Low microwave-amplitude ESR spectroscopy: Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins
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Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h1<0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T2e, is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-I252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T 2e. We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.
|Journal||Journal of Biochemical and Biophysical Methods|
|Number of pages||22|
|Publication status||Published - 31 Aug 2004|
- chromium(III) oxalate, continuous wave, Crox, CW, DL-dithiothreitol, DTT, EDTA, Electron spin resonance, electron spin resonance, electrospray mass spectroscopy, Electrospray MS, ESR, ethylenediamine tetraacetic acid, HEPES, Lipase, Microwave saturation, MTSL