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Research Project
Electron paramagnetic resonance (EPR) is the name given to
the process of resonant absorption of microwave radiation by
paramagnetic ions that have at least one unpaired electron spin
in the presence of a static magnetic field. EPR provides an
alternative to classical techniques for the elucidation of structural
information of proteins that may not be amenable to characterization
by NMR or X-ray crystallography. Double site-directed spin labeling
(D-SDSL) refers to the construction of proteins that contain
a single pair of cysteine residues that can then be covalently
modified with thiol-specific nitroxide spin labels. When two
spin labels are held in proximity to one another by the structural
constraints of a protein, the two unpaired electrons of the
nitroxides experience an electron-electron dipolar interaction
which causes a broadening in the EPR spectrum of the labels.
This spectral broadening is proportional to the distance between
the two spin labels, which permits characterization of the relative
position of secondary structure elements. Used in conjunction
with molecular modeling, EPR and D-SDSL can thus be used to
measure the structural changes of proteins under conditions
relative to function, such as the ligand-induced conformational
change of human estrogen receptor alpha ligand-binding domain
(hER LBD). My project (with Jason Matthews) involves D-SDSL
of the hER LBD for the purpose of characterizing structural
differences between the agonist-bound, antagonist-bound, and
non-liganded conformations using EPR spectroscopy.
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