copper binding to premature galactose oxidase: biogenesis of the tyr-cys cofactor alta howells...
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Copper Binding to Premature Galactose
Oxidase: Biogenesis of the Tyr-Cys Cofactor
Alta HowellsDooley Group
Chemistry and Biochemistry
The Dooley GroupThe Dooley GroupCopper MetalloproteinsCopper Metalloproteins
NO3- NO2- NO N2O N2
Nitrous OxideNitrous Oxide ReductaseReductaseRCH2NH2 + O2 RCHO + NH3 + H2O2
Amine OxidaseAmine Oxidase
RCH2OH + O2 RCHO + H2O2
Galactose OxidaseGalactose Oxidase
Why Study Metalloproteins?
• 1/3 of all proteins require metals for their function–Fe, Cu, Co, Zn
Cytochrome c OxidasePDB: 1OCC
A perfect example…
Copper Proteins• Produces reactive
oxygen speciesH2O2, Superoxide, Hydroxyl
• Genetic diseases• Green house gases• Biotechnology
Galactose Oxidase
RCH2OH + O2 RCHO + H2O2
Implication of GO• Secreted from Polyporus circinatus Fr. • Believed to break down tree lignin• Biomedical applications
Sensor for colon cancerDisaccharide tumors
Bioassays for D-Galactose and Lactose
• Synthesis of carbohydrates
Structure-Function Relationship
StructuStructurere
How the Structure is GeneratedHow the Structure is GeneratedThe pieces that make it a wholeThe pieces that make it a whole
Without those Without those piecespiecesLOSS OF LOSS OF FUNCTIONFUNCTION
H496
Y495
H581
Y272
C228 W290
H2O
Crystal StructureCrystal StructureEssential CofactorsEssential Cofactors CuCu Tyr272-Cys228Tyr272-Cys228
Crystal Structure and Active Site of Galactose Oxidase
Maturation of Galactose Oxidase• Four post-translational modification events:
??
Previous Research
• Tyr-Cys crosslink is formed with Cu and oxygen or excess Cu(II) (Rogers)
• Cu(I) processes at a faster rate than Cu(II) (Whittaker)
Rogers, Melanie.Rogers, Melanie. Biochemistry, Biochemistry, 20082008, 47, 39, 47, 39
Whittaker, Mei M. Whittaker, Mei M. JBCJBC, , 20032003, 278, 22090, 278, 22090
Previous Research
• Cu(II)SO4 titration into Premat-GO in anaerobic condition
• 406 nm band increases over time and then decays
• Yellow complex is formed
Rogers, Melanie.Rogers, Melanie. Biochemistry, Biochemistry, 20082008, 47 (39), 47 (39)
Current Work on Cu(II) Binding
• Binding affinity of Cu(II)
• Titrate Cu(II)(Gly)4 complex into Premat-GO in anaerobic conditions
• Use UV-Vis and CD spectroscopy • Observe the change in abs (UV-Vis) and
molar ellipticity (CD) as we are titrating in Cu(II)
• Rate of formation of certain intermediates
Experiment 1: Cu(II)(Gly)4 to Premat-GO in aerobic conditions
0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.600
200
400
600
800
1000
1200
Mo
lar
Ell
ipti
city
[Cu(II)]mM
Change in Molar Ellipticity at 490nm
375 400 425 450 475 500 525 550
-2500
-2000
-1500
-1000
-500
0
500
1000
1500
Mo
lar
Ell
ipti
city
Wavelength nm
.307mM ApoGO .5:1 Cu:ApoGO 20min. .5:1 Cu:ApoGO 40min. 1:1 Cu:ApoGO 60min. 1:1 Cu:ApoGO 80min. 1.5:1 Cu:ApoGO 100min. 1.5:1 Cu:ApoGO 120min. 2:1 Cu:ApoGO 140min. 2:1 Cu:ApoGO 160min.
