1. The document discusses the covalent and electrostatic attachment of yeast cytochrome c to a fused silica surface and how its structure and function are affected. 2. Experiments examined how the protein's absorption spectra changed when covalently or electrostatically attached to the surface versus in solution under varying conditions like pH and alcohol concentration. 3. The goal was to better understand how the attachment method and external factors influence the protein's conformation and ability to perform its electron transport function.

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

3. Functionality Plays a major role in the electron transport chain in the inner membrane of mitochondria Shuttles electrons between complexes III & IV

9. YCC Free in Solution

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. YCC Covalently Attached

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. 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

15. YCC Electrostatically attached

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

26. Determination of the [YCC] Abs max = 0.0054 pH 4.00, 7mM phosphate buffer Surface Adsorption of covalently anchored YCC

27. Determination of [YCC] ~0.0054 Electrostatically adsorbed Surface Adsorption Isotherm Covalently attached studies done at ~1.00 uM [YCC]

30. Surface adsorption Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M

31. Solution Absorption Alcohol: 1 propanol pH 4.00 Buffer: 7 mM Succinate [YCC] : 1.00E-6M