Electron spin label peptides

The solution structures, dynamics, and interactions of and between biological macromolecules are topics of widespread interest in biochemistry. The chapter on electron spin labels, by Millhauser et al. illustrates how the EPR spectra of the stable nitroxide free radical can be used to address such problems. The chemistry of the nitroxides and their modes of attachment to the host molecules are discussed first. The details of the EPR spectra and of the spin Hamiltonian are then presented, showing how the intrinsic tensorial nature of the EPR spectrum of the reporter group is affected by motion. Such dynamic information is then extracted from some small peptides. The interaction between pairs of nitroxides is used to extract structural information. Finally, an example of Fourier transform EPR is introduced. ... 

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... 

Selected entries from Methods in Enzymology [vol, page(s)] Electron paramagnetic resonance [effect on line width, 246, 596-598 motional narrowing spin label spectra, 246, 595-598 slow motion spin label spectra, 246, 598-601] helix-forming peptides, 246, 602-605 proteins, 246, 595 Stokes-Einstein relationship, 246, 594-595 temperature dependence, 246, 602, 604. 

Fajer, P. G. (2000). Electron spin resonance spectroscopy labeling in proteins and peptides analysis. In Encyclopedia of Analytical Chemistry, (R. Meyers, ed.), pp. 5725-5761. Wiley, Chichester. 

Milov AD, Tsvetkov YD, FormaggioE, OanceaS.TonioloC, Raap J (2004) Solvent effect on the distance distribution between spin labels in aggregated spin labeled trichogin GA IV dimer peptides as studied by pulsed electron-electron double resonance. Phys Chem Chem Phys 6 3596-3603... 

Columbus. L. Hubbell, W.L. A new spin on protein dynamics. Trends Biochem. Sci. 2002. 27 (6). 288-295. Smythe, M.L. Nakaie. C.R. Marshall, C.R. Alpha-helical versus 3(10)-helical conformation of alanine-based peptides in aqueous solution—An electron-spin-resonance investigation. J. Am. Chem. Soc. 1995. 117 (42). 10555- 10562. Miller. W.G. Spin-Labeled Synthetic Polymers. In Spin Labeling 11 Theory and Applications Berliner. L.J.. Ed. Academic Press New York. 1979 173-221. 

Radicals normally do not occur in peptides but they can be generated by irradiation at low temperature with UV, electrons or X-rays, usually at the a-carbon atom, and then can be recognized by ESR. There are some radicals that are stable at ambient temperature, for instance N-oxides with the structural element C-NO-C. In biochemistry, e.g. derivatives of 2,2,5,5-tetramethyl-pyrrolidine-l-oxide are being used as spin labels, conjugated to bioactive compounds. In this way ESR can localize the position of possible receptor sites. For instance, in hormone research the distances in the neurophysin complex between spin-labeled small peptides, models of oxytocin, have been determined [27]. 

A combination of circular dichroism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical crosslinking, and analytical ultracentrifugation studies showed that both the apo- and metallated derivatives of H21(31-mer) form two-stranded a-helical coiled coils in aqueous solution. Further characterization of these derivatives by EPR spin-label experiments helped to determine its three-dimensional backbone structure. In these studies, a Cys-21 mutant of the 31-mer coiled coil, H21/C21(31-mer), was prepared and labeled with a thiol-specific nltroxide spin label (MTSL = l-oxyl-2,2,5,5-tetramethyl-A -pyrroline-3-methyl-methanethiosulfonate) at position 21 of the peptide sequence which is the site of metal substitution in the ET heterodimer. Comparison of the low-temperature, dipolar-broadened spectrum of the spin-labeled dimer with those of magnetically dilute peptide samples yielded a backbone-to-backbone distance that was nearly identical to that of the GCN4 homodimer. Based on these results, computer modeling studies provided an estimate of the metal-to-metal distance in the ET heterodimer of m-m > 25 A. The electron-transfo properties of this system are now being studied by a combination of laser flash-quench and pulse radiolysis techniques. 

Electron Spin Echo Envelope Modulation, or ESEEM, is widely used to investigate structure of the immediate surroundings of paramagnetic species. In combination with site-specific spin labeling and selective deuterium substitution, it becomes a helpful tool to study location of proteins and small peptides within membranes, interaction of small molecules with proteins, supramolecular assemblies, water accessibility to specific regions of proteins and water penetration into membranes, protein folding and secondary structures. In this review, experimental approaches and limitations, theoretical background of the method, and recent applications are discussed. 

At high peptide concentration (peptide/lipid ratio 1 20 mol mol ), the ESEEM amplitudes were found to change substantially the peak amplitude for the spin-label position near the N-terminus is markedly reduced. Thus, by increasing the concentration of peptide its N-terminus becomes immersed deeper in the membrane - the peptide orientation becomes closer to transmembrane. Data from pulse electron-electron double resonance (PELDOR) showed that the peptide aggregates in the form of dimers, in a head to head configuration. " Transmembrane dimers are long enough and apparently can lead to the formation of channels in the membrane. 

An extension of site-directed spin labeling is to monitor H-ESEEM from a deuterated amino acid (valine or leucine) that is introduced at a fixed position, i, in the peptide sequence. The nitroxide spin label is then attached at a cysteine residue that is stepped sj tematically away from the deuterated residue. For an a-helical peptide, H-ESEEM is observed when the spin label is at position z- -3, i.e., in register with the deuterated residue, but not at position i + 2. For a (Tsheet peptide, on the other hand, H-ESEEM is observed when the spin label is at position i -I- 2 on the same side of the p-sheet as the deuterated residue, but not at position i -I- 3 on the opposite side of the p-sheet. Therefore the H-ESEEM amplitude, which is inversely proportional to the sixth power of the distance between electron and nuclear spins, directly reflects the characteristic periodicity of the peptide secondary structure, in the vicinity of the deuterated residue. 

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