Anisotropic spin label mobilities in azurin from 95 GHz electron paramagnetic resonance spectroscopy

Analysis of nitroxide spin label motion in a protein–protein complex using multiple frequency EPR spectroscopy

“…The W-band spectrum of spin labelled Im9 C23 is a direct indication of anisotropic rotational motion of the spin label. Similar effects have been observed previously in the literature [19,42] The spectral shape can be explained if, on the W-band timescale, motional averaging in the xz plane of the spin label is much faster than in both the xy and zy planes, that is, the rotation along the y axis is the fastest. Using the model of axially symmetric anisotropic mobility of spin label we have been able to reproduce the spectral features of the W-band spectrum (Fig.…”

“…Hence, W-band EPR line shapes are sensitive to a different dynamic range than X-band lineshapes. This is valuable in the analysis of spin label dynamics since slow motions that contribute to line shape changes at X-band are less likely to cause averaging effects in the W-band spectrum [15,19].…”

“…By using multiple EPR frequencies, spectra can be obtained that are sensitive to different time scales thus helping the de-convolution of the various motional contributions. The increasing availability of W-band (95 GHz) spectrometers are making this more common [15,[18][19][20]. An alternative approach to this problem has been the addition of a thickening agent, often the polysucrose Ficoll, which is used to increase the solvent viscosity hence slowing down the rotary diffusion of the protein [21].…”

High-field (275 GHz) spin-label EPR for high-resolution polarity determination in proteins

“…The difficulty to dimerize S118C-azurin has been interpreted as evidence for a low solvent accessibility of S118 [7]. Also, mobility studies performed by W-band EPR reveal a significantly reduced mobility for S118C [8], suggesting that S118 is more buried than the other residues. Interestingly, the mobility of the spin label attached to Q12C is lower than that attached to K27C [8], whereas the present study reveals a more apolar/aprotic, i.e., more buried environment for K27C.…”

Accurate long‐range distance measurements in a doubly spin‐labeled protein by a four‐pulse, double electron–electron resonance method

“…[19] Spin labeling of the mutants with the spin label (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSL) was performed as described in Ref. 20.…”