Poster Abstract

Spring 2011 Biomedical Seminar Series

April 8

Incorporation of the non –canonical amino acid, Azidohomoalanine, into N-peptide base inhibitor of HIV entry to create a trimer via click chemistry

Jhanisus Melendez
MBRS-RISE Undergraduate Scholar

Infection of Human Immunodeficiency Virus type-1 (HIV-1) occurs by the fusion of viral membrane with that of the host and release of viral genetic material into the host cell. This fusion process is assisted by the envelope glycoprotein (Env) on the surface of the virus. Env encompasses the subunit glycoprotein 120 (gp120) and glycoprotein 41 (gp41). Fusion of membrane is promoted by the formation of trimer-of-hairpins structure which brings the amino- and carboxyl-terminal regions of the gp41 envelope glycoprotein ectodomain into close proximity. Inhibition of HIV-1 viral infections can be achieved by treatment with peptides derived from the N-amino acid and C-amino acid sequence regions of gp41. Synthetic peptides derived from these two regions (called N and C peptides, respectively) inhibit HIV-1 entry. C-peptides are potent inhibitors of HIV-1 infectivity with activity at nanomolar concentrations. In contrast, N-peptides aggregate and precipitate at high concentrations and at low concentrations they are not trimmers and are less potent than C peptides as viral inhibitors. Work in the Tirrell lab has developed variants of the N-peptide region of gp41 where proteins can accommodate the noncanonical amino acid Azidohomoalanine (Aha). The incorporation of Aha into our designed protein will permit azide alkyne click chemistry. This reaction will facilitate the trimer formation required to trigger the achieve state of the designed protein.


Evidence that residues 753, 755, 942 and 956 form part of the binding site of substrate phosphoenolpyruvate in phosphoenol pyruvate carboxylase

Steve Halaby
MBRS-RISE Undergraduate Scholar

Based on their location with in the tertiary structure, we propose that the residues R753, N755, N942, and I956 contribute to the binding site of the substrate phosphoenolpyruvate on phosphoenolpyruvate carboxylase in maize. Using the web based program Computed Atlas of Surface Topography of proteins, it was determined that pocket 217 houses these residues in addition to residues R231 and R232, which are also believed to be involved in the binding of the substrate. Mutations R753E and N755A displayed a 9 fold increase and a 3 fold increase respectively in the K0.5 PEP for the substrate. R753E displayed a 3 fold reduction in the Vmax, while N755A only showed a 1/3 reduction. Residue N942D showed a two fold increase in K0.5 PEP and roughly a 1/3 reduction in Vmax. Residue I956F showed a dramatic loss of affinity for its substrate and had not achieved Vmax during preliminary studies. These results suggest that these six residues are critical to PEPc’s binding of its substrate.


S- Glutathionylation of Wild Type and Mutant p53

Michael Mendoza
MBRS-RISE Undergraduate Scholar

The ability of a cell to appropriately respond to stresses and maintain its redox potential is essential for life. Stressors that can alter the redox potential in the cell include UV light, reactive oxygen intermediates (ROI), heat shock, ionizing radiation, and DNA damage. Adverse changes in redox potential can alter the tertiary structure of proteins and affect their ability to perform their functions. It has been shown that the tumor suppressor protein p53 is subjected to a reversible form of modification called S-glutathionylation. (Velu et al. 2007) p53 is tetramer protein, containing two identical dimer subunits that together form a fully functional protein which can bind to DNA. Human p53 contains six solvent exposed cysteine residues: Cys 124, Cys 176, Cys 182, Cys 229, Cys 242, and Cys 277 within the DNA binding domain. Maintaining the reduced states of these cysteine residues is vital for p53 to remain functional. It has been shown that Cysteines 124, 141, and 182 are sites for S-glutathionylation. (Velu et al. 2007) It has been hypothesized that modification at these sites can prevent the two p53 dimers from forming a tetramer and prevent binding to consensus DNA. With the importance of the redox states of these cysteines known, it is important to understand the protein structural changes caused by S-glutathionylation. The research I am involved in will consist of purifying wild type and cysteine mutant p53. The mutant p53 proteins will have all cysteine residues in the DNA binding domain replaced with glycine, except for one. The purified protein will then be used in glutathionylation and DNA binding experiments.

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