Electron nuclear double resonance spectroscopy resolution

EPR spectroscopy is used widely in the study of proteins and of lipid-protein interactions.0 It has often been used to estimate distances between spin labels and bound paramagnetic metal ions.g A high-resolution EPR technique that detects NMR transitions by a simultaneously irradiated EPR transition is known as electron-nuclear double resonance (ENDOR).h... 

Electron-Nuclear Double Resonance (ENDOR) Spectroscopy. This observes a spin resonance transition after a nuclear resonance transition has been saturated by a radio-frequency pulse (Fig. 11.65) so as to invert the relative populations of the y.ey. > and y,./) .y> spin states this forces the populations of the aeaN> and fSey > states to be different and thus offers the opportunity to measure hyperfine splittings much more carefully, with better resolution than in standard EPR. 

ESR-related spectroscopies that hold the potential to overcome some resolution limitations and yield more information than the classical ESR approach about the chemical environment of paramagnetic metal ions are the electron—nuclear double resonance (ENDOR) (Kevan and Kispert, 1976) and electron-spin echo envelope modulation (ESEEM) (Kevan and Schwartz, 1979) spectroscopies. Either ENDOR or ESEEM represents by principle a useful tool in extending resolution of the ESR experiment. However, the sensitivity of ENDOR and ESEEM is much lower than that of ESR, and interpretation of ENDOR and ESEEM spectra is not a simple matter, especially if ligands are not well characterized, as is the case for HSs. Both ENDOR and ESEEM techniques have not yet been applied to strictly metal-HS complexes, but the sensitivity and ease of carrying out experiments are improving rapidly, so major scientific activity may be anticipated to occur in this area of ESR spectroscopy. 

Electron paramagnetic resonance (EPR) spectroscopy [1-3] is the most selective, best resolved, and a highly sensitive spectroscopy for the characterization of species that contain unpaired electrons. After the first experiments by Zavoisky in 1944 [4] mainly continuous-wave (CW) techniques in the X-band frequency range (9-10 GHz) were developed and applied to organic free radicals, transition metal complexes, and rare earth ions. Many of these applications were related to reaction mechanisms and catalysis, as species with unpaired electrons are inherently unstable and thus reactive. This period culminated in the 1970s, when CW EPR had become a routine technique in these fields. The best resolution for the hyperfine couplings between the unaired electron and nuclei in the vicinity was obtained with CW electron nuclear double resonance (ENDOR) techniques [5]. 

There are a variety of techniques for the determination of the various parameters of the spin-Hamiltonian. Often applied are Electron Paramagnetic or Spin Resonance (EPR, ESR), Electron Nuclear Double Resonance (ENDOR), Electron Electron Double Resonance (ELDOR), Nuclear Magnetic Resonance (NMR), occassionally utilizing effects of Chemically Induced Dynamic Nuclear Polarization (CIDNP), Optical Detection of Magnetic Resonance (ODMR), Atomic Beam Spectroscopy and Optical Spectroscopy. The extraction of the magnetic parameters from the spectra obtained by application of these and related techniques follows procedures which may in detail depend on the technique, the state of the sample (gaseous, liquid, unordered solid, ordered solid) and on spectral resolution. For particulars, the reader is referred to the general references (D). 

Electron - nuclear double resonance (ENDOR) offers enhanced resolution compared with conventional ESR. This is achieved mainly because the technique is ESR-detected NMR spectroscopy. 

Electron Spin Echo Envelope Modulation (ESEEM) and pulse Electron Nuclear Double Resonance (ENDOR) experiments are considered to be two cornerstones of pulse EPR spectroscopy. These techniques are typically used to obtain the static spin Hamiltonian parameters of powders, frozen solutions, and single crystals. The development of new methods based on these two effects is mainly driven by the need for higher resolution, and therefore, a more accurate estimation of the magnetic parameters. In this chapter, we describe the inner workings of ESEEM and pulse ENDOR experiments as well as the latest developments aimed at resolution and sensitivity enhancement. The advantages and limitations of these techniques are demonstrated through examples found in the literature, with an emphasis on systems of biological relevance. 

Apart from ESEEM methods, electron nuclear double resonance (ENDOR) spectroscopy is the other well-estabhshed technique for measuring nuclear transition frequencies of paramagnetic compounds. We start with a brief discussion of the two standard pulse schemes, Davies and Mims ENDOR, before moving onto 2D sequences aimed at resolution improvement. 

The structures now available at 5-8 A resolution allow differentiation of protein a-helices from double-helical regions of rRNA based on their structural characteristics some protein -sheets can also be seen. The structures of several individual ribosomal proteins have been solved by X-ray crystallography or nuclear magnetic resonance spectroscopy, and the X-ray data on the whole 508 particle have been correlated with the previously determined structures of individual proteins. As expected, the accumulated data fromlEM, cross-linking, and neutron diffraction helped to position these protein stmctures within the ribosome electron density and will continue to direct placement of proteins and specihc regions of rRNA. 

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