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The Effect of New Parameters and Increased Database Size on the Cysteine Oxidation Prediction Program. Megan Riddle with Ricardo Sanchez and Dr. Jamil Momand California State University, Los Angeles August 23, 2007. NH 2. HS. CH 2. C. COOH. H. Overview.

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slide1

The Effect of New Parameters and Increased Database Size on the Cysteine Oxidation Prediction Program

Megan Riddle

with Ricardo Sanchez and Dr. Jamil Momand

California State University, Los Angeles

August 23, 2007

NH2

HS

CH2

C

COOH

H

overview
Overview
  • Cysteine Oxidation Prediction Program (COPP)
    • Oxidation defined
  • Biological Significance of Cysteine Oxidation
    • Effects of oxidation on proteins
  • Summer 2007 Goals
    • Increase database size
    • Add new parameters
  • Methods and Results

NH2

HS

CH2

C

COOH

H

cysteine oxidation prediction program
Cysteine Oxidation Prediction Program
  • Goal: Create a program that will use physicochemical parameters to predict reactive surface cysteine thiols
  • Methods:
    • Gather examples of proteins susceptible to cysteine oxidation
    • Extract parameters from Protein Data Bank
    • Use computer classifier C4.5 to determine rules that will predict if cysteine can become oxidized
cysteine prediction
Cysteine Prediction

Two types of cysteine oxidation:

  • Permanent structural oxidation: cysteines that form permanent disulfide bonds or bind to metals shortly after translation
      • Prediction programs based on sequence already exist  88% accuracy (Martelli et al. 2002)
  • Reactive surface cysteine thiols: cysteines that become oxidized under certain conditions, most reversibly
      • No prediction programs exist  COPP
biological significance oxidation and enzyme function
Biological Significance: Oxidation and Enzyme Function
  • The active sites of glutaredoxin and thioredoxin cycle between reduced and oxidized states

http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/Thioredox/RNA2.GIF

enzyme inactivation via oxidation
Enzyme Inactivation via Oxidation
  • H2O2 inactivates PTEN tumor suppressor protein by causing the formation of a disulfide bond

Lee et al. JBC (2002)

summer 2007 goals
Summer 2007 Goals
  • Increase the size of the COPP database
  • Test new parameters to determine if they affect the rules and accuracy of COPP

NH2

HS

CH2

C

COOH

H

increase database size
Increase Database Size
  • Previously:
    • 85 proteins that undergo non-structural cysteine oxidation
    • 135 cysteines that undergo oxidation
    • 225 cysteines that remain reduced under oxidizing conditions
  • To create an accurate, general set of rules for cysteine oxidation requires a large, unbiased database
methods increase database size
Methods: Increase Database Size
  • Search Entrez for keywords
    • i.e. cysteine and oxidation, sulfenic acid, etc.
  • Look for proteins in Protein Data Bank

60

Potential Proteins

increase database size11
Increase Database Size
  • Do BLASTALL – eliminate proteins with:
    • Identity > 35%
    • E value < 1
    • Conserved cys

31

Potential Proteins

40

123

Cysteines

Oxidize

increase database size12
Increase Database Size

Original Proteins

New Proteins

C4.5/ J48

Rules to Classify Cysteines

results new proteins
Results: New Proteins
  • Increased database size caused reduction in rules
  • Accuracy decreased

Old Rules

New Rules

S1 DISTANCE <= 6: 1 (88.19/17.0)

S1 DISTANCE > 6

| ASA (Å2) <= 1: 0 (136.51/6.51)

| ASA (Å2) > 1

| | N1 DISTANCE <= 5.2: 1 (32.54/9.0)

| | N1 DISTANCE > 5.2

| | | O1 ASA <= 2: 1 (33.0/15.0)

| | | O1 ASA > 2: 0 (71.76/15.76)

S1 DISTANCE <= 6.1: 1 (115.44/28.0)

S1 DISTANCE > 6.1

| ASA (Å2) <= 1.8: 0 (177.51/12.51)

| ASA (Å2) > 1.8

| | N1 DISTANCE <= 5.4: 1 (46.54/17.0)

| | N1 DISTANCE >5.4: 0 (133.51/39.51)

