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Temperature dependent stability study concerning the PDZ domain of TIAM 1 Wild Type Protein

Temperature dependent stability study concerning the PDZ domain of TIAM 1 Wild Type Protein. Allison McClain Dr. David Speckhard. T-cell lymphoma invasion and metastasis-inducing protein (TIAM) 1. TIAM is a protein complex that is found in almost all types of tissues

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Temperature dependent stability study concerning the PDZ domain of TIAM 1 Wild Type Protein

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  1. Temperature dependent stability study concerning the PDZ domain of TIAM 1 Wild Type Protein Allison McClain Dr. David Speckhard

  2. T-cell lymphoma invasion and metastasis-inducing protein (TIAM) 1 • TIAM is a protein complex that is found in almost all types of tissues • Nerve tissue in the brain has a high amount of it • A modulator that activates proteins including Rho GTP-binding and GTPase Rac1 • The proteins that TIAM control, regulate cell polarity, adhesion, migration and metastasis • Rac1 plays a significant role in the development of various cancers TIAM 1 3d structure

  3. Overexpressed TIAM 1 presence in tumor cells is shown to make the cancer more invasive and metastasize easier and quicker • This is why drug therapies target it TIAM 1 is overexpressed, or transfected, on the upper right. Cell is larger and ruffled when compared to normal TIAM 1 cell (left, bottom)

  4. post synaptic density, Drosophila disc large tumor suppressor, zonulaoccludens-1 protein (PDZ) domain • Abundant- over 200 PDZ domains identified • Important domain in the TIAM 1 protein • Main interactive part of TIAM 1 that can be studied well • Binding ability allows PDZ to regulate biological functions of the cell PDZ domain of TIAM 1

  5. Nuclear Magnetic Resonance spectroscopy (NMR) • Basic NMR is 1 dimension, like learned in organic chemistry • Allows for the chemical structure of a known or unknown material to be determined • Relies on the fact that the nuclei of atoms have a spin associated with them • Chemical shifts arise based on the nearby bonds and atoms • The peaks indicate the chemical structure of the unknown material

  6. 1d NMR sample

  7. 2 dimension nmr - HSQC • Defined by two axes instead of one • Used to determine structure of molecules too complicated to be determined by 1d nmr –signals overlap and can’t be read • Results in contour map that plots two frequencies and intensity against each other, instead of frequency against intensity only • 2d is Advantageous • 15 N-HSQC correlates to the hydrogen groups attached to the nitrogen

  8. Resultant NMR peaks are plotted as like the top of a topographical map – uses two different times and if frequency stays the same it can be viewed as a point or the top of a topographical map 1d NMR reading compared to a 2d HSQC NMR reading

  9. Methods • Using transformed BL21 DE3 cells • Start with cells that already have the plasmid that will make PDZ • Grow cells with and add IPTG • Harvest and then lyse cells • Purify PDZ by use of column chromatography • Collect the a 1 d protein NMR • Use SDS-Page to determine if PDZ is detected

  10. After being sure this method works be added in place of • Repeat the previous steps • After collecting and concentrating the protein, HSQC spectra will be collected

  11. 2D NMR • Collect HSQC spectra at different temperatures and analyze the results • Compare peak intensity • Determine where peaks have changed as temperature increases • Change of peaks and peak intensity indicates the protein unfolding and becoming unstable

  12. General Objectives • Determine the stability of PDZ TIAM 1 at different temperatures • Determine the stability of • Loops • Beta Sheets • Alpha Helices • Expecting loops to be the least stable aspect of the protein

  13. Current Loras Research on TIAM • Stability study • Mutant (QM) PDZ in TIAM 1 protein instead of non mutant PDZ • Temperature dependent NMR run and analyzed • Protein-Protein interaction • PDZ and cancer interaction • Students are studying how TIAM1 and TIAM2 interact with certain other proteins

  14. Other objectives: • Will compare the wild type data to a mutant PDZ protein’s data • Our hypothesis is that the non mutated PDZ will be more stable

  15. Sources • http://jcb.rupress.org/content/139/3/797.full.pdf • http://www.biosignaling.com/content/8/1/8 • http://www.ncbi.nlm.nih.gov/pubmed/9204476 • http://www.ncbi.nlm.nih.gov/pubmed/20802514 • http://atlasgeneticsoncology.org/Genes/TIAM1ID42557ch21q22.html • http://www.emory.edu/NMR/mysite06/NMR%20Course/all_book_041410.pdf • http://www-keeler.ch.cam.ac.uk/lectures/2d_a4.pdf • http://www.cryst.bbk.ac.uk/PPS2/projects/schirra/html/2dnmr.htm • http://chem.ch.huji.ac.il/nmr/techniques/2d/2d.html

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