Spin-labeling in High-field EPR

The method of labeling biomolecules with nitroxides was initially developed in the early to mid 1960s, primarily in the laboratories of McConnell (Stanford) and Rozantzev (Institute of Chemical Physics, Russia), as well as others. Since then, spin-labeling has matured into a valuable tool to study local structure and dynamics of complex macromolecules. Over the years, the progress in spinlabeling methodology and applications has been well documented in the literature.  

1 Structure and dynamics of large molecular weight proteins in solution  

2 Membrane and membrane-associated proteins structure, location with respect to the membrane, side-chain dynamics, and interactions with other 

3 Fast conformational transitions of proteins and RNAs in solution, protein 

Electron Paramagnetic Resonance, Volume 18 The Royal Society of Chemistry, 2002 

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In the last decades, EPR has become a very versatile research field, with different subdisciplines. Recent developments, including the introduction of high-field EPR, different pulsed EPR methodologies, spin-labeling techniques, and miniaturization, have enormously increased the number of problems that can be addressed with EPR. At the same time, there is a strong need of new and dedicated theoretical models and calculation tools in order to extract the maximum of information from the obtained data. 

Two methods are useful to measure slow motion spin label spectra saturation transfer (ST-EPR) and two-dimensional electron spin echo spectroscopy (2D ESE). In the ST-EPR experiment, " a cw spectrometer is operated at high microwave power, and this causes partial saturation of the nitroxide spectrum. As the field is scanned, molecular motion carries this saturation to nearby regions of the spectrum, yielding spectra that are very sensitive to the rotational correlation time. One of the great advantages of ST-EPR is that little instrumentation is required beyond the conventional cw EPR instrument. Most any laboratory equipped to perform EPR can also perform ST-EPR. 

Electron nuclear double resonance is a powerful tool for the study of the electronic structure of triplet states because of its high precision. ENDOR linewidths can be as narrow as 10 kHz, which represents an increase in resolution of better than six orders of magnitude over that which can be obtained optically. The technique is particularly useful when combined with hf methods owing to the first-order nature of the hyperfine interaction in the presence of a field. Although such experiments are difficult, the information obtained is unique. Accordingly, the hf EPR (or ODMR) spectrometer has been modified for ENDOR operation in several laboratories. In order to illustrate the power of the method, we discuss here some recent optically detected hf ENDOR experiments on (njr ) benzophenone and its iso-topically labeled derivatives (Brode and Pratt, 1977, 1978a,b). The results, although incomplete, show considerable promise for the ultimate determination of the complete spin distribution in this prototype triplet state. 

Fig. 5.12. The light-induced uptake of spin label TEMPOamine by the thylakoids revealed as the increase in the magnitude of the high field component of the EPR signal from TEMPOamine in a chloroplast suspension containing 16 mM chromium oxalate, the lower curve is in the presence of 20 /xM gramicidin D (after [78]).

Fig. 5.15. The dependence of the magnitude on the reversible light-induced decrease of the high field component of the EPR signal from spin label on TEMPOamine concentration in the suspension of bean chloroplasts functioning under conditions of cyclic electron transport mediated by phenasine metosulphate, photosystem 2 was inhibited by diuron (after [70]).

Figure 15. EPR spectra of the high-spin Fe-NO complex of soybean LOX-1 at two frequencies. The soybean lipoxygenase (LOX-l)-nitric oxide complex (3.2 mM in 0.1 M potassium phosphate, pH 7.0) was sealed under argon in a quartz EPR tube of 0.7 mm ID. Spectra were recorded 9.26 GHz and 94 GHz, and the temperature was 6 K. The original 94 GHz spectrum was scaled to the same g-value scale as the 9.26 GHz spectrum, and the magnetic field units are shown for the X-band spectrum. Because of the scaling, the high-fiequency spectrum is labeled in the figure as if it was recorded at 92.6 GHz, and the corresponding magnetic field scale would be ten times that shown.

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