2005 Pilot Projects
Title: Novel Hemoprotein in Microbial Defense
Investigator: Alan R. Brash, Ph.D., Department of Pharmacology
Description: The objectives of this project were to determine the catalytic activities of novel catalase-related proteins in reactions with peroxide substrates and their role in oxidative defense. Three such “mini-catalase” proteins were studied, from the toxin-producing fungus Fusarium graminearum, from the pathogenic bacteria Mycobacterium avium subsp paratuberculosis and Helicobacter pylori. The main highlights of this project were the successful characterization of unique products formed in reaction of the Fusarium mini-catalase with linoleic and linolenic acid 13S-hydroperoxides. The results suggested that the protein catalyzes formation of the 13-peroxyl radical, and from this various unusual products arose that have since been characterized by LC-MS and NMR. An analysis has also been completed involving the role of a distal heme residue in determining the reaction of prototypical mini-catalase, coral allene oxide synthase, with hydrogen peroxide versus fatty acid hydroperoxide.
Title: Polymerase Interactions with Acrolein-Modified DNA
Investigator: Martin Egli, Ph.D., Department of Biochemistry
Description: Using the 18mer with sequence 3’-CCC CCT TCC TAG A-g-HO-PdG-C GCT-5’ and g-HOPdG at position 5, Dr. Egli has conducted initial crystallization experiments with a binary Dpo4 complex at various pH values (dC oppositeg-HO-PdG). The sequence context around the adduct corresponds to that in DNAs containing g-HOPdG that were investigated in the Rizzo, Stone, and Lloyd laboratories in connection with intra- and inter-strand crosslinks (5’-…Cg-HO-PdG…-3’; see underlined portion of the above template sequence). Crystallizations were set up at low (ca. 5), neutral, and high pH (9) to potentially isolate the g-HO-PdG adduct in the cyclic (pH 5) and open-chain aldehydic forms (pH 9). The observation that crystals grown at neutral pH appeared to be of lower quality is not unexpected, since one would expect the adduct to be present as a mixture of the cyclic and linear forms at the polymerase active site. Crystals grown at pH 9 are not suitable for diffraction experiments thus far. By comparison, crystals obtained at pH 5 are of better quality. Template-primer construct and protein were annealed at low pH, leading to the conclusion that the equilibrium between the cyclic and linear forms lies essentially on the side of the former. Diffraction experiments at the APS with these crystals revealed a resolution limit of around 3.5 Å. However, the diffraction frames showed reflections that were split into multiple spots, rendering indexing and subsequent extraction of a useful dataset impossible. It is unclear whether the encountered problems were related to some degree to the particular sequence. For example, in the crystallized sequence the adduct is part of a 5’-CG-3’ step that enables inter-strand crosslink formation. Presence of even small amounts of crosslinked material would most likely negatively affect the quality of crystals.
Title: Mechanism of Eukaryotic DNA Replication Initiation
Investigator: Brandt F. Eichman, Ph.D., Assistant Professor, Department of Biological Sciences
Description: Initiation of eukaryotic DNA replication entails the coordinated action of more than 20 proteins that function to recognize replication origins, melt and locally unwind the DNA, and assemble the proteins necessary for coordinated DNA synthesis at the replication fork. Mcm10 and Cdc45 are the first two proteins recruited to replication origins at the onset of S-phase and are essential for DNA unwinding and replication fork assembly. Dr. Eichman took a structural approach to investigate the molecular basis for Mcm10 and Cdc45 interactions with DNA and with other replication components as a first step toward dissecting the architectures, mechanics, and specific functions of the multiprotein replication machinery. His lab identified structural domains of Xenopus laevis Mcm10 and Cdc45 using limited proteolysis, mass spectrometry, and Edman degradation. A library of deletion mutant proteins encompassing different combinations of these stable domains was cloned, over-expressed, and purified to homogeneity in milligram quantities. This library of soluble Mcm10 and Cdc45 proteins enabled Dr. Eichman’s lab to begin a comprehensive structure-function analysis of Mcm10 and Cdc45. Mcm10 truncation mutants are well-folded and primarily alpha-helical, as demonstrated by circular dichroism spectroscopy. A highly conserved region of Mcm10 binds DNA with a 10-fold preference for single- versus double-stranded DNA, suggesting that Mcm10 is directly involved in origin melting. Furthermore, Dr. Eichman’s lab has demonstrated that an N-terminal domain of Mcm10 forms a hexameric complex in solution. Interactions between this N-terminal domain and Mcm2-7 hexameric helicase are currently under investigation, and crystallization experiments of both Mcm10 and Cdc45 have begun.
Title: Use of Magnetic Resonance Spectroscopy to Assess Manganese Neurotoxicity
Investigator: Vanessa A. Fitsanakis, Ph.D., Research Instructor, Department of Pediatrics
Description: In this in vivo study, Dr. Fitsanakis exposed male Sprague-Dawley rats to a relatively low-dose of manganese (Mn) (3 mg [54 µmol] Mn /kg body mass) for a period of 12 weeks. In order to ascertain the effect of treatment on the striatum, the main target of Mn exposure, data from either 31P or 3H magnetic resonance spectra was later collected. Initial experiments with phantoms of various phosphate (ATP, ADP, AMP) and carbon compounds (glutamate, Y-aminobutyric acid, N-acetyl aspartate [NAA]) provided data for coil and protocol refinement necessary to confirm basic in vivo parameters. Preliminary in vivo scans suggested an early change in the levels of phosphates, implying a shift from normal metabolism to glycolysis. The data also supported potential changes in the level of NAA, a neuronal marker, often associated with cell viability. Interestingly, it appears that alterations in phosphate peaks may occur at a much earlier time point than anticipated, demanding a re-evaluation of the scanning times for animals in the study. Various data processing methods of preliminary spectra have also been conducted to determine the best approach to data analysis for animals in the study. Although manganese, a paramagnetic metal, could potentially cause peak broadening in both spectra, it appears that this phenomenon may affect the 3H spectra more than 31P. However, it is anticipated that changes in both spectra are still meaningful and likely reflect actual changes in brain metabolism. Finally, arrangements were made with the imaging center to include several PET scans to determine a potential timeline for dopaminergic involvement in manganese neurotoxicity.