Covalent Versus Electrostatic Attachment of Yeast  Cytochrome c to a Fused Silica Surface By  Sheetal Mistry Department of...
Yeast cytochrome c <ul><li>In yeast cells </li></ul><ul><li>Similar in function to some cytochromes in eukaryotic cells </...
Functionality Plays a major role in the electron transport chain in the inner membrane of mitochondria Shuttles electrons ...
Methods: <ul><li>1.  Solution Absorption </li></ul><ul><li>2.  ATR (Attenuated Total Internal  </li></ul><ul><li>  Reflect...
Solution Absorption 10   µ M [YCC], 7mM Succinate Buffer, pH 4.00   Soret Band <ul><li>Soret peak at 408 nm </li></ul><ul>...
Conformation <ul><li>Three dimensional structure </li></ul><ul><li>Primary, Secondary, and Tertiary </li></ul><ul><li>Heli...
ATR spectroscopy Cheng, Y.-Y.; Lin, S. H.; Chang, H.-C.; Su, M.-C.: Probing Adsorption, Orientation and Conformational Cha...
Experiments: <ul><ul><ul><ul><li>YCC  free  in solution </li></ul></ul></ul></ul><ul><ul><ul><ul><li>YCC  covalently  atta...
YCC  Free  in Solution
Solution Absorption Wavelength 20% alcohol 60% alcohol Alcohol Denaturation pH Denaturation Solution at pH 6.9, 3.2, 2.9  ...
YCC  Covalently  Attached
How is YCC covalently  tethered on silica? (3-aminopropyl)-trimethoxylsilane + N[ γ -maleimidobutyryloxy]sulfosuccinimide ...
pH Dependent Surface Adsorption Shift of the Soret band maximum: Free YCC ,  Surface bound YCC Conditions:  7mM phosphate ...
Surface Alcohol Denaturing <ul><li>Shifts of the Soret band maximum: </li></ul><ul><li>Surface bound YCC </li></ul><ul><li...
YCC  Electrostatically  attached
Yeast Cytochrome c <ul><ul><li>-disulfide linkage.  </li></ul></ul><ul><ul><li>-Dimerization of YCC </li></ul></ul>dimer M...
Why work with monomer? <ul><li>Dimer has different functionality </li></ul><ul><li>Want to compare with the covalently tet...
Method to retain monomer <ul><li>1.  Treatment with iodoacetate: </li></ul><ul><li>Reaction: </li></ul>+ Iodoacetate YCC
2. Size Exclusion Chromatography: <ul><li>Separate molecules of different sizes  </li></ul><ul><li>Heavy molecules elute r...
3.  Gel Electrophoresis:   Molecular   HCC   YCC   Weight   1μg   1μg   Marker  15μL   15μL  Method to retain monomer Dime...
Procedure to get the data: <ul><li>1.  Make samples </li></ul><ul><li>2.  Kinetic study  </li></ul><ul><li>3.  Surface was...
Encountered Problems at Step 2 <ul><li>Right after Taiwan </li></ul>Proteins did not stick to the  Surface…. Does not look...
Several Factors could play a role <ul><li>[YCC] </li></ul><ul><li>[Buffer] </li></ul><ul><li>[alcohol] </li></ul><ul><li>[...
Kinetic Study <ul><li>Proteins stick to the surface  </li></ul><ul><li>longer </li></ul><ul><li>See the monolayer  </li></...
Adsorption Isotherm pH 4.0, 7mM Succinate Buffer <ul><li>purpose:   know the concentration  </li></ul><ul><li>at which the...
Determination of the [YCC] Abs max = 0.0054 pH 4.00,  7mM phosphate buffer Surface Adsorption of covalently anchored YCC
Determination of [YCC] ~0.0054 Electrostatically adsorbed Surface Adsorption Isotherm Covalently attached studies done at ...
Future Direction <ul><li>Denaturation studies: </li></ul><ul><ul><li>Variation in pH </li></ul></ul><ul><ul><li>Variation ...
Special Thanks To <ul><ul><li>Dr. Geoffrey C. Hoops  </li></ul></ul><ul><ul><li>Dr. Todd A. Hopkins </li></ul></ul><ul><ul...
