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

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]

Cytochrome c ,[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

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

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

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],[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

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

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

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

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]

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

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