title: Low microwave-amplitude ESR spectroscopy: measuring spin-relaxation interactions of moderately immobilized spin labels in proteins.
abstract: 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-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.
authors.affiliation: Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 91, Stockholm, Sweden. eva@biochem.kth.se
authors.forename: Eva M K
authors.initials: EM
authors.lastname: Hedin
authors.forename: Karl
authors.initials: K
authors.lastname: Hult
authors.forename: Ole G
authors.initials: OG
authors.lastname: Mouritsen
authors.forename: Pernille
authors.initials: P
authors.lastname: Høyrup
mesh_terms: D001203:Ascomycota / Q000201:enzymology; D001703:Biophysics / Q000379:methods*; D003497:Cyclic N-Oxides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008049:Lipase / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008698:Mesylates / Q000737:chemistry; D008872:Microwaves; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels; D013997:Time Factors; D014867:Water / Q000737:chemistry
chemical_list: D003497:Cyclic N-Oxides; D008698:Mesylates; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D013113:Spin Labels; D014867:Water; C034248:(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate; D008049:Lipase; D003545:Cysteine