Study of the Folding and Unfolding of Proteins Adsorbed to a Fused Silica Surface By  Sheetal Mistry Department of Chemist...
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><li>II.  Types of Experiments </li></ul></ul>...
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><ul><ul><ul><li>Structure </li></ul></ul></ul...
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><ul><ul><ul><li>Structure </li></ul></ul></ul...
Cytochrome  c <ul><li>Water soluble peripheral protein </li></ul><ul><li>Resides in intermembrane space of mitochondria </...
Cytochrome  c Heme
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><ul><ul><ul><li>Structure </li></ul></ul></ul...
General Function Voet, Donald; Voet, Judith; Pratt, Charlotte.  Fundamentals of Biochemistry Upgrade Ed.  2001:501
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><li>II.  Types of Experiments </li></ul></ul>...
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><li>II.  Types of Experiments </li></ul></ul>...
Solution Spectroscopy Soret Band <ul><li>Soret peak at 408 nm </li></ul><ul><li>Used to measure unfolding </li></ul><ul><u...
Heme <ul><li>Prosthetic Group </li></ul><ul><li>Iron complex in porphyrin ring </li></ul><ul><li>Liganding interactions wi...
Conformation <ul><li>Three dimensional structure </li></ul><ul><li>Primary, Secondary, and Tertiary </li></ul><ul><li>Heli...
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><li>II.  Types of Experiments </li></ul></ul>...
ATR spectroscopy Cheng, Y.-Y.; Lin, S. H.; Chang, H.-C.; Su, M.-C.: Probing Adsorption, Orientation and Conformational Cha...
Example Surface Spectra 1   µ M [YCC], 7mM Succinate Buffer, pH 4.00
Solution and Surface 1   µ M [YCC], 7mM Succinate Buffer, pH 4.00   Solution Surface
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><li>II.  Types of Experiments </li></ul></ul>...
Amino acid sequence: <ul><li>HCC:   GDVEKGKKIFVQK C AQ C HTVEKG </li></ul><ul><li>YCC:   TEFKAGSAKKGATLFKTR C LQ C HTVEKG ...
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><li>II.  Types of Experiments </li></ul></ul>...
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><li>II.  Types of Experiments </li></ul></ul>...
Studies on Horse Cytochrome  c <ul><ul><li>Surface Coverage Study </li></ul></ul><ul><ul><ul><li>Denaturation with [alcoho...
Studies on Horse Cytochrome  c <ul><ul><li>Surface Coverage Study </li></ul></ul><ul><ul><ul><li>Denaturation with [alcoho...
Alcohol Study in Solution <ul><li>[Succinate Buffer] = 10 mM  </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7...
Alcohol Study in Solution <ul><li>[Succinate Buffer] = 10 mM  </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7...
Alcohol Study in Solution <ul><li>[Succinate Buffer] = 10 mM  </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7...
Studies on Horse Cytochrome  c <ul><ul><li>Surface Coverage Study </li></ul></ul><ul><ul><ul><li>Denaturation with [alcoho...
Alcohol Study on Surface <ul><li>[Succinate Buffer] = 10 mM  </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 ...
  Alcohol Study on Surface <ul><li>[Succinate Buffer] = 10 mM  </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4....
<ul><li>Two types of Interactions: </li></ul><ul><ul><li>1.  Surface and protein </li></ul></ul><ul><ul><li>2.  Protein an...
Adsorption Isotherm <ul><li>The adsorption isotherm shows  </li></ul><ul><li>that the surface coverage of  </li></ul><ul><...
Alcohol Study on Surface <ul><li>[Succinate Buffer] = 10 mM  </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 ...
Alcohol Study on Surface *Note the spectrum for 0.1µM is enlarged <ul><li>[Succinate Buffer] = 10 mM  </li></ul><ul><li>[N...
Studies on Horse Cytochrome  c <ul><ul><li>Surface Coverage Study </li></ul></ul><ul><ul><ul><li>Denaturation with [alcoho...
Surface vs. Solution: [Succinate Buffer] = 10 mM  pH = 4.7 [NaCl] = 150 mM *Note the spectrum for 0.1 µM is enlarged Surfa...
Surface vs. Solution: [Succinate Buffer] = 10 mM  pH = 4.7 [NaCl] = 150 mM *Note the spectrum for 0.1 µM is enlarged <ul><...
Surface vs. Solution: [Succinate Buffer] = 10 mM    60% n-propanol [NaCl] = 150 mM pH = 4.7
Outline <ul><ul><li>I.  Introduction to Cytochrome  c </li></ul></ul><ul><ul><li>II.  Types of Experiments </li></ul></ul>...
Experiments: <ul><ul><ul><ul><li>YCC  free  in solution </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Proteins denature...
