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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 AngelesAugust 23, 2007
HS CH2 C COOH
H
NH2
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
HS CH2 C COOH
H
NH2
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
Oxidation of Cysteines
Sanchez (2007)
Cysteine Prediction
Two types of cysteine oxidation:1. 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)
2. Reactive surface cysteine thiols: cysteines that become oxidized under certain conditions, most reversibly
– No prediction programs exist COPP
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
• H2O2 inactivates PTEN tumor suppressor protein by causing the formation of a disulfide bond
Lee et al. JBC (2002)
Summer 2007 Goals
1. Increase the size of the COPP database
2. Test new parameters to determine if they affect the rules and accuracy of COPP
HS CH2 C COOH
H
NH2
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 1. Search Entrez for keywords
• i.e. cysteine and oxidation, sulfenic acid, etc.
2. Look for proteins in Protein Data Bank
Potential Proteins
Increase Database Size
3. Do BLASTALL – eliminate proteins with:• Identity > 35%• E value < 1• Conserved cys
Potential Proteins
Cysteines Oxidize
Increase Database Size
C4.5/ J48
Original Proteins
Rules to Classify Cysteines
New Proteins
Results: New Proteins
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)
6Å
+
5.2Å
-
Sanchez (2007)
• Increased database size caused reduction in rules
• Accuracy decreasedOld Rules New Rules
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
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 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
• 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?
-S CH2 C COOH
H
NH2
H
Methods: New Parameters • PCE: Protein Continuum Electrostatics
– Calculates Electrostatic Potential
Coordinates Electrostatic 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
Miteva et al. NAR (2005)http://bioserv.rpbs.jussieu.fr/cgi-bin/PCE-Pot
New Parameters• PROPKA
– Calculates pKa
Li et al. Proteins (2005)http://propka.ki.ku.dk/
New Parameters
C4.5/ J48
Electrostatic Potential and pKa Data
Original Data
Rules to Classify Cysteines
Results: New Parameters
• New parameters caused an alteration in the final rule
• The accuracy is similar
Old ParametersS1 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)
79.0698% Accuracy
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)
New Parameters
78.6469% Accuracy
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. . .
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
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Results: New Proteins
• Increased database size caused reduction in rules
• Accuracy decreased
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)
81.8% Accuracy 79.1% Accuracy
Old Rules New RulesS1 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-SH HS-Cys