Covalent Versus Electrostatic Attachment of Yeast Cytochrome c to a Fused Silica Surface
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Transcript of Covalent Versus Electrostatic Attachment of Yeast Cytochrome c to a Fused Silica Surface
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Covalent Versus Electrostatic Attachment of Yeast
Cytochrome c to a Fused Silica Surface
By Sheetal Mistry
Department of Chemistry, Butler UniversityIndianapolis, IN 46208
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Yeast cytochrome c• In yeast cells
• Similar in function to some cytochromes in eukaryotic cells
• Water soluble peripheral protein
• Resides in intermembrane space of mitochondria
• Positively charged at pH 7 ~since pI at 10.7
• Located near negatively charged phospholipid bilayer surface
• Heme (red) consists of Iron
• Sovlent exposed Cysteine (yellow)
Yeast Cytochrome c
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Functionality
Plays a major role in the electron transport chain in the inner membrane of mitochondria
Shuttles electrons between complexes III & IV
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Methods:
1. Solution Absorption
2. ATR (Attenuated Total Internal Reflection)
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Solution Absorption
10 µM [YCC], 7mM Succinate Buffer, pH 4.00
Soret Band
300 400 500 600
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Abs
orba
nce
Wavelength (nm)
• Soret peak at 408 nm
• Used to measure unfolding
•Soret band shifts left
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Conformation
• Three dimensional structure• Primary, Secondary, and
Tertiary• Helices maximize hydrogen
bonds• Conformation is considered
“native” in solution under physiological conditions (pH≈7)
Cox, M., Nelson, D. Principles of Biochemistry 2000:194
Process of Denaturation:• Temperature change• pH change• Chemical change
- Urea - Alcohol
Tertiary
Primary
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ATR spectroscopy
Cheng, Y.-Y.; Lin, S. H.; Chang, H.-C.; Su, M.-C.: Probing Adsorption, Orientation and Conformational Changes ofCytochrome c on Fused Silica Surfaces with the Soret Band. J. Phys. Chem. A pp. 10687, 107(49) 2003
• ATR (Attenuated Total internal Reflection)
• Only detects proteins on surface
DetectorPrism
θ
Glass plateO-ring
Sample solution
To detector
Light
Source
• Quartz prism
• Hydrophilic surface
• Negatively charged (similar to phospholipid bilayer)
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Experiments:
1. YCC free in solution
3. YCC covalently attached
3. YCC electrostatically attached
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YCC Free in Solution
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Solution Absorption
Wavelength20% alcohol60% alcohol
Alcohol Denaturation pH Denaturation
Solution at pH 6.9, 3.2, 2.9 and 1.9 (from right to left)
Proteins denature at higher [alcohol] and at lower pH
wavelength
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YCC Covalently Attached
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How is YCC covalently tethered on silica?
Si
H3CO
H3CO
OCH3
NH2
(3-aminopropyl)-trimethoxylsilane
+ N
O
OS
O
O-Na+O
O
N
O
O
O
N[γ-maleimidobutyryloxy]sulfosuccinimide ester
Si
H3CO
H3CO
OCH3
NH
N
O
O
O
+
Si
H3CO
H3CO
OCH3
NH
N
O
O
O
SiO2
SiO2
SiO2
YCC
sGMBS
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pH Dependent Surface Adsorption
Shift of the Soret band maximum: Free YCC , Surface bound YCC
Conditions: 7mM phosphate buffer
YCC on surface takes longer to unfold than the solution
YCC on surface and in solutiondenature as the pH is lowered
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Surface Alcohol Denaturing
Shifts of the Soret band maximum:(b) Surface bound YCC(c) Free YCC
Methanol1-propanol
Conditions: 7mM phosphate buffer, pH 4.00
,
,
• YCC in solution denatures with respect to change in [alcohol]
• YCC on surface denatures partially
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YCC Electrostatically attached
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Yeast Cytochrome c
-disulfide linkage.
-Dimerization of YCC
dimerMonomer
Significance of Sulfur
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Why work with monomer?
