Related Experiment Videos
Phosphorylation-dependent changes in structure and dynamics in ERK2 detected by SDSL and EPR
Andrew N Hoofnagle1, James W Stoner, Thomas Lee
1School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Biophysical Journal
|December 26, 2003
Summary
Mitogen-activated protein kinases (MAPKs) undergo conformational changes upon phosphorylation. Electron paramagnetic resonance reveals altered side-chain mobilities in specific interdomain regions of extracellular signal-regulated protein kinase-2, indicating functional regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Mitogen-activated protein kinases (MAPKs) are crucial signaling enzymes regulated by phosphorylation.
- Previous studies indicated significant conformational changes in MAPKs upon activation, distant from phosphorylation sites.
Purpose of the Study:
- To investigate the conformational dynamics of extracellular signal-regulated protein kinase-2 (ERK2) in response to diphosphorylation.
- To precisely map changes in protein mobility using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy.
Main Methods:
- Site-directed spin labeling (SDSL) was employed at specific amino acid residues (101, 105-109, 111, 112) of ERK2.
- Electron paramagnetic resonance (EPR) spectroscopy was utilized to measure the mobility and conformational state of the spin labels.
- Data analysis focused on anisotropic hyperfine splitting and rotational diffusion rates.
Main Results:
- Phosphorylation did not alter the anisotropic hyperfine splitting, consistent with crystallographic data showing no global structural change.
- EPR revealed no significant changes in spin label mobility at positions 101, 111, and 112.
- Diphosphorylation induced subtle yet significant alterations in rotational diffusion at residues 105-108 and affected the proportion of probes in a constrained state at residues 105, 107, and 109.
Conclusions:
- EPR spectroscopy confirms localized, nanosecond-timescale changes in side-chain mobility within the interdomain region of ERK2.
- These findings highlight specific conformational adjustments distal to the phosphorylation sites, contributing to the understanding of MAPK regulation.