Surface adsorption Alcohol:  1 propanol pH 4.00 Buffer:  7 mM Succinate [YCC] : 1.00E-6M
Solution Absorption Alcohol:  1 propanol pH 4.00 Buffer:  7 mM Succinate [YCC] : 1.00E-6M
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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

    1. 1. Covalent Versus Electrostatic Attachment of Yeast Cytochrome c to a Fused Silica Surface By Sheetal Mistry Department of Chemistry, Butler University Indianapolis, IN 46208
    2. 2. Yeast cytochrome c <ul><li>In yeast cells </li></ul><ul><li>Similar in function to some cytochromes in eukaryotic cells </li></ul><ul><li>Water soluble peripheral protein </li></ul><ul><li>Resides in intermembrane space of mitochondria </li></ul><ul><li>Positively charged at pH 7 </li></ul><ul><li>~since pI at 10.7 </li></ul><ul><li>Located near negatively charged </li></ul><ul><li>phospholipid bilayer surface </li></ul><ul><li>Heme (red) consists of Iron </li></ul><ul><li>Sovlent exposed Cysteine (yellow) </li></ul>Yeast Cytochrome c
    3. 3. Functionality Plays a major role in the electron transport chain in the inner membrane of mitochondria Shuttles electrons between complexes III & IV
    4. 4. Methods: <ul><li>1. Solution Absorption </li></ul><ul><li>2. ATR (Attenuated Total Internal </li></ul><ul><li> Reflection) </li></ul>
    5. 5. Solution Absorption 10 µ M [YCC], 7mM Succinate Buffer, pH 4.00 Soret Band <ul><li>Soret peak at 408 nm </li></ul><ul><li>Used to measure unfolding </li></ul><ul><ul><li>Soret band shifts left </li></ul></ul>
    6. 6. Conformation <ul><li>Three dimensional structure </li></ul><ul><li>Primary, Secondary, and Tertiary </li></ul><ul><li>Helices maximize hydrogen bonds </li></ul><ul><li>Conformation is considered “native” in solution under physiological conditions (pH≈7) </li></ul>Cox, M., Nelson, D. Principles of Biochemistry 2000:194 <ul><li>Process of Denaturation : </li></ul><ul><li>Temperature change </li></ul><ul><li>pH change </li></ul><ul><li>Chemical change </li></ul><ul><ul><li>- Urea </li></ul></ul><ul><ul><li>- Alcohol </li></ul></ul>Tertiary Primary
    7. 7. ATR spectroscopy Cheng, Y.-Y.; Lin, S. H.; Chang, H.-C.; Su, M.-C.: Probing Adsorption, Orientation and Conformational Changes of Cytochrome c on Fused Silica Surfaces with the Soret Band. J. Phys. Chem. A pp. 10687, 107 (49) 2003 <ul><li>ATR (Attenuated Total internal </li></ul><ul><li>Reflection) </li></ul><ul><li>Only detects proteins on surface </li></ul>Detector Light Source <ul><ul><li>Quartz prism </li></ul></ul><ul><ul><li>Hydrophilic surface </li></ul></ul><ul><ul><li>Negatively charged (similar </li></ul></ul><ul><ul><li>to phospholipid bilayer) </li></ul></ul>Prism θ Glass plate O-ring Sample solution To detector
    8. 8. Experiments: <ul><ul><ul><ul><li>YCC free in solution </li></ul></ul></ul></ul><ul><ul><ul><ul><li>YCC covalently attached </li></ul></ul></ul></ul><ul><ul><ul><ul><li>3. YCC electrostatically attached </li></ul></ul></ul></ul>
    9. 9. YCC Free in Solution
    10. 10. Solution Absorption Wavelength 20% alcohol 60% 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
    11. 11. YCC Covalently Attached
    12. 12. How is YCC covalently tethered on silica? (3-aminopropyl)-trimethoxylsilane + N[ γ -maleimidobutyryloxy]sulfosuccinimide ester + SiO 2 SiO 2 SiO 2 YCC sGMBS
    13. 13. 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 solution denature as the pH is lowered
    14. 14. Surface Alcohol Denaturing <ul><li>Shifts of the Soret band maximum: </li></ul><ul><li>Surface bound YCC </li></ul><ul><li>Free YCC </li></ul>Methanol 1-propanol Conditions: 7mM phosphate buffer, pH 4.00 , , <ul><li>YCC in solution denatures with </li></ul><ul><li>respect to change in [alcohol] </li></ul><ul><li>YCC on surface denatures </li></ul><ul><li>partially </li></ul>
    15. 15. YCC Electrostatically attached
