Surface Coverage Dependent Renaturation of Oxidized Cytochrome c Adsorbed to a Fused Silica Surface S. A. Mistry, S. A. Ho...
Cytochrome c <ul><li>In most eukaryotic cells </li></ul><ul><li>Water soluble peripheral protein </li></ul><ul><li>Resides...
Cyt c Function Voet, Donald; Voet, Judith; Pratt, Charlotte.  Fundamentals of Biochemistry Upgrade Ed.  2001:501   Plays a...
Solution Absorption 20   µ M [Cyt c] in 10mM pH 4.7 Succinate Buffer Cox, M., Nelson, D.  Principles of Biochemistry   200...
Solution Alcohol Denatured <ul><li>At 20 μM, native cyt  c  has soret  </li></ul><ul><li>location at 408nm  </li></ul><ul>...
ATR spectroscopy of cyt  c  on fused silica surface Cheng, Y.-Y.; Lin, S. H.; Chang, H.-C.; Su, M.-C.: Probing Adsorption,...
Alcohol denatured Isotherm <ul><li>The adsorption isotherm shows  </li></ul><ul><li>that the surface coverage of  </li></u...
Surface Spectra: <ul><li>Denatured 20 μM cyt  c  has soret location at 405 nm  </li></ul><ul><li>0.1 μM cyt  c  at 401 nm ...
Surface adsorbed vs. Solution: [Succinate Buffer] = 10 mM  pH = 4.7 [NaCl] = 150 mM *Note the spectrum for 0.1 µM is enlar...
Surface adsorbed vs. Solution: [Succinate Buffer] = 10 mM   60% n-propanol [NaCl] = 150 mM  pH = 4.7 <ul><li>Concentration...
Protein-Protein Interactions: <ul><ul><ul><li>At low [cyt c] the proteins adsorb to the surface with little change to thei...
Conclusion <ul><li>Protein-protein interactions are an important  </li></ul><ul><li>factor in the conformation of cyt  c  ...
Special Thanks To: <ul><ul><li>Dr. Todd A. Hopkins </li></ul></ul><ul><ul><li>Dr. Geoffrey C. Hoops </li></ul></ul><ul><ul...
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Surface Coverage Dependent Renaturation of Oxidized Cytochrome c Adsorbed to a Fused Silica Surface

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  • Surface Coverage Dependent Renaturation of Oxidized Cytochrome c Adsorbed to a Fused Silica Surface

    1. 1. Surface Coverage Dependent Renaturation of Oxidized Cytochrome c Adsorbed to a Fused Silica Surface S. A. Mistry, S. A. Hocker, V. S. Fahrenbach, T. L. Benz, G. C. Campanello G. C. Hoops, T. A. Hopkins, M.-C. Su Department of Chemistry, Butler University, Indianapolis, IN 46208
    2. 2. Cytochrome c <ul><li>In most 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 consists of Iron </li></ul><ul><li>Two states: oxidized Fe(III) and </li></ul><ul><li>reduced Fe(II) </li></ul>Heme Tryptophan Tyrosine
    3. 3. Cyt c Function Voet, Donald; Voet, Judith; Pratt, Charlotte. Fundamentals of Biochemistry Upgrade Ed. 2001:501 Plays a major role in the electron transport chain in the inner membrane of mitochondria Shuttles electrons between complexes III & IV
    4. 4. Solution Absorption 20 µ M [Cyt c] in 10mM pH 4.7 Succinate Buffer Cox, M., Nelson, D. Principles of Biochemistry 2000:194 Soret Band <ul><li>Soret peak at 408 nm </li></ul><ul><li>Used to measure unfolding </li></ul><ul><li>Conformation is considered </li></ul><ul><li>“ native” in solution under </li></ul><ul><li>physiological conditions (pH≈7) </li></ul>
    5. 5. Solution Alcohol Denatured <ul><li>At 20 μM, native cyt c has soret </li></ul><ul><li>location at 408nm </li></ul><ul><li>n -propanol denatured cyt c at 400nm </li></ul><ul><li>Protein degree of denaturation is not dependent on [cyt c ] </li></ul><ul><li>20µM [cyt c], 0% n-propanol </li></ul><ul><li>20µM [cyt c], 60% n-propanol </li></ul><ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>
    6. 6. ATR spectroscopy of cyt c on fused silica surface 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
    7. 7. Alcohol denatured 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>
    8. 8. Surface Spectra: <ul><li>Denatured 20 μM cyt c has soret location at 405 nm </li></ul><ul><li>0.1 μM cyt c at 401 nm </li></ul><ul><li>Degree of denaturation of proteins is dependent on [cyt c] </li></ul>*Note the spectrum for 0.1µM is enlarged <ul><li>20µM [cyt c], 60% n -propanol </li></ul><ul><li>0.1µM [cyt c], 60% n -propanol </li></ul><ul><li>[Succinate Buffer] = 10 mM </li></ul><ul><li>[NaCl] = 150 mM </li></ul><ul><li>pH = 4.7 </li></ul>
    9. 9. Surface adsorbed 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 [cyt c ], surface </li></ul><ul><li>adsorbed proteins are more </li></ul><ul><li>denatured than at higher [cyt c ] </li></ul><ul><li>Denatured proteins in the solution </li></ul><ul><li>are renatured at the surface </li></ul>
    10. 10. Surface adsorbed vs. Solution: [Succinate Buffer] = 10 mM 60% n-propanol [NaCl] = 150 mM pH = 4.7 <ul><li>Concentration dependence: </li></ul><ul><li>In solution cyt c proteins are “completely” </li></ul><ul><li>denatured </li></ul><ul><li>At all [cyt c ] the surface adsorbed proteins </li></ul><ul><li>are less denatured than in solution </li></ul><ul><li>The degree of denaturation of surface </li></ul><ul><li>adsorbed proteins depends on </li></ul><ul><li>concentration </li></ul>
    11. 11. Protein-Protein Interactions: <ul><ul><ul><li>At low [cyt c] the proteins adsorb to the surface with little change to their state of denaturation </li></ul></ul></ul><ul><ul><ul><li>As the [cyt c] increases the proteins are renaturing on the surface due to increasing protein - protein interactions </li></ul></ul></ul><ul><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></ul>
    12. 12. Conclusion <ul><li>Protein-protein interactions are an important </li></ul><ul><li>factor in the conformation of cyt c </li></ul><ul><li>adsorbed to fused silica surface </li></ul><ul><li>Future Direction </li></ul><ul><li>Increase signals of low concentration spectra via multiple reflection ATR absorbance spectroscopy using a multi pass cell </li></ul><ul><li>Surface Fluorescence to indicate conformational changes </li></ul>
    13. 13. Special Thanks To: <ul><ul><li>Dr. Todd A. Hopkins </li></ul></ul><ul><ul><li>Dr. Geoffrey C. Hoops </li></ul></ul><ul><ul><li>Dr. Meng-Chih Su </li></ul></ul><ul><ul><li>Scott A. Hocker </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>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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