Graduate Program

Chemistry

Degree Name

Master of Science (MS)

Semester of Degree Completion

2009

Thesis Director

Gopal Periyannan

Thesis Committee Member

Barbara Lawrence

Thesis Committee Member

Daniel Sheeran

Thesis Committee Member

Zhiqing Yan

Abstract

A novel zinc metalloprotease gene isolated from Caulobacter crescentus (CCM) was cloned and overexpressed in Escherichia coli. A protease database search using MEROPS predicted this protein is a homolog of Glutamate Carboxypeptidase II (GCP II), a zinc metalloprotease found in humans and many other organisms. GCP II is considered as a suitable target for treating stroke, neurological disorders and a biomarker for prostate cancer. Chromatographic purification of CCM produced pure protein in milligram quantities. Molecular weight detern1ination using MALDI-TOF showed 51,582 Da and the extinction coefficient was found to be 33,007 M"1cm·1. Metal analysis and substrate profiling showed neither sufficient metal content nor enzyme activity, although fluorescence spectroscopy indicates the protein is expressed in the properly folded form. Due to rapid degradation of the CCM, even at low temperatures, some characetrization experiments did not produce expected results.

The degradation properties of CCM were thoroughly investigated on the basis of protein half-life dependency on primary structure and other external factors such as presence of contaminants. The correlation between the in vivo stability of a protein and its dipeptide composition is indicated by the Protein Instability Index (PII). CCM has a PII of 41.01, whereas the unstable proteins have a PII > 40. A dipeptide composition based PII calculator was designed and made available to public. An in vitro protein stability study of CCM and other candidate proteins was carried out to compare PII values and the corresponding degradation pattern. We altered the PII by changing the dipeptide composition by point mutations to monitor any change in the stability. Our results indicate that caution should be exercised for the use of PII as an indicator for in vitro stability prediction of proteins.

Homology modeling was used to obtain the three dimensional structures of CCM and CBM (GCP II from Caenorhabditis briggsae). We have investigated the structural similarity between human Glutamate Carboxypeptidase II (GCP II) and three other proteins (VpAp, CCM, and CBM) from three different organisms: Vibrio proteolytica (VpAp), Caulobacter crescentus (CCM) and Caenorhabditis briggsae (CBM) using homology modeling. These three target proteins have sequence similarity to various domains and the full-length of GCP II. A clear structural evolution leading to the fulllength human GCP II emerges from the analysis of previously solved VpAp structure the homology modeled structures of CCM and CBM. A molecular docking-based substrate profiling of the modeled structures, with the help of existing substrate information of GCP II and VpAp, will be a proof of the predicted structures and is proposed.

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