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Limited Proteolysis

Limited Proteolysis. Kyle Arrington , Syna Daudfar, Shiana Ferng , Tyler Foutch , Jay Mitchell, Siddharth Pandya, and Arvin Jandu Fall 2011 - BIOC463A. Test the effects of DTT on the stability of Alkaline Phosphatase (AP) during tryptic digestion. Purpose. AP is very stable

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Limited Proteolysis

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  1. Limited Proteolysis Kyle Arrington , Syna Daudfar, ShianaFerng, Tyler Foutch, Jay Mitchell, Siddharth Pandya, and Arvin Jandu Fall 2011 - BIOC463A

  2. Test the effects of DTT on the stability of Alkaline Phosphatase (AP) during tryptic digestion Purpose

  3. AP is very stable • Denatures at high temperatures • Disulfide bonds • Dithiothreitol (DTT): Reduces disulfide bonds Background

  4. Disulfide Bonds in Asymmetric Unit of AP

  5. Hypothesis: AP with intact disulfide bonds is resistant to proteolytic digestion (via trypsin). In the presence of DTT, AP will be digested. Overview of Experiment

  6. Materials

  7. Methods Overview

  8. Expt 1: Methods

  9. Experiment 1: Results

  10. Expt 2: Methods

  11. Experiment 2: Results

  12. Inconclusive results • (+) DTT experiment = Control • Trypsin:AP Ratio • Trypsin autolysis • [DTT] • Trypsin reconstitution error Discussion

  13. Trypsin Gold • Modified K residues • Increasing [DTT] Future Directions

  14. 1M. Sone, S. Kishigami, T. Yoshihisa, and K. Ito (1997) J. Biol. Chem. 272, 6174 - 6178. Roles of Disulfide Bonds in Bacterial Alkaline Phosphatase. 2Hazzard, James T. "Limited Proteolysis." Biochemistry 463a. 21 Jan. 2011. Web. 21 Nov. 2011. 3Derman, A. I., and Beckwith, J. (1991) J. Bacteriol. 173, 7719 –7722 4 Bessette PH, Åslund F, Beckwith J, Georgiou G. Efficient folding of proteins with multiple disulfide bonds in the Escherichiacoli cytoplasm . Proc Natl Acad Sci U S A. 96:13703-13708. (1999). 5 Rietsch A., Belin D., Martin N., Beckwith J. 1996. An in vivo pathway for disulfide bond isomerization in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 93:13048–13053. 6J.E. Coleman, Structure and mechanism of alkaline phosphatase. Annu. Rev. Biophys. Biomol. Struct.,  21  (1992), pp. 441–483.  7 Stec B, Holtz KM, Kantrowitz ER. A revised mechanism for the alkaline phosphatase reaction involving three metal ions. J Mol Biol. 2000;299:1303–1311. References

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