FY2010 - Oak Ridge National Laboratory

Director’s R&D Fund—
Neutron Sciences
structure in solution is directly measured. It is expected that this research will provide unique insight into
the mechanism of IDP function while simultaneously making advances in the use of neutron scattering for
the study of biological systems through the combination with osmotic stress.
Mission Relevance
This research project aims to make progress in biomedicine and is consistent with the DOE mission to
promote scientific and technological innovations that improve quality of life. This project also takes
particular advantage of the unique neutron facilities within the Neutron Scattering Science Division
(NSSD) and supporting laboratories at ORNL. We are using the BioSANS instrument at the High Flux
Isotope Reactor (HFIR). For future SANS experiments, we will carry out deuterated protein expression in
the Biodeuteration Facility at the Center for Structural and Molecular Biology (CSMB). Also, we are
working with Kunlun Hong in the Center for Nanophase and Materials Science (CNMS) for deuterated
polymer synthesis to be used for neutron experiments. We also strive for educational outreach, and this
project has been enhanced by the participation of three undergraduate students: Laura Grese (UT), Zac
Enderson (Georgia Tech), and Amanda DeBuhr (UT), along with two graduate students working through
the Laboratory for Conformational Diseases and Therapeutics (V. Berthelier), UT Graduate School of
Medicine: Tatiana Perevozchikova and Dimitriy Smolensky. Overall, this research fosters collaborations
and should assist in positioning ORNL at the forefront of neutron scattering applications in biological and
biomedical research.
For the long-term development of this research project, we have identified a funding opportunity from a
National Institutes of Health R01/R21 grant.
Results and Accomplishments
The major scientific accomplishments of the research project for FY 2010 have been (1) the continued
development of our combined SANS and osmotic stress approach for studying protein hydration,
conformation, and protein-protein interactions; (2) SANS characterization of an IDP pair that undergoes
coupled folding and binding; and (3) the preparation, identification, and characterization of our CREB
binding protein (CBP) fragments that contain IDP regions.
We are developing a combined SANS and osmotic stress approach to directly correlate protein structure
and structural transitions with the associated hydration and energetics. Our first SANS studies were on the
preferential hydration of hexokinase (HK) monomer and dimer states by osmolytes. Building upon this
work, we began probing the modulation of the HK monomer-to-dimer transition using osmotic stress.
With beamtime on BioSANS (Jan. 20–22, 2010 / IPTS-1282), we explored the pH dependence of this
transition with SANS and found a transition midpoint of pH 6.8. Using pH 7.4 (68% monomer) and
deuterated osmolytes contrast matched in D 2 O buffer, we perturbed the HK equilibrium toward the dimer
state and found that osmotic pressures up to 70 atm were required to begin to induce changes. These
studies are continuing and have been instructive toward applying our SANS and osmotic stress method to
understand the hydration properties and structural transitions in IDPs.
Using circular dichroism (CD) spectroscopy and SANS, we investigated the structure and binding
interaction properties between the 59 residue IDP region of CBP: nuclear-receptor co-activator-binding
domain (NCBD) and its binding partner, activator for thyroid hormone and retinoid receptors (ACTR),
which also is an IDP. CD indicates that NCBD alone retains some α-helical secondary structure, while
ACTR alone contains more random coil. With the BioSANS (Oct 17–20, 2009 / IPTS-1809) we
characterized the NCBD/ACTR complex and the structure of ACTR alone. The SANS structure on
NCBD/ACTR complex is in good agreement with the NMR structure, while SANS on ACTR reveals the
expanded nature of the unbound, unfolded state. In this way, we gain further insight into the structural
flexibility of these IDP regions.
50