-

+

Cys-S

H

Cys-S

H

O-Q/D

N-K/R/H

H

S-Cys

5.2Å

81.8% Accuracy

79.1% Accuracy

Sanchez (2007)

methods parameters already used by copp
Methods: Parameters already used by COPP
  • S1 DISTANCE distance to nearest sulfur atom
  • S1 ASA area exposed to the surface
  • N1 DISTANCE distance to the nearest +nitrogen atom
  • N1 DONOR nitrogen’s parent side chain
  • N1 ASA area exposed to the surface
  • O1 DISTANCE distance to the nearest -oxygen
  • O1 DONOR oxygen’s parent side chain
  • O1 ASA area exposed to the surface
  • ASA exposed surface of S in question
  • CLASS class: 0 if reduced; 1 otherwise
methods parameters already used by copp15
Methods: Parameters already used by COPP
  • S1 DISTANCE distance to nearest sulfur atom
  • S1 ASA area exposed to the surface
  • N1 DISTANCE distance to the nearest +N atom
  • N1 DONOR nitrogen’s parent side chain
  • N1 ASA area exposed to the surface
  • O1 DISTANCE distance to the nearest -oxygen
  • O1 DONOR oxygen’s parent side chain
  • O1 ASA area exposed to the surface
  • ASA exposed surface of S in question
  • CLASS class: 0 if reduced; 1 otherwise
new parameters
New Parameters
  • pKa: acid dissociation constant
    • How easily can the S lose a proton?
  • Electrostatic Potential: potential energy per unit charge
    • How well stabilized is the charged S after the proton is lost?

NH2

-

H

S

CH2

C

COOH

H

methods new parameters
Methods: New Parameters
  • PCE: Protein Continuum Electrostatics
    • Calculates Electrostatic Potential

CoordinatesElectrostatic Potential

7.854 0.668 -0.602 -10.725 4.683 1.223 3.305 -25.413 3.330 8.072 3.708 -19.335 2.256 -11.243 9.879 -21.887 14.014 7.907 3.298 -13.670

http://bioserv.rpbs.jussieu.fr/cgi-bin/PCE-Pot

Miteva et al. NAR (2005)

new parameters18
New Parameters
  • PROPKA
    • Calculates pKa

http://propka.ki.ku.dk/

Li et al. Proteins (2005)

new parameters19
New Parameters

Electrostatic Potential and pKa Data

Original Data

C4.5/ J48

Rules to Classify Cysteines

results new parameters
Results: New Parameters
  • New parameters caused an alteration in the final rule
  • The accuracy is similar

Old Parameters

New Parameters

S1 DISTANCE <= 6.1: 1 (115.44/28.0)

S1 DISTANCE > 6.1

| ASA (Å2) <= 1.8: 0 (177.51/12.51)

| ASA (Å2) > 1.8

| | N1 DISTANCE <= 5.4: 1 (46.54/17.0)

| | N1 DISTANCE >5.4: 0 (133.51/39.51)

S1 DISTANCE <= 6.1: 1 (115.44/28.0)

S1 DISTANCE > 6.1

| ASA (Å2) <= 1.8: 0 (177.51/12.51)

| ASA (Å2) > 1.8

| | pKa of S0 <= 8.75: 1 (74.29/32.0)

| | pKa of S0 > 8.75: 0 (105.76/26.76)

79.0698% Accuracy

78.6469% Accuracy

conclusions
Conclusions
  • New Proteins:
    • A larger database results in a more general, but less accurate, set of rules
  • New Parameters:
    • A low pKa value correlates with oxidation, but does not improve the accuracy of COPP
  • Future Goals:
    • Make COPP publicly available
    • Modify COPP to predict type of oxidation
with many thanks to
With many thanks to. . .

Dr. Jamil Momand, Ricardo Sanchez, and the rest of the Momand lab

SoCalBSI fellow students and mentors

California State University at Los Angeles

Funding from:

LA Orange County Biotechnology Center

results new proteins26
Results: New Proteins
  • Increased database size caused reduction in rules
  • Accuracy decreased

Cys-SH

HS-Cys

Old Rules

New Rules

S1 DISTANCE <= 6: 1 (88.19/17.0)

S1 DISTANCE > 6

| ASA (Å2) <= 1: 0 (136.51/6.51)

| ASA (Å2) > 1

| | N1 DISTANCE <= 5.2: 1 (32.54/9.0)

| | N1 DISTANCE > 5.2

| | | O1 ASA <= 2: 1 (33.0/15.0)

| | | O1 ASA > 2: 0 (71.76/15.76)

S1 DISTANCE <= 6.1: 1 (115.44/28.0)

S1 DISTANCE > 6.1

| ASA (Å2) <= 1.8: 0 (177.51/12.51)

| ASA (Å2) > 1.8

| | N1 DISTANCE <= 5.4: 1 (46.54/17.0)

| | N1 DISTANCE >5.4: 0 (133.51/39.51)

81.8% Accuracy

79.1% Accuracy

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