UC Davis

Visualization of biological macromolecules in three-dimensions provides us information to understand cell biology at the atomic level. X-ray macromolecular crystallography is a significant biophysical method for determining the structures of biomolecules including proteins, nucleic acids, and drugs. To-date, RCSB Protein Databank contains over 195,325 biomolecular atomic structures, with 86.4 % of these structures determined by X-ray diffraction.X-ray crystallography is routinely used to explore the detailed molecular mechanism of enzymes and to determine how substrates interact with its targeted protein and what changes might improve it. The main goal of macromolecular crystallization is to produce well-ordered crystals that do not have impurities and are large enough to provide high quality X-ray diffraction pattern. If such crystals are obtained, diffraction data can be collected using an X-ray radiation beam. This thesis describes the results of three projects that use crystallographic data to decipher the cellular function of aldose reductase (AR), a microcystin-binding nanobody, and aldehyde deformylating oxygenases (ADOs). Chapter 1 describes the crystallographic complex of aldose reductase (AR) with novel inhibitors (AT-001, AT-003, and AT-007, AT-00# collectively) design by Applied Therapeutics, Inc.. Besides a 1:1 stoichiometry of inhibitor to AR observed for AT-00# inhibitors, a second unexpected structure was determined for AT-001 with a 2:1 stoichiometry. Chapter 2 investigates the structural elucidation of nanobody A2.3 forming a microcystin-LR bound complex. The biophysical characteristic of the complex is discussed, with the identification of key amino acid residues that are important for binding and their role in binding for other microcystin variants. Chapter 3 discusses results from the comparative analysis of protein structural alignment of ferritin-like enzymes, in efforts to elucidate structural determinates that allow for ADO activity. A pool of 16 proteins was used for the analysis, 3 of which were solved in our lab. The biophysical characteristics of the 3 proteins are discussed in-depth within the chapter.