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Henry Jakubowski Chemistry Department College of St. Benedict/St. John's University |
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Professor of Chemistry, 2002 Visiting Professor of Chemistry Ph.D. University of Iowa, 1986 B.S. - State University of New York at Albany, 1975 Email: hjakubowski@csbsju.edu Phone: 320-363-5354 |
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CSB/SJU Links |
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Low Molecular Weight Protein Tyrosine Phosphatase (LMW PTP) Enzymes that cleave phosphate groups from proteins are abundant in all cells. These enzymes, called phosphatases, regulate the activity of phosphorylated proteins, rendering the dephosphorylated proteins either active or inactive. Phosphatases are key proteins in the regulation of cells growth and metabolic state. I am studying human low molecular protein tyroysl phosphatase (PTP). Dr. McIntee (Chemistry Dept) and I are collaborating to study the natural protein substrates of this enzyme and to develop drugs to inhibit its activity. The protein is made from a cDNA inserted into a plasmid, PGEX-6P1 which is used to transform E. Coli. Gene expression is induced by addition of IPTG, which activated the lac promoter, leading to the formation of a GST-PTP fusion protein. The active site of the enzyme, where the chemical cleavage of the phosphate group occurs, must bind phosphate groups which are covalently attached to target proteins. The activity of the enzyme can be monitored easily in solution using p-nitrophenol phosphate which is cleaved by the enzyme to produce a yellow solution. An active site cysteine (Cys 12) acts as a nucleophile in the cleavage reaction. The enzyme binds phosphate in the active site, which competitively inhibits the enzyme.
My research group has made a series of mutations in the DNA for PTP and we are presently studying the effect of these mutants on its activity. The mutants can be divided into two groups: 1. Active site mutant: The active site Cys 12 (C12) is replaced for a Ser (C12S). This makes the enzyme catalytically inactive, unable to cleave phosphate groups from proteins. However, this mutant will still be able to bind phospho-proteins. We will use this mutant in future research to bind to natural phospho-proteins in epithelial and fat cells, and to identify the binding sites on those phospho-proteins for PTP. 2. Nonactive site mutant: PTP contains two tryptophans (W). We have made two different mutants, changing a single W to phenyalanine (F) in each one, thereby producing two mutants that contain only one W residue. We have changed W49, an amino acid that is near the active site and which fluoresces, to F which does not fluoresce. We have also made a second mutant to change W39, located on the opposite side from the active site, to a phenylalanine. Fluorescence from the mutant with only a single W at position 49 will be sensitive to the environment of the active site, while the other mutant, containing a single W at position 39, will be used to detect changes in protein structure away from the active site and serve as a control. The activity of the two mutant proteins, as measured by cleavage of p-nitrophenyl phosphate and fluorescence, in the presence and absence of different inhibitors of the enzyme, will be used to better understand how the structure of the enzyme influences its activity. In addition, the stability and unfolding of the protein will be studied used fluorescence from the single tryptophan-containing mutants. We have also made a double mutant: C12S/W39F, producing a protein that can not cleave phosphates from target proteins (C12S) and which has a single W at position 49 (W39F) which will be used to monitor phospho-protein binding to the double mutant by monitoring fluorescence changes in W49. Additionally, we are studying the protein and its interaction with inhibitors through in silico computer modeling of the protein uisng VMD/NAMD and Autodock. Applications of Fluorescence Measurements in BiochemistryMy second project involves using the spectrofluorometer to study a range of biological questions. When molecules absorb UV or visible light, electrons are excited to higher energy levels. The excited electrons can �relax� back to lower energy levels by losing energy through collisions or by emitting photons of lower energy than the original excitation photons. This emission is called fluorescence. In contrast to simple absorbance properties, fluorescence emission is extremely sensitive to the environment of the flourophore, the molecule that emits. The properties of biological molecules can be studied using fluorescence. Two types of fluorophores are used. Intrinsic fluorophores are part of the actual molecule (for example a tryptophan side chain in a protein). Extrinsic fluorophores (like fluorescein) can be attached (covalently or noncovalently) to proteins, lipid aggregates and DNA. We develop research projects using fluorescence to study the structural transition and binding interactions of proteins, lipids, and DNA. My Research GroupIn the summer of 2008 I have two research students, Rob Hvalicek (SJU) and Yang Lin from Southwest University (SWU), Beibei, Chongqing, Peoples' Republic of China. Here's a list of some of my past research students and a bit about what they are presently doing;
Grants-Net: Supported by HHMI and AAAS 11/24/2015 |
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