TY - JOUR
T1 - Low microwave-amplitude ESR spectroscopy
T2 - Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins
AU - Hedin, Eva M.K.
AU - Hult, Karl
AU - Mouritsen, Ole G.
AU - Høyrup, Pernille
PY - 2004/8/31
Y1 - 2004/8/31
N2 - 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.
AB - 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.
KW - chromium(III) oxalate
KW - continuous wave
KW - Crox
KW - CW
KW - DL-dithiothreitol
KW - DTT
KW - EDTA
KW - Electron spin resonance
KW - electron spin resonance
KW - electrospray mass spectroscopy
KW - Electrospray MS
KW - ESR
KW - ethylenediamine tetraacetic acid
KW - HEPES
KW - Lipase
KW - Microwave saturation
KW - MTSL
UR - http://www.scopus.com/inward/record.url?scp=3242710304&partnerID=8YFLogxK
U2 - 10.1016/j.jbbm.2004.05.002
DO - 10.1016/j.jbbm.2004.05.002
M3 - Journal article
C2 - 15262447
AN - SCOPUS:3242710304
SN - 0165-022X
VL - 60
SP - 117
EP - 138
JO - Journal of Biochemical and Biophysical Methods
JF - Journal of Biochemical and Biophysical Methods
IS - 2
ER -