Graduate Opportunities at
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Oak Ridge National
Laboratory
Wm. Brad O’Dell
Department of Structural and Molecular Biochemistry
NC State University
September 18, 2015
Cellulose Degrading Enzymes
Horn et al. Biotechnology for Biofuels 2012 5:45
A variety of enzymes function together to break down cellulose
to soluble sugars for conversion to ethanol.
Two activities are key:
• Hydrolytic
• Oxidative
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Polysaccharide Monooxygenases
Eibinger et al. J. Biol. Chem. (2014) 289:52
Neurospora crassa PMO-2 is a model cellulose oxidizing enzyme.
• Mononuclear Cu(I/II) metalloprotein
• Requires inputs of electrons and molecular oxygen
• Oxidizes C1’ of glycosidic bond leading to chain disruption
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Polysaccharide Monooxygenases
Kim et al. PNAS (2013) 111:1
Phillips et al. ACS Chem. Biol. (2011) 6
Neurospora crassa PMO-2 is a model cellulose oxidizing enzyme.
• Mononuclear Cu(I/II) metalloprotein
• Requires inputs of electrons and molecular oxygen
• Oxidizes C1’ of glycosidic bond leading to chain disruption
• Multiple catalytic mechanisms proposed
• Protonation states likely influence the mechanism
4
Polysaccharide Monooxygenases
Neurospora crassa PMO-2 is a model cellulose oxidizing enzyme.
• Mononuclear Cu(I/II) metalloprotein
• Requires inputs of electrons and molecular oxygen
• Oxidizes C1’ of glycosidic bond leading to chain disruption
• Multiple catalytic mechanisms proposed
• Protonation states likely influence the mechanism
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We are using protein crystallography to gain insights into the
PMO catalytic mechanism.
Neutron Protein Crystallography
X-ray crystallography is poorly
suited for studying protonation.
Protein crystallography with
neutron radiation can locate the
positions of hydrogen (1H/2H)
atoms.
NPC has technical challenges:
• Large crystal volumes
• 2H-labeled protein
Gardberg et al. Acta Crystallogr. D 2010 66:5
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NPC at Oak Ridge National Lab
IMAGINE
Neutron Sciences at ORNL operates the
only single-crystal neutron protein
diffractometers in North America.
• IMAGINE, High Flux Isotope Reactor
• MaNDi, Spallation Neutron Source
The BioDeuteration Laboratory at ORNL is
dedicated to producing 2H-labeled proteins
for neutron scattering experiments.
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BioDeuteration Laboratory
ORNL Graduate Opportunities Program
“ORNL is extending its energy campus to top research
universities to enable the brightest graduate students to
participate in research at ORNL while earning a Ph.D. degree
at their home institution.”
• Duke
• Vanderbilt
• Florida State
• Virginia Tech
• University of Florida
• University of Virginia
• Illinois Urbana-Champaign • Ohio State
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• Georgia Tech
• Wisconsin at Madison*
• University of Memphis
• Penn State*
• NC State University
• University of Maryland*
• Purdue
• Nanyang University*
GO! experience at ORNL
Research benefits
• Produced large quantities of
natural-abundance protein for
crystallization trials
• Increased crystal volume
approaching NPC requirements
• Proposed and received ~96
hours of discretionary
beamtime for different test
experiments
• Isotopic labeling experiments
upcoming
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GO! experience at ORNL
Mentorship and experience
• Specialized experience at the bench and beamline
• Abundant and accessible scientists and post-docs
• Internal seminars and journal clubs
Professional Development
• Trainee member, SNS/HFIR Users Group Executive
Committee
• Instrument/technique workshops
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Acknowledgements
Flora Meilleur
Energy and the Environment Group
Biology and Soft Matter Division
Neutron Sciences Directorate
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