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...
Method to retain monomer <ul><li>1.  Treatment with iodoacetate: </li></ul><ul><li>Reaction: </li></ul>+ Iodoacetate YCC +...
Size Exclusion Chromatography: <ul><li>Separate molecules of different sizes  </li></ul><ul><li>Heavy molecules elute rapi...
Is it really a monomer?...
Gel Electrophoresis HCC   1μg     Dimer ~24,000g/mol Monomer~12,000g/mol YCC freshly dissolved  10  μ g YCC uncapped  (6 m...
Procedure to get the data: <ul><li>1.  Make samples </li></ul><ul><ul><li>Buffer </li></ul></ul><ul><ul><li>Water </li></u...
Encountered Problems at Step 2 Intensity proportional to  Number of proteins on  surface @ 409 nm
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>Take the data when  </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 Covalently attached studies done at ~1.00 uM [YCC] Electrostatically adsorbed Surface Adsor...
Denaturation Study <ul><li>Variation in [n-propanol]  alcohol: </li></ul><ul><li>-  On Surface </li></ul><ul><li>-  In Sol...
Alcohol Study on Surface Alcohol:  1 propanol pH 4.00 Buffer:  7 mM Succinate [YCC] : 1.00E-6M = 1.00 uM
Alcohol Study in Solution Alcohol:  1 propanol pH 4.00 Buffer:  7 mM Succinate [YCC] : 1.00E-6M
Surface and Solution Alcohol:  1 propanol pH 4.00 Buffer:  7 mM Succinate [YCC] : 1.00E-6M
Conclusions: <ul><li>Horse Cytochrome c: </li></ul><ul><li>Under denaturing conditions (60% alcohol): </li></ul><ul><li>At...
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...
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Study of the folding and unfolding of proteins adsorbed to a fused silica surface

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  • Study of the folding and unfolding of proteins adsorbed to a fused silica surface

    1. 1. Study of the Folding and Unfolding of Proteins Adsorbed to a Fused Silica Surface By Sheetal Mistry Department of Chemistry, Butler University Indianapolis, IN 46208
    2. 2. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    3. 3. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><ul><ul><ul><li>Structure </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Function </li></ul></ul></ul></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    4. 4. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><ul><ul><ul><li>Structure </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Function </li></ul></ul></ul></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    5. 5. Cytochrome c <ul><li>Water soluble peripheral protein </li></ul><ul><li>Resides in intermembrane space of mitochondria </li></ul><ul><li>Located near negatively charged phospholipid bilayer surface </li></ul><ul><li>Positively charged at pH 7.00 </li></ul><ul><ul><li>pI = 10.7 </li></ul></ul>
    6. 6. Cytochrome c Heme
    7. 7. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><ul><ul><ul><li>Structure </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Function </li></ul></ul></ul></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    8. 8. General Function Voet, Donald; Voet, Judith; Pratt, Charlotte. Fundamentals of Biochemistry Upgrade Ed. 2001:501
    9. 9. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    10. 10. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    11. 11. Solution Spectroscopy 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>1 µ M [YCC], 7mM Succinate Buffer, pH 4.00
    12. 12. Heme <ul><li>Prosthetic Group </li></ul><ul><li>Iron complex in porphyrin ring </li></ul><ul><li>Liganding interactions with </li></ul><ul><ul><li>Methionine 80 </li></ul></ul><ul><ul><li>Histidine 18 </li></ul></ul>Graphic derived from PDB file 1AKK Banci et al., Biochemistry , v 36, pp 98679877, 1997 .
