Date of Award

12-18-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Dr. Irene T. Weber

Second Advisor

Dr. Jenny J. Yang

Third Advisor

Dr. Robert W. Harrison

Fourth Advisor

Dr. Stuart A. Allison

Abstract

HIV-1 protease is an important enzyme for the maturation of infectious virions and has been an effective drug target for HIV/AIDS. Protease inhibitors were successfully designed based on the structural data for AIDS therapy. Nonetheless, the drug resistant PR variants are selected rapidly during therapy. Mutation L76V is associated with drug resistance and shows opposite effects on different protease inhibitors. Kinetics and stability of PRL76V were studied and high-resolution crystal structures of PRL76V with inhibitors were solved to identify structural changes. HIV-1 PRP51 variant is a multiple mutant selected for resistance to darunavir in the laboratory in vitro and it is useful to investigate the mechanisms of HIV-1 resistance to darunavir. The crystal structures of an inactive form of PRP51 have been determined: a darunavir bound structure and a ligand free structure. The kinetics and crystal structures of these drug resistant mutants provide the information to understand drug resistance mechanisms and hints to design novel inhibitors.

Ca2+ is a ubiquitous signaling molecule in regulating numerous biological functions. Calcium biosensor CatchER was designed by site-directed mutagenesis in the fluorescent sensitive location of chromophore. Crystal structures of CatchER in the absence of Ca2+, complexed with Ca2+ and also with Gd3+ were determined to investigate the calcium binding site and mechanisms of chromophore in response to Ca2+. Metal ions were identified in the designed calcium-binding site and structures showed the metal ion induced changes related to changes in optical properties. The structural information can be useful for the optimization and design of biosensors.

Available for download on Thursday, November 27, 2014

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