Donovan Brown

Mentors: Borden Lacy, Eric Skaar, Walter Chazin
Home Institution: Pennsylvania State University

Andy Dorfeuille

Mentor: Walter Chazin
Home Institution: Morehouse College

Andy was featured in the August 2, 2018 Vanderbilt Reporter.

Research Abstract:
DNA replication is the basis for biological inheritance. Most new DNA is synthesized by proofreading polymerases, however, these polymerases cannot initiate the synthesis of a new strand of DNA. This functional gap is filled by the essential enzyme called primase. Primase creates a small DNA primer that can be extended by other polymerases. Recently, we have shown that a domain of primase, p58C, contains a redox-active iron-sulfur cluster that can engage in redox switching.  The redox state of primase’s iron-sulfur cluster modulates its DNA binding affinity. We propose that this plays a role in primase’s ability to initiate and terminate primers. Our research is designed to better understand the role redox switching plays in replication. The goal of my project is to create and biophysically characterize a redox deficient tyrosine to phenylalanine triple mutant in the p58C subunit of human primase. The mutant p58C protein will be studied by Fluorescence Anisotropy to assess DNA binding affinity and Circular Dichroism to determine thermal stability of the subunit, relative to the native species.  Furthermore, X-Ray Crystallography will be used to determine the structure of the mutant, and other biophysical techniques to investigate the properties of a redox deficient variant. This variant will then be tested electrochemically and phenotypically by collaborators at Caltech. This work will inform the biological relevance of iron-sulfur cluster redox in primase activity.  As DNA replication is essential to all living beings, we view this research as foundational for future research.

Anna Lauko

Mentor: Carlos Lopez
Home Institution: Carleton College
Research Abstract:
In multicellular organisms, apoptosis, or programmed cell death, is a tightly regulated process initiated under specific biochemical conditions. A hallmark of cancer cells, apoptosis evasion promotes tumor growth and resistance to therapies. Cellular commitment to death is regulated at multiple levels along the TNF-receptor-induced signal cascade, notably by the formation of signaling complex II in the cytoplasm. Although a vast body of work exists detailing the role of complex II in cell death, the dynamic mechanisms that drive its formation are difficult to measure experimentally and thus largely unknown. Computer modeling of the dynamics of complex II assembly could shed light on the requirements that lead to TNF-receptor driven apoptosis. Because the complex is assembled of multiple proteins, we hypothesize that its abundance is likely to be low and exhibit bursts. Therefore, classical kinetic modeling approximations, which fail to account for the intrinsic stochasticity and biochemical gradients present in cells, may not adequately represent the behavior of this system. To address this limitation, we will instead evaluate multiple approaches to modeling complex II assembly: a classical chemical kinetic model, a model incorporating stochasticity, and a stochastic model with spatial resolution. We believe that varying the reaction rates and initial conditions, along with multiple levels of chemical reaction detail, will provide deep insight into how protein interactions control apoptosis execution. If successful, this work could lead to novel methods to induce or avoid cell death in toxicology or cancer treatments.

Jonathan Lee

Mentor: Michael Stone
Home Institution: Universidad de Puerto Rico
Research Abstract:
Abasic sites, or AP sites, are one of the most common DNA modifications and can produce various types of biological consequences if not repair correctly. They have also been found to form interstrand crosslinks with the exocyclic amino group of DNA bases that are in close proximity of itself. Interstrand cross-links in a DNA duplex are highly toxic to cells since these lesions interfere with the cells ability to extract information from the DNA in terms of replication and transcription and has been used as a form of cancer therapy. Our laboratory hopes to better understand the chemistry of the N4‐amino‐2′‐deoxycytidine (dC*) -AP site crosslinks in a DNA duplex. Understanding the chemistry of the alpha versus beta configuration of the 2’ deoxyribosyl ring of the AP site, which is still unknown, and the conformation of the N4 proton, would give us better insight into possible base recognition and repair. Another goal that our laboratory hopes to achieve is to acquire a better understanding of the structure of this dC*-AP crosslink. All of this would be achieved by using Nuclear Magnetic Resonance spectroscopy (NMR). This research would greatly improve the understanding of the recognition of dC*-AP crosslinks in cells, knowledge that could be used to improve current chemotherapy in cancer patients.

Kathleen McClanahan

Mentor: Brian Bachmann
Home Institution: University of Arkansas
Research Abstract:
New antibiotics are in high demand due to the increasing prevalence of antibiotic-resistant infections. The everninomicins, an antibiotic class first isolated in the 1960s but never utilized clinically, show great promise as a treatment for these serious infections. This class displays potent action against drug-resistant gram-positive bacteria due to a novel mode of action. We propose to harness the everninomicin biosynthetic machinery to make unnatural analogues with greater efficacy and safety.

