2004 Tobacco Settlement Fund-supported Participants-Department of Biological Sciences - Carnegie Mellon University

2004 Tobacco Settlement Fund-supported Participants

Elizabeth FaulElizabeth Faul, Gettysburg College
(Mentor: Dr. Gordon Rule)

Isolation of Class Mu Glutathione S-Transferase and Analysis via NMR Study
Glutathione S- transferases (GST) are large, 50-kilo Dalton proteins that detoxify carcinogenic compounds, such as epoxide intermediates. GSTs work by catalyzing the addition of glutathione to endogenous or xenobiotic substrates. Once glutathione binds to the active site of the enzyme, it is deprotonated, making it nucleophilic. The final product, 1-(S-glutathionyl)-2,4-dinitrobenzene, is formed as the carcinogenic substrate's electrophilic functional groups attacks glutathione. However, the mechanism by which the specific interaction between GST and the hydrophobic substrate occurs within the cell is incomplete. To further investigate the structural properties of GST while in solution, both the methyl and amide groups of the protein are examined using NMR. A transformation introduces a class mu GST expression vector into the Escherichia coli AMP cells. The cells are subsequently grown in a specific, isotopically labeled media. The incorporation of a deuterated water source into the growth media is especially critical because of the size of GST. An abundance of naturally occurring hydrogen will lead to broadening of the proton NMR signal. Use of 15N labeled ammonium sulfate allows for observation of solvent exposed amide groups in the protein via NMR. To target specific carbon residues, namely alanine, isoleucine-delta, valine and leucine for visualization in the NMR spectrum it is necessary to grow the cells in the presence of 13C-labeled alanine, alpha-ketobutyrate, and alpha-ketoisovalerate, respectively. A glutathione affinity column and an anion exchange column were used to purify the enzyme. Structural properties and atomic interactions within the protein are found by 2-dimensional HSQC and both 3D and 4D NOESY NMR. To supplement the data obtained from the NMR, thereby further characterizing the structural properties of GST, a filamentous phage can be included in the protein sample. The phage infects Pseudomonas aeruginosa and is isolated using PEG (8000 average molecular weight) precipitation and a KBr gradient. The phage aligns in the magnetic field and provides an axis and predictable protein orientation from which the protein bond angles can be measured.

Connor MurphyConnor Murphy, Dickinson College
(Mentors: Drs. Jon Minden and William Eddy)

Difference Gel Electrophoresis (DIGE) to Determine Protein Differences Between Bovine and Rabbit Serum
Serum is the liquid component of the blood. Some of the common proteins found in serum are albumin (~50% of total concentration), immunoglobulins (~14%), clotting factors, complement factors, and transferrin. Less than 1% of the total serum protein is made up of thousands of different proteins, some of which may change in abundance depending on the physiological changes and disease states. Difference gel electrophoresis (DIGE) is a method used to differentiate between two separately labeled protein samples on the same acrylamide resolving gel. Two protein samples are each fluorescently labeled a different color, and an image of each fluorescent label is taken separately. The images are overlaid and compared for spot differences. We predicted that by using only SDS-PAGE and DIGE we would be able to qualitatively discern bovine from rabbit serum based on the different serum proteomes. The first goal of the project was to remove albumin and Immunoglobin light and heavy chains from the serum proteome because they mask the proteins of interest. Samples of whole bovine and rabbit serum were labeled with 2 different fluorescent dyes (Cy3-maleamide and Cy5-maleamide). The proteins were separated by preparative SDS-PAGE, and the albumin, Ig heavy, and Ig light chains were excised. Our second objective was to recover the remaining proteins from the gel and concentrate them. This was achieved by whole-gel elution, followed by a concentrating step using centrifugation. Our final goal was to use DIGE to compare the two labeled samples. The concentrated samples were loaded onto a 2D gel. The 2D gel was imaged for both Cy3 and Cy5 separately and the resultant images were compared. An SDS-PAGE running samples from before and after the procedure showed significant reduction in the albumin present; it also revealed proteomic differences between the two labeled samples.

