Browsing by Author "Kinsley, Nichole"
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- ItemRestrictedArginine 15 stabilizes an SNAr reaction transition state and the binding of anionic ligands at the active site of human glutathione transferase A1-1(Elsevier, 2010) Gildenhuys, Samantha; Dobreva, Marina; Kinsley, Nichole; Sayed, Yasien; Burke, Jonathan; Pelly, Stephen; Gordon, Graeme P; Sayed, MuhammedArg15, conserved in class Alpha GSTs (glutathione transferases), is located at the interface between the G- and H-sites of the active site where its cationic guanidinium group might play a role in catalysis and ligand binding. Arg15 in human GSTA1-1 was replaced with a leucine and crystallographic, spectroscopic, thermodynamic and molecular docking methods were used to investigate the contribution made by Arg15 towards (i) the binding of glutathione (GSH) to the G-site, (ii) the pKa of the thiol group of GSH, (iii) the stabilization of an analog of the anionic transition state of the SNAr reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, and, (iv) the binding of the anionic non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to the H-site. While the R15L mutation substantially diminishes the CDNB–GSH conjugating activity of the enzyme, it has little effect on protein structure and stability. Arg15 does not contribute significantly towards the enzyme's affinity for GSH but does determine the reactivity of GSH by reducing the thiol's pKa from 7.6 to 6.6. The anionic σ-complex formed between GSH and 1,3,5-trinitrobenzene is stabilized by Arg15, suggesting that it also stabilizes the transition state formed in the SNAr reaction between GSH and CDNB. The trinitrocyclohexadienate moiety of the σ-complex binds the H-site where the catalytic residue, Tyr9, was identified to hydrogen bond to an o-nitro group of the σ-complex. The affinity for ANS at the H-site is decreased about 3-fold by the R15L mutation implicating the positive electrostatic potential of Arg15 in securing the organic anion at this site.
- ItemRestrictedA modeled structure of an aptamer-gp120 complex provides insight into the mechanism of HIV-1 neutralization(American Chemical Society, 2010) Joubert, Marisa K; Kinsley, Nichole; Capovilla, Alexio; Sewell, B Trevor; Jaffer, Mohamed A; Khati, MakobetsaThe HIV-1 envelope glycoprotein, gp120, is a key target for a class of drugs called entry inhibitors. Here we used molecular modeling to construct a three-dimensional model of an anti-gp120 RNA aptamer, B40t77, alone and in complex with gp120. An initial model of B40t77 was built from the predicted secondary structure and then subjected to a combination of energy minimization and molecular dynamics. To model the B40t77-gp120 complex, we docked the B40t77 predicted structure onto the CD4-induced epitope of the gp120 crystal structure. A series of gp120 point mutations in the predicted B40t77-gp120 interface were measured for their binding affinity for B40t77 by surface plasmon resonance. According to the model, of the 10 gp120 amino acids that showed a reduction in the level of binding when mutated to alanine, all of them are modeled as making direct contact with B40t77 as part of a hydrogen bonding network. Comparison by electron microscopy of the B40t77-gp120 complex with gp120 alone revealed that only the longest dimension of the complex significantly increased in length, in a manner consistent with the predicted model. Binding assays revealed that B40t77 can weaken the binding of gp120 to the monoclonal antibodies B6, B12, and 2G12, none of which have binding sites that overlap with B40t77, as well as strengthen the binding to the antibody 19b. Thus, B40t77 may induce distant conformational changes in gp120 that disrupt its association with host cells and may suggest a mechanism for aptamer neutralization of HIV-1.