    13. 13. 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
    14. 14. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    15. 15. 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><ul><ul><li>above pH ~ 3.00 </li></ul></ul>Prism θ Glass plate O-ring Sample solution To detector
    16. 16. Example Surface Spectra 1 µ M [YCC], 7mM Succinate Buffer, pH 4.00
    17. 17. Solution and Surface 1 µ M [YCC], 7mM Succinate Buffer, pH 4.00 Solution Surface
    18. 18. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><ul><ul><ul><li>Horse Heart Cytochrome c (HCC) </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Yeast Cytochrome c (YCC) </li></ul></ul></ul></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    19. 19. Amino acid sequence: <ul><li>HCC: GDVEKGKKIFVQK C AQ C HTVEKG </li></ul><ul><li>YCC: TEFKAGSAKKGATLFKTR C LQ C HTVEKG </li></ul><ul><li> </li></ul><ul><li> GKHKTGPNLHGLFGRKTGQAPGFTYTDAN </li></ul><ul><li> GPHKVGPNLHGIFGRHSGQAQGYSYTDAN </li></ul><ul><li> KNKGITWEETLMEYLENPKKYIPGTKMI </li></ul><ul><li> IKKNVLWDENNMSEYLTNPXKYIPGTKM </li></ul><ul><li>FAGIKKKTEREIDLIAYLKKATNE </li></ul><ul><li>AFGGLKKEKDRNDLITYLKKA C E </li></ul>102
    20. 20. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    21. 21. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><ul><ul><ul><li>~Surface Coverage studies </li></ul></ul></ul></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    22. 22. Studies on Horse Cytochrome c <ul><ul><li>Surface Coverage Study </li></ul></ul><ul><ul><ul><li>Denaturation with [alcohol] in solution </li></ul></ul></ul><ul><ul><ul><li>2. Denaturation with [alcohol] on surface </li></ul></ul></ul><ul><ul><ul><li>3. Comparison between the denatured proteins in solution and on the surface </li></ul></ul></ul>
    23. 23. Studies on Horse Cytochrome c <ul><ul><li>Surface Coverage Study </li></ul></ul><ul><ul><ul><li>Denaturation with [alcohol] in solution </li></ul></ul></ul><ul><ul><ul><li>2. Denaturation with [alcohol] on surface </li></ul></ul></ul><ul><ul><ul><li>3. Comparison between the denatured proteins in solution and on the surface </li></ul></ul></ul>
    24. 24. Alcohol Study in Solution <ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>20µM [HCC], 0% n-propanol (native state) ~409 nm
    25. 25. Alcohol Study in Solution <ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>20µM [HCC], 60% n-propanol (denatured state) ~400nm
    26. 26. Alcohol Study in Solution <ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>20µM [HCC], 0% n-propanol 20µM [HCC], 60% n-propanol
    27. 27. Studies on Horse Cytochrome c <ul><ul><li>Surface Coverage Study </li></ul></ul><ul><ul><ul><li>Denaturation with [alcohol] in solution </li></ul></ul></ul><ul><ul><ul><li>2. Denaturation with [alcohol] on surface </li></ul></ul></ul><ul><ul><ul><li>3. Comparison between the denatured proteins in solution and on the surface </li></ul></ul></ul>
    28. 28. Alcohol Study on Surface <ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>20µM [HCC], 0% n -propanol ~409 nm
    29. 29. Alcohol Study on Surface <ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>20µM [cyt c ], 60% n -propanol ~405 nm
    30. 30. <ul><li>Two types of Interactions: </li></ul><ul><ul><li>1. Surface and protein </li></ul></ul><ul><ul><li>2. Protein and protein </li></ul></ul><ul><li> </li></ul>
    31. 31. Adsorption Isotherm <ul><li>The adsorption isotherm shows </li></ul><ul><li>that the surface coverage of </li></ul><ul><li>cyt c reaches a saturation level </li></ul><ul><li>at 15-20 µM bulk concentration </li></ul><ul><li>Choose 0.1 uM [HCC] </li></ul>
    32. 32. Alcohol Study on Surface <ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>0.1µM [cyt c], 60% n -propanol ~ 400 nm *Note the spectrum for 0.1µM is enlarged
    33. 33. Alcohol Study on Surface *Note the spectrum for 0.1µM is enlarged <ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>20µM [cyt c], 60% n -propanol 0.1µM [cyt c], 60% n -propanol
    34. 34. Studies on Horse Cytochrome c <ul><ul><li>Surface Coverage Study </li></ul></ul><ul><ul><ul><li>Denaturation with [alcohol] in solution </li></ul></ul></ul><ul><ul><ul><li>2. Denaturation with [alcohol] on surface </li></ul></ul></ul><ul><ul><ul><li>3. Comparison between the denatured proteins in solution and on the surface </li></ul></ul></ul>
    35. 35. Surface vs. Solution: [Succinate Buffer] = 10 mM pH = 4.7 [NaCl] = 150 mM *Note the spectrum for 0.1 µM is enlarged Surface Adsorption
    36. 36. Surface vs. Solution: [Succinate Buffer] = 10 mM pH = 4.7 [NaCl] = 150 mM *Note the spectrum for 0.1 µM is enlarged <ul><li>At lower bulk [HCC], surface </li></ul><ul><li>adsorbed proteins are more </li></ul><ul><li>denatured than at higher [HCC] </li></ul><ul><li>Denatured proteins in the solution </li></ul><ul><li>are renatured at the surface </li></ul>
