Mitogen-activated protein kinases are regulated by occupancy at two phosphorylation sites near the active site cleft. Previous studies using hydrogen exchange to investigate the canonical mitogen-activated protein kinase, extracellular signal-regulated protein kinase-2, have shown that phosphorylation alters backbone conformational mobility >10 A distal to the site of phosphorylation, including decreased mobility within amino acids 102-105 and increased mobility within 108-109. To further describe changes after enzyme activation, site-directed spin labeling at amino acids 101, 105-109, 111, 112 and electron paramagnetic resonance spectroscopy were used to investigate this region. The anisotropic hyperfine splitting of the spin labels in glassy samples was unchanged by phosphorylation, consistent with previous crystallographic studies that indicate no structural change in this region. At positions 101, 111, and 112, the mobility of the spin label was unchanged by diphosphorylation, consistent with little or no conformational change. However, diphosphorylation caused small but significant changes in rotational diffusion rates at positions 105-108 and altered proportions of probe in a motionally constrained state at positions 105, 107, and 109. Thus, electron paramagnetic resonance indicates reproducible changes in nanosecond side-chain mobilities at specific residues within the interdomain region, far from the site of phosphorylation and conformational change.
Electron paramagnetic resonance spectra of the S3 radical center in ultramarine blue over a factor of about 2500 in frequency (258 MHz to 670 GHz) reveal a substantially Lorentzian shape, without resolution of g anisotropy. Variable temperature measurements found that the line width is independent of temperature, within experimental uncertainty, up to about 90 K at 9.5 GHz and between ca. 5 K and room temperature at 95 and 217 GHz, as expected for an exchange-narrowed signal. Analysis of the increase in the low-temperature line width as a function of frequency above 9 GHz is consistent with an exchange interaction of about 2. 10 -2 K. The line width increases as frequency is decreased from 2.7 GHz to 258 MHz which is attributed to the contribution from nonsecular terms that has been denoted the "10/3" effect.
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