Download Huang, David, Center for Structural Biochemistry

Center for Structural Biochemistry
Montpellier, France
David Huang
2015
Protein Preparation, Purification, and
Crystallization
 Preparation – Plasmids were transformed into
competent cells and the specific protein was
expressed and collected.
 Purification – Protein was purified using a nickel
affinity column and a gel filtration column.
 Crystallization – Purified proteins were prepared
with various solvent and ligands on
crystallography plates. Crystals were collected
after a few days.
Protein Preparation, Purification, and
Crystallization
Data Collection, Structure
Determination, and Analysis
 Data Collection – Electron density data were collected
from the protein crystals using x-ray crystallography.
 Structure Determination – The electron density data
was used to determine the structure of the proteins in
complex with the ligands using the COOT software.
 Analysis – The specific hydrogen bonds or hydrophobic
interactions around the ligand binding pocket were
analyzed after the structure had been determined.
Data Collection, Structure
Determination, and Analysis
Most Rewarding Aspect of My Work
Experience
 I was able to travel to Grenoble to use the ESRF
(European Synchroton Radiation Facility), a large
particle accelerator that allows us to collect well
resolved x-ray diffraction data.
 Collecting good x-ray diffraction data and solving
these structures was the culmination of all the
weeks I spent in the lab doing bench work
preparing proteins and crystals.
European Synchrotron Radiation
Facility
How My Work Impacted the
Organization
 I helped to solve the structures of PXR (pregnane
X receptor) in complex with the ligands
oxadiazon, pretilachlore, fipronil, fenvalerate, αzearalenol, 4-tert-octylphenol and 2 other
unnamed synthetic ligands.
 Solving these structures give us a better
understanding of how the protein binds to
different ligands and will help us with structure
based rational drug design.
Ligands
Pretilachlore
Fenvalerate
Oxadiazon
α-Zearalenol
Fipronil
4-tert-octylphenol
Academic Choices and Career Plans
 This internship helped me solidify my interest in
doing research and made me certain that I want
to continue doing research in graduate school.
 Since I still prefer chemistry research over
structural biochemistry research, I hope to work
in the relevant field of synthesizing ligands and
drug molecules based on the structural data of
proteins.
Cultural Awareness/Personal Growth
 Beyond Montpellier, I traveled to Sete, St.Guilhem,
Grenoble, Paris, and Barcelona and experienced
French and Spanish culture through food and travel.
 I met a lot of international students and learned their
perspective on American politics and education.
 I learned to be truly independent by surviving in a
country that spoke a foreign language, working at a 9
to 6 job everyday, and cooking all meals for myself.
Travel
Travel
Travel
Acknowledgments
 Princeton and IIP
 Luisa Duarte-Silva
 Centre de Biochimie Structurale
 Dr. William Bourguet
 Dr. Vanessa Delfosse
 Dr. Deborah Harrus