    37. 37. Surface vs. Solution: [Succinate Buffer] = 10 mM 60% n-propanol [NaCl] = 150 mM pH = 4.7
    38. 38. Outline <ul><ul><li>I. Introduction to Cytochrome c </li></ul></ul><ul><ul><li>II. Types of Experiments </li></ul></ul><ul><ul><li>a. Solution Spectroscopy </li></ul></ul><ul><ul><li>b. Surface Spectroscopy </li></ul></ul><ul><ul><li>III. Two types of cytochrome c </li></ul></ul><ul><ul><li>IV. Experiments </li></ul></ul><ul><ul><li>a. Horse cytochrome c </li></ul></ul><ul><ul><li>b. Yeast cytochrome c </li></ul></ul><ul><ul><li>V. Conclusions </li></ul></ul>
    39. 39. Experiments: <ul><ul><ul><ul><li>YCC free in solution </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Proteins denature at higher [alcohol] and at lower pH </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><li>YCC covalently attached </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>YCC on surface takes longer to unfold than the solution </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>YCC on surface denatures partially </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><li>YCC electrostatically attached </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Alcohol Study: </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>- Solution </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>- Surface </li></ul></ul></ul></ul></ul>
    40. 40. YCC Electrostatically attached
    41. 41. 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
    42. 42. Method to retain monomer <ul><li>1. Treatment with iodoacetate: </li></ul><ul><li>Reaction: </li></ul>+ Iodoacetate YCC + Dimer Crestfield, A.M.,Moore, S., & Stein, W. H (1963) J. Biol. Chem. 238, 622-627 Christopher B., Strottmann M. J., Stellwagen E.; Biochemistry ; 1985 ; 24 (14); 3459-3464 Conditions: Tris-HCl pH 8.6 0.1 M iodoacetate
    43. 43. Size Exclusion Chromatography: <ul><li>Separate molecules of different sizes </li></ul><ul><li>Heavy molecules elute rapidly </li></ul><ul><li>Dimer (2 x 12,588 g/mol) </li></ul><ul><li>Monomer (12,588 g/mol) </li></ul>Separation of Monomer and Dimer
    44. 44. Is it really a monomer?...
    45. 45. Gel Electrophoresis HCC 1μg Dimer ~24,000g/mol Monomer~12,000g/mol YCC freshly dissolved 10 μ g YCC uncapped (6 mths) 1 μ g YCC uncapped (4 mths) 1 μ g YCC capped /purified 1 μ g
    46. 46. Procedure to get the data: <ul><li>1. Make samples </li></ul><ul><ul><li>Buffer </li></ul></ul><ul><ul><li>Water </li></ul></ul><ul><ul><li>Proteins </li></ul></ul><ul><ul><li>Denaturants (alcohol) </li></ul></ul><ul><ul><li>pH </li></ul></ul><ul><li>Surface washing </li></ul><ul><li>Kinetic study </li></ul><ul><li>Scans </li></ul><ul><li>5. Data analysis </li></ul>
    47. 47. Encountered Problems at Step 2 Intensity proportional to Number of proteins on surface @ 409 nm
    48. 48. 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. Solution: Tried using diluted soap for rinsing the surface
    49. 49. Kinetic Study <ul><li>Proteins stick to the surface </li></ul><ul><li>longer </li></ul><ul><li>Take the data when </li></ul><ul><li>see the monolayer </li></ul>For every sample: 1. Kinetic run 2. Take scan
    50. 50. 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 7
    51. 51. Determination of the [YCC] Abs max = 0.0054 pH 4.00, 7mM phosphate buffer Surface Adsorption of covalently anchored YCC
    52. 52. Determination of [YCC] ~0.0054 Covalently attached studies done at ~1.00 uM [YCC] Electrostatically adsorbed Surface Adsorption Isotherm
    53. 53. Denaturation Study <ul><li>Variation in [n-propanol] alcohol: </li></ul><ul><li>- On Surface </li></ul><ul><li>- In Solution </li></ul>
    54. 54. Alcohol Study on Surface Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M = 1.00 uM
    55. 55. Alcohol Study in Solution Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M
    56. 56. Surface and Solution Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M
    57. 57. Conclusions: <ul><li>Horse Cytochrome c: </li></ul><ul><li>Under denaturing conditions (60% alcohol): </li></ul><ul><li>At low [cyt c] the proteins adsorb to the surface with little change to their state of denaturation </li></ul><ul><li>As the [cyt c] increases the proteins are renaturing on the surface due to increasing protein - protein interactions </li></ul><ul><li>When the surface is saturated (>15-20 µM) the protein-protein interactions remain constant and protein renaturation reaches a limit </li></ul><ul><li>Yeast Cytochrome c: </li></ul><ul><li>Stabilization in monomeric form by treating with iodoacetate </li></ul><ul><li>Shows distinctly different unfolding behavior than horse </li></ul><ul><ul><li>Spectroscopic phenomenon </li></ul></ul><ul><ul><li>Reverse soret band behavior </li></ul></ul>
    58. 58. 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 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>

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