Department of Biochemistry and Molecular Biology
Oklahoma State University, Stillwater, OK, B.S., Biochemistry and Molecular Biology, 1993
Oklahoma State University, PhD, Biochemistry and Molecular Biology, 1999
Texas A&M University, College Station, TX, Postdoctoral Research Associate, Biochemistry and Biophysics, 1999-2003
University of Kansas Medical Center, Kansas City, KS, Biochemistry and Molecular Biology, Assistant Professor, 2004 - 2010
University of Kansas Medical Center, Biochemistry and Molecular Biology, Associate Professor, 2010 - 2017.
University of Kansas Medical Center, Biochemistry and Molecular Biology, Professor, 2017 - present.
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Dissecting Molecular Mechanisms of Allosteric Regulation
Research in my lab focuses on understanding the mechanisms of allosteric regulation. Metabolic and signal transduction pathways need to be regulated to enable organisms to respond to ever changing environmental conditions. Allostery is often a key component to providing this necessary regulation. In fact, Monod (Nobel Prize winner) found allosteric regulation so important to biological functions that he referred to it as " the second secret to life".
At the single protein level, allosteric regulation is the altered functions that result when a regulatory molecule binds to a protein at a site distinct from the protein’ s active site. What is not well understood is how the binding of the effector molecule is communicated through the protein to alter the active site. If we could understand the mechanisms within proteins that give rise to allostery, then modulating allosteric properties would be useful to an enormous number of biological applications.
I want to understand both the molecular and thermodynamic mechanisms of allostery. Pyruvate kinase from human liver (L-PYK) is allosterically activated by Fru-1,6-BP and allosterically inhibited by ATP and Ala. This enzyme is also inhibited upon phosphorylation. This system is significant to biology due to the required regulation of L-PYK to maintain liver homeostasis between glycolysis and gluconeogenesis. This is also a great model system to study allosteric regulation. Using this system we have the opportunity to compare and contrast multiple allosteric mechanisms.
Instead of limiting our thoughts to a limited number of assumed confirmations, we consider allosteric regulation using thermodynamic arguments that are free of presumed conformational states. This approach also offers the theory for quantifying a "magnitude" of how much allosteric regulation is present instead of treating this regulation as on-or-off; plus-or-minus; all-or-none. At the current time we are focusing our attention on the areas of L-PYK that must by necessity play roles in allosteric regulation: the active site and the effector binding sites. We are using mutagenesis and ligand analogs to answer which coordinating interactions between protein and ligand contribute to the allosteric communication. Based on our previous results, our hypothesis is that most of the coordinating interactions contribute to ligand binding, but only a very limited number of the coordinating interactions contribute to the allostery.
The results from this study will be used to direct future mutagenesis studies as a way of "tracing" which residues in the protein communicate the allosteric signal. To the same goal, we are also initiating D/H-exchange mass spectrometry experiments that will identify what regions of the protein have altered flexibilities due to the allosteric signal.
Current tools we are using in the lab include: X-ray crystallography, solution X-ray scattering, D/H-exchange mass spectrometry, enzyme kinetics, steady state fluorescence, UV difference spectra, protein purification, site-directed mutagenesis, DNA isolation and cloning, hybrid proteins, thermodynamic linkage-analysis, E.coli and yeast protein expression systems, and gene knock-out procedures in E. coli. We also have experiments designed that will incorporate: single molecule fluorescence, time resolved fluorescence, FRET, second derivative spectra, analytical ultracentrifuge, light scattering, filter binding assays, protein denaturation, isothermal calorimitry, circular dichrosim, and chemical labeling.
Aron W. Fenton, Ph.D.