licata@lsu.edu
LiCata Lab Homepage
Our laboratory studies proteins: most particularly protein-DNA and protein-ligand interactions,
along with protein folding and stability. Our work is primarily focused on the energetics and
global structural attributes of these processes and how they are controlled.
We utilize several different model systems to address questions in these areas, including the
Type 1 DNA polymerases from Escherichia coli and Thermus aquaticus, the mouse adipocyte lipid
binding protein, and the classic allosteric enzyme aspartate transcarbamylase.
The Type 1 DNA polymerases from E. coli and T. aquaticus are a homologous mesophilic-thermophilic
pair of proteins. Their molecular structures and basic catalytic functions are highly homologous,
yet the T. aquaticus enzyme works near the boiling point of water, while the E. coli enzyme is
irreversibly destroyed by exposure to 50oC. We directly and comparatively examine the DNA binding,
protein folding, and global structural characteristics of these two species of DNA polymerases with
the goal of delineating the exact differences and similarities between the two proteins in order
to understand what features are necessary to achieve high temperature stability and activity for
a DNA polymerase. We are in the midst of an extensive comparative characterization of the DNA
binding properties and thermodynamic stabilities of the wildtype polymerases along with
their “Klenow” and “Klentaq” large fragment domains. These studies include characterizations
of DNA binding using fluorescence anisotropy and titration calorimetry, characterizations
of stability using thermal and chemical denaturation methods, and characterization of
structural and conformational changes using small angle X-ray scattering, analytical
ultracentrifugation, and electrophoretic methods. We have just initiated site-directed
mutagenesis and directed evolution studies aimed at elucidating the
detailed structural features differentiating the two species of polymerase.
Mammalian adipocyte lipid binding protein (ALBP) is found in high concentrations in adipose cells,
and has been strongly implicated in the development of Type 2 diabetes, obesity, and atherosclerosis.
Our laboratory is studying the stability and lipid binding properties of ALBP, with emphasis on the
control of these processes by electrostatics and environmental (solution) conditions. We have
recently identified significant salt based regulatory effects on both the lipid binding and
the stability of ALBP, and have begun characterizing the thermodynamic linkages among these
processes (lipid binding, stability, and salt). We have also constructed site directed mutants
of the protein and begun mapping the surface residues responsible for the regulation of the
protein by ions.
Our laboratory is also characterizing the association and dissociation kinetics of both the
polymerase-DNA and ALBP-lipid systems, in order to more precisely define the origins of solvent
control in these systems. Protein-ligand equilibria and kinetics have long been known to be
effected by nearly every aspect of their environment, including pH, salt, osmotic pressure,
temperature, et cetera. As part of one ongoing project, our mapping of the effects of different
environmental variables will soon be extended to examining the effects of gravity on the kinetics
and equilibria of these reactions
E. coli
aspartate transcarbamylase (ATCase) is one of the most extensively studied enzymes in
biochemistry. It has served, and continues to serve in our laboratory as a benchmark for
different studies, particularly our analytical ultracentrifugation and small angle X-ray
scattering structural studies. We are, however, also investigating various hypotheses for
explaining the mechanism of substrate inhibition in ATCase, and the mechanism of transmission
of heterotropic allosteric regulatory signals within the enzyme.
Datta, K, and LiCata, VJ, 2003, Salt dependence of DNA binding by Thermus aquaticus and Escherichia coli DNA polymerases, J. Biol. Chem. 278: 5694-5701.
Joubert, AM, Byrd, AS, and LiCata, VJ, 2003, Global conformations, hydrodynamics, and X-ray scattering properties of Taq and Escherichia coli DNA polymerases in solution, J. Biol. Chem. 278: 25341-25347.
Karantzeni, I., Ruiz, C.R., Liu, C.-C., and LiCata, V.J., 2003, Comparative thermal denaturation of Thermus aquaticus and Escherichia coli Pol 1 Type DNA polymerases, Biochem. J. 374, 785-792.
Schoeffler, A.J., Ruiz, C.R., Joubert, A.M., Yang, X., and LiCata, V.J., 2003, Salt modulates the stability and lipid binding affinity of the adipocyte lipid binding protein, J. Biol. Chem. 278, 33268-33275.