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

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

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

4. Disulfide Bonds in Asymmetric Unit of AP

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

6. Materials Materials 0.5 µg/µL of alkaline phosphatase (AP) in 50 mM Tris-HCl at pH 8 100 µg Trypsin in 1 mM HCl 9% acetonitrile (ACN) in ammonium bicarbonate (ambic) Trypsin in ACN & ambic used in experiment 50 mM or 70 mM DTT in water

7. Methods Overview Control: AP + TrypsinAP + DTT + Trypsin 1. Trypsin + AP, incubate in 37 ⁰ C1. AP+ DTT, incubate in 37 ⁰ C for 1 hr, then add trypsin 2. Collect timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min 3. Run SDS-PAGE gel and stain

8. Expt 1: Methods Control: AP + TrypsinAP + DTT + Trypsin Ratio: 2:1 (v/v) AP:trypsinRatio: 1. 25uL of trypsin + 50uL AP, incubated in 37 ⁰ C for 1 hr 1. 25uL of AP+ 4 uL of 50 mM DTT, incubated in 37 ⁰ C for 1 hr, then 50uL of trypsin added 2. Timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min 3. Quench reaction in boiling H 2 O immediately for 5 min, add 24 uL LD and boil again, 5 min 4. Load 15% SDS-PAGE gel and run for 1 hr at 150V, then stain with Coomassie O/V, destain in 50/10 methanol/acetic acid, then 10/10 methanol/acetic acid

9. Experiment 1: Results

10. Expt 2: Methods Control: AP + TrypsinAP + DTT + Trypsin Ratio: 10:1 (w/w) AP:trypsin 1. 100 uL of 0.5 ug/uL AP + 100 uL of 0.02 ug/uL trypsin, incubate in 37 ⁰ C for 1 hr 1. 100 uL of 0.5 ug/uL AP + 4 uL of 70 mM DTT, incubate in 37 ⁰ C for 1 hr, then + 100 uL of 0.02 ug/uL trypsin, incubate in 37 ⁰ C for 1 hr 2. Collect 8 uL timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min 3. Quench reaction in boiling H 2 O immediately for 5 min, add 24 uL LD and boil again, 5 min 5. Load 15% SDS-PAGE gel and run for 1 hr at 150V, then stain with Coomassie O/V, destain in 50/10 methanol/acetic acid, then 10/10 methanol/acetic acid 4. Load 15% SDS-PAGE gel and run for 1 hr at 150V, then stain with Coomassie O/V, destain in 50/10 methanol/acetic acid, then 10/10 methanol/acetic acid

11. Experiment 2: Results

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

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

14. References 1 M. Sone, S. Kishigami, T. Yoshihisa, and K. Ito (1997) J. Biol. Chem. 272, 6174 - 6178. Roles of Disulfide Bonds in Bacterial Alkaline Phosphatase. 2 Hazzard, James T. "Limited Proteolysis." Biochemistry 463a. 21 Jan. 2011. Web. 21 Nov. 2011. 3 Derman, 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 Escherichia coli 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. 6 J.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.