We are studying the structures of selected proteins by single crystal X-ray diffraction. In all of these studies, we are collaborating closely with colleagues at Colorado State in areas such as biochemistry, microbiology, and physiology. The proteins currently of interest include actin depolymerizing factor (ADF) and the ADF complex with monomeric actin (G-actin), glutaminase, and hexokinase (especially in complex with inorganic polyoxoanions). Diffraction data are collected on a state of the art R-Axis-IV X-ray diffractometer in the Department of Biochemistry and Molecular Biology at Colorado State, using the most modern X-Stream cryocooling instrumentation to minimize damage to the crystals by the X-radiation. Brief descriptions of each of our main projects of interest are as follows.
Actin Depolymerization Factor (ADF). We are particularly interested in the structure of actin depolymerizing factor (ADF) and its complex with monomeric actin (G-actin). ADF is an actin monomer sequestering, F-actin severing protein, and in collaboration with Professor James Bamburg (Department of Biochemistry and Molecular Biology), we are studying several mutants of ADF as well as the complex between these mutants and G-actin. The ADF mutants vary remarkably in their degree of activity, and we believe that determination of their structures, especially in complexation with G-actin, will help elucidate the mechanism by which ADF activity is regulated as well as its detailed mode of binding to G-actin.
Glutaminase. Our studies on the structure of the enzyme glutaminase are being carried out in collaboration with Professor Norman Curthoys (Department of Biochemistry and Molecular Biology). In the brain, control of glutamate concentration is critical to avoidance of neuronal cell toxicity. Recent studies have implicated glutaminase as an important contributor to the neurotoxic glutamate levels in stroke-damaged brain tissue. We anticipate that determination of the molecular structure of glutaminase will aid in the development of an effective drug therapy that could limit the progressive neuronal atrophy caused by stroke.
Hexokinase.Professor Debbie Crans (Department of Chemistry) and her co-workers have shown that a number of inorganic polyoxometalate anions (such as decavanadate, V10O286–) of varying sizes and charges inhibit the first step in the metabolism of glucose, which involves catalysis of phosphorylation by the enzyme hexokinase. Furthermore, the magnitude of the inhibitory effect depends on the charge and size of the polyoxometalate anion. This result demands structural investigation in order to understand its origin and to map out the interactions between proteins and these complex inorganic species, and we are actively collaborating with Professor Crans and her group toward that end. As a result, we are giving heavy emphasis to the determination of the structures of yeast hexokinase B (hkB) in complexation with polyoxometalate anions, so that we can understand the origin(s) of these inhibitory effects. For comparison purposes, we also plan to attempt to co-crystallize hkB in the presence of substrates (sugars, nucleotides and nucleotide analogues). The following figure shows a model (created by our colleague, Dr. Terry Gray) of what the structure of the complex between decavanadate and hkB may look like.