Concentration= .307mM Premature GO ~ 20.9mg/ml
Experiment 2: Cu(II)(Gly)4 to premat-GO in anaerobic conditions
0.00 0.05 0.10 0.15 0.20 0.25-1000
-500
0
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1000
1500
2000
2500
3000
3500
Mo
lar
Elli
ptic
ity
[Cu(II)]mM
Change in Molar Ellipticity at 425 nm
0.8 : 1 (Cu : protein)0.8 : 1 (Cu : protein)Issues:Issues:• Signal to noise ratioSignal to noise ratio• Protein precipitationProtein precipitation
300 350 400 450 500 550 600 650 700 750 800-5000
-4000
-3000
-2000
-1000
0
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4000
0 mol Cu: 1mol ApoGO .2 mol Cu: 1 mol ApoGO .4 mol Cu: 1 mol ApoGO .6 mol Cu: 1 mol ApoGO .8 mol Cu: 1 mol ApoGO 1 mol Cu: 1 mol ApoGO 1.5 mol Cu: 1 mol ApoGO 2 mol Cu: 1 mol ApoGO
Mo
lar
Ell
ipti
city
Wavelength nm
Protein Concentration= .247mM Premature GO ~ 16.8mg/mL
Experiment 3: Cu(II)(Gly)4 complex to Premat-
GO with anaerobic conditions
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
-600
-400
-200
0
200
400
600
800
1000
1200
1400
1600
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2200
Mo
lar
Elli
ptic
ity
[Cu(II)]mM
Change in Molar Ellipticity at 425nm
425nm425nm
Peaks at a ratio ofPeaks at a ratio of.5 mol Cu(II): 1 mol Premat-GO.5 mol Cu(II): 1 mol Premat-GO
Issues:Issues:• Signal to noise ratioSignal to noise ratio• Incubation timeIncubation time• Temperature affecting kineticsTemperature affecting kinetics
375 400 425 450 475 500 525 550 575 600 625
-2000
-1500
-1000
-500
0
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1000
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Concentration= .241mM Premature GO ~ 16.4 mg/mL
Mo
lar
Ell
ipti
city
Wavelength nm
ApoGO .1 Cu(II) : 1 ApoGO .2 Cu(II) : 1 ApoGO .3 Cu(II) : 1 ApoGO .4 Cu(II) : 1 ApoGO .5 Cu(II) : 1 ApoGO .6 Cu(II) : 1 ApoGO .7 Cu(II) : 1 ApoGO .8 Cu(II) : 1 ApoGO
Experiment 4: Determining Incubation Time
Incubation on ice (4 C)
At 25 C
Intermediate formationIntermediate formationin 20 minutesin 20 minutes
Intermediate Intermediate formationformationin 40 minutesin 40 minutes
300 400 500 600 700 800 900 1000
0.00
0.05
0.10
0.15
Abs
orba
nce
Wavelength nm
Abs ApoGO Abs Time 0 Abs 10 mins Abs 20 mins Abs 30 mins Abs 40 mins Abs 50 mins Abs 60 mins Abs 70 mins Abs 80 mins Abs 90 mins Abs
0.5 mol Cu(II) : 1 mol ApoGOConc. Protein = 11.3mg/mL
400 500 600 700 800 900
0.00
0.03
0.06
0.09
0.12
0.15
0.18
0.21
Ab
sorb
an
ce
Wavelength nm
Buffer ApoGO Abs Time 0 Abs 5 mins. Abs 10 mins. Abs 15 mins. Abs 20 mins. Abs 25 mins. Abs 30 mins. Abs 35 mins. Abs 40 mins. Abs 45 mins. Abs 50 mins. Abs 55 mins. Abs 60 mins. Abs Buffer + Cu
0.5 mol Cu(II) : 1 mol ApoGOConc. Protein = 17.7mg/mL
Issues:• O2 contamination
Current Research Summary
What has been achieved…
• Cu(II)(Gly)4 binds to premat-GO
• Yellow complex reaches maximum at 0.5:1 (Cu : Protein)
• Incubation time: 20 minutes at 25C
To obtain quality data…• Reaction temperature• Max protein concentration• Maintain anaerobic
condition
AcknowledgementsAcknowledgements