T. Scott NowickiT. Scott Nowicki, Dickinson College
(Mentor: Dr. Robert Murphy)

Effects of the Oncogene H-Ras on Subcellular Location Patterns
Proteomics, which is the study of all of the proteins expressed in a given cell type, utilizes "mass-screening" approaches to study many aspects of a cell's proteome (structure, location, function, interaction, etc.). One such mass-screening method that has been rarely used is the systematic description of the subcellular location patterns for all of the proteins within a given cell (a field otherwise known as location proteomics). The location pattern of a protein is vital to determining its function within a cell (e.g. what organelles it is associated with, if it forms a complex with another protein). H-Ras is an oncogene which is mutated in 30% of human cancers. The oncogenic form of H-Ras creates hyperactive H-Ras proteins that cause overactive cell proliferation, which in turn gives rise to cancerous tumors. Studying the effect of mutant H-Ras on subcellular location patterns can yield important information on the exact mechanism by which this ontogeny is associated with cancer. I examined the subcellular location patterns of a number of proteins in NIH 3T3 cells and compared them with NIH 3T3 cells that were transfected with a mutant form of H-Ras . The proteins in question were randomly labeled with Green Fluorescent Protein using a technique called CD-tagging, which enabled me to study their subcellular location patterns. This task involved collecting 3D time-lapse videos using spinning disk confocal microscopy and extracting numerical features associated with the fluorescence patterns for each type of protein. These numerical features could then be compared using statistical tests. The results indicated a significant change in subcellular location pattern upon H-Ras expression for five different proteins: "Serum deprivation response protein," which inhibits cell division in the absense of growth serum, "ribosomal protein S6," which is a major substrate of various protein kinases in the ribosome, "annexin A5," a calcium-dependent phospholipid binding protein, "ATP synthase isoform 1," which produces ATP for the cell, and "t-complex testis expressed 1 (tctex1)," a cytoplasmic dynein light-chain subunit.   When analyzing the location patterns for each of these proteins in the normal and + H-Ras phenotypes, they were found to be statistically distinguishable. Initial analysis using a Fourier Transform algorithm has also shown the + H-Ras tctex1 line to be significantly different from its - H-Ras counterpart. These results not only indicate that the above proteins are good subjects for further study regarding their activity in cancerous cells, but also demonstrate the potential diagnostic applications of automated analysis of subcellular patterns as applied to cancer.

Ashly Smolyn, Dickinson College
(Mentor: Dr. Jon Minden)

Difference Gel Electrophoresis (DIGE) to Determine Protein Differences Between Bovine and Rabbit Serum
Serum is the liquid component of the blood. Some of the common proteins found in serum are albumin (~50% of total concentration), immunoglobulins (~14%), clotting factors, complement factors, and transferrin. Less than 1% of the total serum protein is made up of thousands of different proteins, some of which may change in abundance depending on the physiological changes and disease states. Difference gel electrophoresis (DIGE) is a method used to differentiate between two separately labeled protein samples on the same acrylamide resolving gel. Two protein samples are each fluorescently labeled a different color, and an image of each fluorescent label is taken separately. The images are overlaid and compared for spot differences. We predicted that by using only SDS-PAGE and DIGE we would be able to qualitatively discern bovine from rabbit serum based on the different serum proteomes. The first goal of the project was to remove albumin and Immunoglobin light and heavy chains from the serum proteome because they mask the proteins of interest. Samples of whole bovine and rabbit serum were labeled with 2 different fluorescent dyes (Cy3-maleamide and Cy5-maleamide). The proteins were separated by preparative SDS-PAGE, and the albumin, Ig heavy, and Ig light chains were excised. Our second objective was to recover the remaining proteins from the gel and concentrate them. This was achieved by whole-gel elution, followed by a concentrating step using centrifugation. Our final goal was to use DIGE to compare the two labeled samples. The concentrated samples were loaded onto a 2D gel. The 2D gel was imaged for both Cy3 and Cy5 separately and the resultant images were compared. An SDS-PAGE running samples from before and after the procedure showed significant reduction in the albumin present; it also revealed proteomic differences between the two labeled samples.