Preliminary Refolding Studies of the Nuclear Hormone RXRa Receptor

Speaker: Susan Kamman

Abstract: This summer our lab has taken on an entirely new field of research,
studying the kinetics of the folding/unfolding mechanisms of the retinoic
acid receptor "X" receptor a. Our primary emphasis in the early stages of
research has been to learn how to purify and concentrate the receptor in
levels sufficient for spectrometric experimentation, to test whether or not
the folding mechanism is reversible, and to gain an understanding of the
fluorescence properties of the protein.
Retinoic acids belong to a superfamily of ligand activated
transcription factors, meaning they bind to an agonist, 9-cis retinoic
acid, which initiates binding to DNA and the beginning of DNA
transcription. The receptor mediates gene expression, cell
differentiation, vertebrate development, and homeostasis. Previous
research has found retinoic acid receptors to be very flexible and highly
functional proteins, with the ability to form homodimers and also
heterodimers and tetramers with any other member of the nuclear hormone
receptor family. This provides for many permutations, each of which leads
to a conformational change in the protein structure and the initiation of a
different signaling pathway for gene expression.
This summer we have completed many preparations of the ligand
binding domain of the RXRa receptor, which was cloned by Dr. Schimerlik and
Dr. Broderick before the summer began, in order to find the most efficient
way to concentrate and purify the protein. Our protocol uses a nickel
chelated column, which binds the protein which has previously been tagged
with a 6 hisitidine residue. Histidine has a high affinity for the charged
nickel. We have been making many minor changes to the buffers we use to
wash the protein, in hopes of preventing precipitation of the hydrophobic
receptor.
We have used the purified receptor in many fluorescence spectroscopy
titrations in order to determine if the protein refolds in the presence of
guanidine HCl and/or urea. We successfully concentrated the protein to
sufficient levels for both fluorescence and circular dichroism (CD)
experimentation. Also, we were able to completely refold the protein in
the presence of 6M guanidine HCl, and have been running numerous titrations
in order to obtain baseline values for fluorescence spectroscopy
experimentation. These preliminary refolding studies have shown us that
long term research on this receptor is feasible and that the studies are
headed in the right direction.