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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
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Cytochrome  c ,[object Object],[object Object],[object Object],[object Object],[object Object]
Cytochrome  c Heme
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
General Function Voet, Donald; Voet, Judith; Pratt, Charlotte.  Fundamentals of Biochemistry Upgrade Ed.  2001:501
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Solution Spectroscopy Soret Band ,[object Object],[object Object],[object Object],1   µ M [YCC], 7mM Succinate Buffer, pH 4.00
Heme ,[object Object],[object Object],[object Object],[object Object],[object Object],Graphic derived from PDB file 1AKK Banci et al.,  Biochemistry , v 36, pp 98679877,  1997 .
Conformation ,[object Object],[object Object],[object Object],[object Object],Cox, M., Nelson, D.  Principles of Biochemistry   2000:194 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Tertiary Primary
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
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 ,[object Object],[object Object],[object Object],Detector Light Source ,[object Object],[object Object],[object Object],[object Object],[object Object],Prism θ Glass plate O-ring Sample solution To detector
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 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Amino acid sequence: ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],102
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Studies on Horse Cytochrome  c ,[object Object],[object Object],[object Object],[object Object]
Studies on Horse Cytochrome  c ,[object Object],[object Object],[object Object],[object Object]
Alcohol Study in Solution ,[object Object],[object Object],[object Object],20µM [HCC],  0% n-propanol  (native state) ~409 nm
Alcohol Study in Solution ,[object Object],[object Object],[object Object],20µM [HCC],  60% n-propanol  (denatured state) ~400nm
Alcohol Study in Solution ,[object Object],[object Object],[object Object],20µM [HCC], 0% n-propanol 20µM [HCC], 60% n-propanol
Studies on Horse Cytochrome  c ,[object Object],[object Object],[object Object],[object Object]
Alcohol Study on Surface ,[object Object],[object Object],[object Object],20µM [HCC], 0%  n -propanol ~409 nm
  Alcohol Study on Surface ,[object Object],[object Object],[object Object],20µM [cyt  c ], 60%  n -propanol ~405 nm
[object Object],[object Object],[object Object],[object Object]
Adsorption Isotherm ,[object Object],[object Object],[object Object],[object Object],[object Object]
Alcohol Study on Surface ,[object Object],[object Object],[object Object],0.1µM  [cyt c], 60%  n -propanol ~ 400 nm *Note the spectrum for 0.1µM is enlarged
Alcohol Study on Surface *Note the spectrum for 0.1µM is enlarged ,[object Object],[object Object],[object Object],20µM [cyt c], 60%  n -propanol 0.1µM [cyt c], 60%  n -propanol
Studies on Horse Cytochrome  c ,[object Object],[object Object],[object Object],[object Object]
Surface vs. Solution: [Succinate Buffer] = 10 mM  pH = 4.7 [NaCl] = 150 mM *Note the spectrum for 0.1 µM is enlarged Surface Adsorption
Surface vs. Solution: [Succinate Buffer] = 10 mM  pH = 4.7 [NaCl] = 150 mM *Note the spectrum for 0.1 µM is enlarged ,[object Object],[object Object],[object Object],[object Object],[object Object]
Surface vs. Solution: [Succinate Buffer] = 10 mM    60% n-propanol [NaCl] = 150 mM pH = 4.7
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Experiments: ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
YCC  Electrostatically  attached
Yeast Cytochrome c ,[object Object],[object Object],dimer Monomer Significance of Sulfur
Method to retain monomer ,[object Object],[object Object],+ 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
Size Exclusion Chromatography: ,[object Object],[object Object],[object Object],[object Object],Separation of Monomer and Dimer
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 mths)  1  μ g YCC uncapped  (4 mths)  1  μ g YCC capped /purified  1  μ g
Procedure to get the data: ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Encountered Problems at Step 2 Intensity proportional to  Number of proteins on  surface @ 409 nm
Several Factors could play a role ,[object Object],[object Object],[object Object],[object Object],[object Object],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
Kinetic Study ,[object Object],[object Object],[object Object],[object Object],For every sample: 1.  Kinetic run 2.  Take scan
Adsorption Isotherm pH 4.0, 7mM Succinate Buffer ,[object Object],[object Object],[object Object],[object Object],[object Object],K ad  YCC = 1.3 E 6 K ad  HCC = 1.3 E 7
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 Adsorption Isotherm
Denaturation Study ,[object Object],[object Object],[object Object]
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: ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Special Thanks To ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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Study of the Folding and Unfolding of Proteins Adsorbed to a Fused Silica Surface

  • 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
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  • 8. General Function Voet, Donald; Voet, Judith; Pratt, Charlotte. Fundamentals of Biochemistry Upgrade Ed. 2001:501
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  • 16. Example Surface Spectra 1 µ M [YCC], 7mM Succinate Buffer, pH 4.00
  • 17. Solution and Surface 1 µ M [YCC], 7mM Succinate Buffer, pH 4.00 Solution Surface
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  • 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.
  • 37. Surface vs. Solution: [Succinate Buffer] = 10 mM 60% n-propanol [NaCl] = 150 mM pH = 4.7
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  • 44. Is it really a monomer?...
  • 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.
  • 47. Encountered Problems at Step 2 Intensity proportional to Number of proteins on surface @ 409 nm
  • 48.
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  • 51. Determination of the [YCC] Abs max = 0.0054 pH 4.00, 7mM phosphate buffer Surface Adsorption of covalently anchored YCC
  • 52. Determination of [YCC] ~0.0054 Covalently attached studies done at ~1.00 uM [YCC] Electrostatically adsorbed Surface Adsorption Isotherm
  • 53.
  • 54. Alcohol Study on Surface Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M = 1.00 uM
  • 55. Alcohol Study in Solution Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M
  • 56. Surface and Solution Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M
  • 57.
  • 58.