    16. 16. Yeast Cytochrome c <ul><ul><li>-disulfide linkage. </li></ul></ul><ul><ul><li>-Dimerization of YCC </li></ul></ul>dimer Monomer Significance of Sulfur
    17. 17. Why work with monomer? <ul><li>Dimer has different functionality </li></ul><ul><li>Want to compare with the covalently tethered proteins </li></ul><ul><ul><li>No chances for these proteins to dimerize as the sulfur is covalently bound to the surface </li></ul></ul>
    18. 18. Method to retain monomer <ul><li>1. Treatment with iodoacetate: </li></ul><ul><li>Reaction: </li></ul>+ Iodoacetate YCC
    19. 19. 2. Size Exclusion Chromatography: <ul><li>Separate molecules of different sizes </li></ul><ul><li>Heavy molecules elute rapidly </li></ul><ul><li>Sephadex G-50 </li></ul><ul><li>Dimer (2 x 12,588 g/mol) </li></ul><ul><li>Monomer (12,588 g/mol) </li></ul>Method to retain monomer
    20. 20. 3. Gel Electrophoresis: Molecular HCC YCC Weight 1μg 1μg Marker 15μL 15μL Method to retain monomer Dimer ~24,000g/mol Monomer~12,000g/mol
    21. 21. Procedure to get the data: <ul><li>1. Make samples </li></ul><ul><li>2. Kinetic study </li></ul><ul><li>3. Surface washing </li></ul><ul><li>Scans </li></ul><ul><li>5. Data analysis </li></ul>
    22. 22. Encountered Problems at Step 2 <ul><li>Right after Taiwan </li></ul>Proteins did not stick to the Surface…. Does not look like a Kinetic scan Intensity proportional to number of proteins on surface
    23. 23. Several Factors could play a role <ul><li>[YCC] </li></ul><ul><li>[Buffer] </li></ul><ul><li>[alcohol] </li></ul><ul><li>[NaCl] </li></ul><ul><li>pH </li></ul>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.
    24. 24. Kinetic Study <ul><li>Proteins stick to the surface </li></ul><ul><li>longer </li></ul><ul><li>See the monolayer </li></ul><ul><li>Take the data when </li></ul><ul><li>see the monolayer </li></ul>For every sample: 1. Kinetic scan 2. Record the time (monolayer) 3. Take data
    25. 25. Adsorption Isotherm pH 4.0, 7mM Succinate Buffer <ul><li>purpose: know the concentration </li></ul><ul><li>at which the covalently anchored </li></ul><ul><li>studies were done </li></ul><ul><li>Surface saturation around </li></ul><ul><li>10 µM YCC concentration </li></ul>K ad YCC = 1.3 E 6 K ad HCC = 1.3 E 6
    26. 26. Determination of the [YCC] Abs max = 0.0054 pH 4.00, 7mM phosphate buffer Surface Adsorption of covalently anchored YCC
    27. 27. Determination of [YCC] ~0.0054 Electrostatically adsorbed Surface Adsorption Isotherm Covalently attached studies done at ~1.00 uM [YCC]
    28. 28. Future Direction <ul><li>Denaturation studies: </li></ul><ul><ul><li>Variation in pH </li></ul></ul><ul><ul><li>Variation in alcohol </li></ul></ul><ul><ul><ul><li>Methanol </li></ul></ul></ul><ul><ul><ul><li>1-propanol </li></ul></ul></ul><ul><li>Methods: </li></ul><ul><ul><li>Solution Absorption </li></ul></ul><ul><ul><li>ATR Spectroscopy </li></ul></ul>For Electrostatic Attachment :
    29. 29. Special Thanks To <ul><ul><li>Dr. Geoffrey C. Hoops </li></ul></ul><ul><ul><li>Dr. Todd A. Hopkins </li></ul></ul><ul><ul><li>Dr. Meng-Chih Su </li></ul></ul><ul><ul><li>Victoria Fahrenbach </li></ul></ul><ul><ul><li>Tara Benz </li></ul></ul><ul><ul><li>Greg Campanello </li></ul></ul><ul><ul><li>Carrie Ann Hedge </li></ul></ul><ul><ul><li>Ken Clevenger </li></ul></ul><ul><ul><li>Butler Summer Institute </li></ul></ul><ul><ul><li>Holcomb Undergraduate Grants </li></ul></ul><ul><ul><li>Lilly Endowment </li></ul></ul><ul><ul><li>Butler University </li></ul></ul><ul><ul><li>Department of Chemistry </li></ul></ul><ul><ul><li>Collaborators: </li></ul></ul><ul><ul><li>Y.-Y. Cheng, S. H. Lin, and H.-C. Chang </li></ul></ul><ul><ul><li>Institute of Atomic and Molecular Sciences, </li></ul></ul><ul><ul><li>Academia Sinica </li></ul></ul>
    30. 30. Surface adsorption Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M
    31. 31. Solution Absorption Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M

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