and 130 GHz) EPR to determine the motion of the protein. It was found that the restricted dynamics can be described as fast libration in a cone with a correlation time of >10 9 s. Several dynamically nonequivalent sites were observed that indicate conformational substates of local protein structure. 

Newer Aspects of the Synthesis and Chemistry of Nitroxide Spin Labels J. F. W. Keana, Chem. Rev., 1978, 78, 37-64. 

There have been many investigations of phosphotransferases by NMR and EPR methods.681,682 One approach is to use paramagnetic ions such as Mn2+, Cu2+, or Cr3+ to induce nuclear relaxation in substrate and coenzyme molecules at active sites of enzymes. Flavin radicals and specifically introduced nitroxide spin labels can serve as well. Paramagnetic ions greatly increase the rate of magnetic relaxation of nearby nuclei (Chapter 3, Section I). Thus, small amounts of... 

Such studies on creatine kinase (Eq. 12-31) utilized both a bound Mn2+ ion and a nitroxide spin label to estimate distances of various protons from the nitroxide.683 Together with EPR measurements (Box 8-C), which gave the Mn2+-nitroxide distance, a model of the ATP Mn2 complex in the active site was constructed. Additional EPR experiments on Mn2+ complexes with ATP and ADP containing 170 in the a, (3, or y phospho groups showed that in the enzyme ATP creatine complex the metal ion is bound to all three phospho groups of ATP. It remained coordinated with the two phospho groups of ADP and also that of the phospho-creatine product in the enzyme ADP creatine-P complex as well as in the transition state, which is pictured occurring via a metaphosphate ion.684... 

Figure 26 also depicts the distances calculated from fluorescence quenching experiments by enzyme-bound Co2+ on the s-adenosine moiety of s-ATP adenylylated glutamine synthetase (e-AMP-GS). Additional data were gathered by an EPR method that measured the decrease in EPR amplitude of the nitroxide spin-labeled adenylylated enzyme (TEMPO-AMP-GS) owing to enzyme-bound Mn2+ (122). Distances between Mn2+ and the N—O of the spin label are also shown in Fig. 26. 

Barhate, N., et al. (2007). A nucleoside that contains a rigid nitroxide spin label A fluorophore in disguise. Angew. Chem. Int. Ed. Engl. 46, 2655—2658. 

Edwards, T. E., and Sigurdsson, S. T. (2007). Site-specific incorporation of nitroxide spin-labels into 2,-positions of nucleic acids. Nat. Protoc. 2, 1954—1962. 

Hustedt, E. J., and Beth, A. H. (2000). Structural Information from CW-EPR spectra of dipolar coupled nitroxide spin labels. In Biological Magnetic Resonance Distance Measurements in Biological Systems by EPR, (L. J. Berliner, S. S. Eaton, and G. R. Eaton, eds.), Vol. 19. Kluwer Academic/Plenum Publisher, New York. 

Since malonate (Structure I) is able to fulfill the role of the anion in transferrin, it seemed reasonable to see whether spin-labeled derivatives of malonate could serve as probes of the active sites. Two such spin-labled derivatives were prepared and tentatively identified as having structures II (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonamide) and III (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonate). Similar results were obtained with each (Figure 3). Upon mixing Fe(III), transferrin, and II at low pH, and then raising the pH to near-neutrality with C02-free ammonia, the characteristic orange-red color of the ternary Fe-transferrin-anion complex is promptly displayed. However, the anticipated EPR signal of the nitroxide spin-label is not observed, presumably because it is broadened beyond detectability by its proximity... 

Hustedt, EJ. and Beth, A.H. (Structural information from CW-Esr spectra of dipolar coupled nitroxide spin labels, in Berliner, L, Eaton, S., and Eaton, G. (eds.),, Magnetic Resonance in Biology, V. 18, Kluwer Academic Publishers. Dordrecht, pp. 155-184... 

Functional groups attached to solvent-swollen polymer chains exhibit free rotational motion as indicated by electron spin resonance rotational correlation times 132-134) These studies using nitroxide spin labels covalently bound to polystyrene matrices indicated that the mobility of the substituent is a function of the cross-link density and degree of swelling. The rotational correlation time of nitroxide within 2% cross-linked beads was about 100 times shorter in dichloromethane or benzene than in ethanol, and 2-3 times longer than nitroxide bound to non-cross-linked polystyrene. The latter observation shows that the heterogeneous reaction involving 2% cross-linked polystyrene is 2-3 times slower than the same reaction in solution. 

---