Spin labels for

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... 

Based on our current understanding of ribosomal protein synthesis, several strategies have been developed to incorporate amino acids other than the 20 standard proteinogenic amino acids into a peptide using the ribosomal machinery . This allows for the design of peptides with novel properties. On the one hand, such a system can be used to synthesize nonstandard peptides that are important pharmaceuticals. In nature, such peptides are produced by nonribosomal peptide synthetases, which operate in complex pathways. On the other hand, non-natural residues are a useful tool in biochemistry and biophysics to study proteins. For example, incorporation of non-natural residues by the ribosome allows for site-specific labeling of proteins with spin labels for electron paramagnetic resonance spectroscopy, with... 

There is much evidence, usually based upon ESR studies with spin labels, for conformational changes in the protein during the enzyme cycle. Enzyme activity shows a discontinuity in the Arrhenius plot, once attributed to phase transitions in the membrane phospholipid, but which is similar to discontinuities in the Arrhenius plot for rotational mobility and which have been rationalized in terms of a conformational change in the ATPase.142... 

Gannett, P. M., et al. (2002). Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides. Nucleic Acids Res. 30, 5328—5337. 

Simultaneously with the refinement of the EC structure, additional data on the conformation of taxol bound to MT came from ss-NMR REDOR measurements [84]. As introduced above, the REDOR experiment exploits the dipolar interaction between nuclear spins to determine distances between atoms closer than 12 A. A PTX analogue with suitable spin labeling for REDOR was prepared and complexed to MT. Labeling of the amide carbonyl and C3 methine with 13C, and position p- of the 2-O-benzoyl with 19F (Fig. 9, compound 3), allowed the measurement of the two 19F-13C distances I and II (Fig. 9), that could serve, in principle, to discriminate among candidates for PTX conformation. The respective values of 9.8 A 0.5 A and 10.3 A 0.5 A were interpreted by the authors as supportive of the hydrophobi-cally collapsed polar conformation for PTX [84]. 

Oxidation of 2,2,4,4-tetraalkyloxazolidines gives stable nitroxide radicals (265) which are used as spin labels for probing biomolecular structures (69ACR17, B-76MI41800). The stearic acid derivative 12-doxylstearic acid (266) is commercially available. 

Makinen, M. W., Mustafi, D., and Kasa, S. (1998) ENDOR of spin labels for structure determination from small molecules to enzyme reaction intermediate, in Berliner, L. (eds.), Biological Magnetic Resonance 14, Spin Labeling Next Millennium, Plenum Press, N. Y. ppl81-249. 

In addition to the isotopically labeled Mannich bases, it is worth mentioning the paramagnetic Mannich base 492, which represents a very good spin-label for the protein SH group,as well as the cyclic Mannich base deriving from 1,-DOPA (493), which exhibits much enhanced optical rotatory power with respect to the open chain precursor allowing it to be more precisely estimated. --... 

Figure 8 Introduction of paramagnetic spin labels for second site screening. Paramagnetic spin labels drastically alter the relaxation properties of binding components. The large gyromagnetic ratio of unpaired electrons amplifies the relaxation via dipole-dipole interaction, which is represented by the arrows. Stars at the edges of the binding ligand illustrate the increased transverse relaxation...

Spin labeling Ligand or protein None Spin label for either ligand or protein Orientation, simultaneous binding 10 M0 M X y" ... 

Nitroxides are persistent free radicals [1] which can often be isolated and handled as kinetically stable species. Nitroxides react rapidly with carbon free-radical intermediates [2] with well-characterized rate constants [3], and can thus be used as kinetic and mechanistic probes, as well as to trap carbon radicals in synthetic processes. They are easily oxidized or reduced, and thus have a rich redox chemistry that has been utilized for a variety of oxidations. As nitroxides have an unpaired electron, they are paramagnetic and thus ESR active, making them valuable as spin labels for biomolecules [4] and as spin traps for transient radicals [5]. In addition, nitroxides have been developed as organic ferromagnetic materials [6]. The synthesis of nitroxides has been reviewed in 1994 [7]. This review will focus on the synthetic applications of nitroxides. 

Constantine KL (2001) Evaluation of site-directed spin labeling for characterizing protein-ligand complexes using simulated restraints. Biophys J 81 1275-1284... 

Sajid M, Jeschke G, Wiebcke M, Godt A (2009) Conformationally unambiguous spin labeling for distance measurements. Chemistry 15 12960-12962... 

To date, the most commonly used spin labels for membrane proteins are pyrroline-type nitroxide radicals. Several labeling strategies and spin labels are available at present. However, the extreme specificity for the free thiol of cysteines, a stoichiometric reaction at most sites, and the relatively small size and flexibility of the modified side chain make the (l-oxyl-2,2,5,5-tetramethyl-A3-p3Troline-3-methyl) methanethiosulfonate spin label (MTSSL) the most popular choice for SDSL applications. 3 -(2-lodoacetamido)-2,2,5,5-tetramethyl-1 -pyrrolidinyloxy radical labels (lAP) are also used in combination with cysteines. The advantage of LAP vs MTSSL is that the covalent bond with the cysteine is irreversibly formed and this may be more suitable for particular situations in which the MTSSL tends to be released from the labeled sites. 

Raitsimring AM, Gunanathan C, Potapov A, Efremenko I, Martin JM, Milstein D, Goldfarb D (2007) Gd3-t complexes as potential spin labels for high field pulsed EPR distance measurements. J Am Chem Soc 129(46) 14138-14139... 

Nitrogen-15 and Deuterium Substituted Spin Labels for Studies of Very Slow Rotational Motion... 

ENDOR of Spin Labels for Structure Determination From Small Molecules to Enzyme Reaction Intermediates... 

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