Limited ProteolysisKyle Arrington , Syna Daudfar, Shiana Ferng, Tyler Foutch, Jay Mitchell, Siddharth

Pandya, and Arvin Jandu

Fall 2011 - BIOC463A

Purpose

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

Background

AP is very stable◦Denatures at high temperatures

Disulfide bondsDithiothreitol (DTT): Reduces disulfide

bonds

Disulfide Bonds in Asymmetric Unit of AP

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.

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

Methods Overview

Control: AP + Trypsin AP + DTT + Trypsin

1. Trypsin + AP, incubate in 37⁰C 1. AP+ DTT, incubate in 37⁰C for 1 hr, then add trypsin

2. Collect timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min

2. Collect timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min

3. Run SDS-PAGE gel and stain 3. Run SDS-PAGE gel and stain

Expt 1: Methods

Control: AP + Trypsin AP + DTT + Trypsin

Ratio: 2:1 (v/v) AP:trypsin Ratio:

1. 25uL of trypsin + 50uL AP, incubated in 37⁰C for 1 hr

1. 25uL of AP+ 4 uL of 50 mM DTT, incubated in 37⁰C for 1 hr, then 50uL of trypsin added

2. Timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min

2. Timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min

3. Quench reaction in boiling H2O immediately for 5 min, add 24 uL LD and boil again, 5 min

3. Quench reaction in boiling H2O 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

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

Experiment 1: Results

Expt 2: Methods

Control: AP + Trypsin AP + DTT + Trypsin

Ratio: 10:1 (w/w) AP: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 37⁰C for 1 hr

1. 100 uL of 0.5 ug/uL AP + 4 uL of 70 mM DTT, incubate in 37⁰C for 1 hr, then + 100 uL of 0.02 ug/uL trypsin, incubate in 37⁰C for 1 hr

2. Collect 8 uL timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min

2. Collect 8 uL timepoints: 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min

3. Quench reaction in boiling H2O immediately for 5 min, add 24 uL LD and boil again, 5 min

3. Quench reaction in boiling H2O 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

Experiment 2: Results

Discussion

Inconclusive results◦(+) DTT experiment = Control

Trypsin:AP RatioTrypsin autolysis[DTT]Trypsin reconstitution error

Future Directions

Trypsin Gold◦Modified K residues

Increasing [DTT]

References

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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 –77224 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.

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.