• Dimer has different functionality
• Want to compare with the covalently tethered proteins– No chances for these proteins to dimerize as
the sulfur is covalently bound to the surface
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Method to retain monomer
1. Treatment with iodoacetate:
Reaction:
I
OH
O
+
IodoacetateYCC
OH
O
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2. Size Exclusion Chromatography:
• Separate molecules of different sizes
• Heavy molecules elute rapidly
• Sephadex G-50• Dimer (2 x 12,588 g/mol)• Monomer (12,588 g/mol)
Method to retain monomer
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3. Gel Electrophoresis: Molecular HCC YCC Weight 1μg 1μg
Marker 15μL 15μL
Method to retain monomer
Dimer ~24,000g/mol
Monomer~12,000g/mol
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Procedure to get the data:
1. Make samples
2. Kinetic study
3. Surface washing
4. Scans
5. Data analysis
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Encountered Problems at Step 2
Right after Taiwan
Proteins did not stick to the Surface…. Does not look like a Kinetic scan
Intensity proportional to numberof proteins on surface
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Several Factors could play a role
1. [YCC]
2. [Buffer]
3. [alcohol]
4. [NaCl]
5. pH
Result: Found that by using the base bath, the surface was getting too basic and was not allowing proteins to stick to the surface.
Tried using diluted soap by rinsing the surface several times and turned Out to be a success.
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Kinetic Study
-2 0 2 4 6 8 10 12 14 16-0.002
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
Abs
orb
ance
Time (min)
Time Scan 2 uM YCC, pH 4.00, 7mM succinate buffer• Proteins stick to the surface
longer
• See the monolayer
• Take the data when see the monolayer
For every sample: 1. Kinetic scan 2. Record the time (monolayer) 3. Take data
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Adsorption Isotherm
pH 4.0, 7mM Succinate Buffer
Abs vs. [ ]
0.0000
0.0020
0.0040
0.0060
0.0080
0.0100
0.0120
0.0140
0 2 4 6 8 10 12
[ YCC] uM
Abs
• purpose: know the concentration at which the covalently anchored studies were done
• Surface saturation around 10 µM YCC concentration
Kad YCC = 1.3 E6
Kad HCC = 1.3 E6
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Determination of the [YCC]
Abs max = 0.0054
350 400 450 500 550 600
0.000
0.002
0.004
0.006
0.008
0.010
A
bs
wavelength
pH 4.00, 7mM phosphate buffer
Surface Adsorption of covalently anchored YCC
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Determination of [YCC]
~0.0054
Electrostatically adsorbedSurface Adsorption Isotherm
Covalently attached studies done at ~1.00 uM [YCC]
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Future Direction
Denaturation studies:– Variation in pH– Variation in alcohol
• Methanol• 1-propanol
Methods:– Solution Absorption– ATR Spectroscopy
For Electrostatic Attachment :
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Special Thanks To
Dr. Geoffrey C. Hoops
Dr. Todd A. Hopkins
Dr. Meng-Chih Su
Victoria Fahrenbach
Tara Benz
Greg Campanello
Carrie Ann Hedge
Ken ClevengerButler Summer InstituteHolcomb Undergraduate GrantsLilly Endowment
Butler University Department of Chemistry
Collaborators:Y.-Y. Cheng, S. H. Lin, and H.-C. ChangInstitute of Atomic and Molecular Sciences,Academia Sinica
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Surface adsorption
0 20 40 60 80
402
403
404
405
406
407
408
409
410
S
ore
t M
axim
um
(n
m)
% Alcohol
Alcohol: 1 propanolpH 4.00Buffer: 7 mM Succinate[YCC] : 1.00E-6M
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Solution Absorption
0 20 40 60 80398
399
400
401
402
403
404
405
406
407
408
409
410
So
ret M
axim
um (
nm)
% Alcohol
Alcohol: 1 propanolpH 4.00Buffer: 7 mM Succinate[YCC] : 1.00E-6M