We aim to elucidate the biosynthetic pathway of the aromatic moiety dichloroisoeverninic acid (DCIE), a substructure implicated in the antibiotic activity of the everninomicins. An acyl transferase (AT) and a polyketide synthase (PKS) are involved in the biosynthesis of the orsellinic acid (OSA) core of DCIE, but little is known about the mechanism of this process.  In vitro biochemical studies will elucidate the critical interactions between the AT and PKS involved in DCIE biosynthesis. Additional biochemical turnover assays will focus on utilizing the AT to attach unnatural substrates to the everninomicin core to produce novel analogues. This project will uncover the vital interactions between enzymes involved in everninomicin biosynthesis and allow access to analogs with increased antibacterial activity. Through this work we hope to revitalize this powerful class of molecules for clinical use.

Nicolas Robalin

Mentor: Gary Sulikowski
Home Institution: University of Notre Dame
Research Abstract:
With the rise of antibiotic resistance in bacteria, the development of inhibitory compounds has grown in importance. The creation of compounds to limit antibiotic resistance is a promising field of chemical biology. The formation of biofilms is a key way in which bacteria resist antibiotics. Aurachin C 1-10 (Aurachin C Analogue, N-Hydroxy-2-n-decyl-3-methyl-4-quinolone) has been shown to be a potent inhibitor against the formation of biofilms in E. Coli. Aurachin C 1-10 inhibits two enzymes, cytochrome bo and cytochrome bd, which are vital oxidases in the aerobic respiratory chains of E. Coli. Unfortunately, Aurachin C is a scarce compound in nature. Therefore, we hope to find a more efficient pathway for synthesis of Aurachin C 1-10 due to the compound’s promise as a bacterial inhibitor. The hope of the project is to reach Aurachin C 1-10 starting with aniline through a 6-step synthesis. The synthesis of the compound will further help study the inhibitory effects of quinolone derivatives on the formation of biofilms.

Caroline Stanton

Clostridium difficile is the leading cause of healthcare-associated infective diarrhea and can be fatal in the elderly and those who have diminished gut microbiota due to antibiotic use. Traditional treatments often include antibiotics, which are not always effective and can contribute to recurrence. C. difficile infection is known to be a toxin-mediated disease and while the toxins are the major components of its pathogenesis, other less-studied proteins should also be investigated to determine their functions and potential as drug targets. The proteins I intend to investigate are C9YJA5 and C9YJB7, both of which have unknown structures and functions in C. difficile. However, C9YJA5 has been identified as a C. difficile spore-coat protein and is homologous to methyltransferases from other species, making it a potential immunotherapy target. C9YJB7 may be downregulated in a toxin-deficient strain of C. difficile, suggesting that it could be linked to toxicity. I have recombinantly expressed these proteins in E. coli, purified them using affinity and size exclusion chromatography, and performed preliminary crystallization screens. By trying different protein concentrations and crystallization conditions, I hope to identify favorable conditions for crystal formation. I will then collect X-ray diffraction data from these optimized crystals to elucidate the protein structures. I also plan to pursue other biophysical assays such as dynamic light scattering to further characterize these proteins. This structural information will allow for better understanding of the proteins’ functions and their potential as therapeutic targets for more efficient treatment of C. difficile infection.

Morgan Vanderwall

Mentor: Lauren Buchanan
Home Institution: Whitworth University
Research Abstract:
Amyloids are highly organized assemblies of cross-β-sheet peptide aggregates that can form long, unbranched fibrils. Cytotoxic amyloids are associated with the onset of many human diseases such as Alzheimer’s, Parkinson’s, Huntington’s or type II diabetes. Recently, amyloids have been observed in normal biological function, including the storage and release of hormones. The cholecystokinin tetrapeptide hormone (CCK-4) has been shown to reversibly form functional amyloid assemblies in response to changes in pH. CCK-8 is the octapeptide derived from the C-terminus of the cholecystokinin protein and contains the CCK-4 sequence required for complete biological activity. We plan to utilize 2D infrared spectroscopy to characterize the aggregation behavior of CCK-8 with varying pH. 2D IR spectroscopy, when combined with isotope labeled samples, can provide single residue resolution of structural changes. The determination of CCK-8’s aggregation behavior will ideally provide greater insight to the formation of functional amyloids, and potentially cytotoxic amyloids as well.