Low microwave-amplitude ESR spectroscopy: Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins
2004 (English)In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 60, no 2, 117-138 p.Article in journal (Refereed) Published
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 (h(1) < 0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T-2e, 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-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-1252C), 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.
Place, publisher, year, edition, pages
2004. Vol. 60, no 2, 117-138 p.
electron spin resonance, microwave saturation, MTSL, crox, lipase, electron-paramagnetic-resonance, thermomyces-lanuginosa lipase, bacteriorhodopsin mutants, progressive saturation, membrane transporter, linewidth analysis, loop region, dynamics, liquids, conformation
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
IdentifiersURN: urn:nbn:se:kth:diva-23667DOI: 10.1016/j.jbbm.2004.05.002ISI: 000223463400003ScopusID: 2-s2.0-3242710304OAI: oai:DiVA.org:kth-23667DiVA: diva2:342366
QC 20100525 QC 201109232010-08-102010-08-102011-09-23Bibliographically approved