Covalent Versus Electrostatic Attachment of Yeast Cytochrome c to a Fused Silica Surface By Sheetal Mistry Department of Chemistry, Butler University Indianapolis, IN 46208

Yeast cytochrome c In yeast cells Similar in function to some cytochromes in eukaryotic cells Water soluble peripheral protein Resides in intermembrane space of mitochondria Positively charged at pH 7 ~since pI at 10.7 Located near negatively charged phospholipid bilayer surface Heme (red) consists of Iron Sovlent exposed Cysteine (yellow) Yeast Cytochrome c

In yeast cells

Similar in function to some cytochromes in eukaryotic cells

Water soluble peripheral protein

Resides in intermembrane space of mitochondria

Positively charged at pH 7

~since pI at 10.7

Located near negatively charged

phospholipid bilayer surface

Heme (red) consists of Iron

Sovlent exposed Cysteine (yellow)

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

Methods: 1. Solution Absorption 2. ATR (Attenuated Total Internal Reflection)

1. Solution Absorption

2. ATR (Attenuated Total Internal

Reflection)

Solution Absorption 10 µ M [YCC], 7mM Succinate Buffer, pH 4.00 Soret Band Soret peak at 408 nm Used to measure unfolding Soret band shifts left

Soret peak at 408 nm

Used to measure unfolding

Soret band shifts left

Conformation Three dimensional structure Primary, Secondary, and Tertiary Helices maximize hydrogen bonds Conformation is considered “native” in solution under physiological conditions (pH≈7) Cox, M., Nelson, D. Principles of Biochemistry 2000:194 Process of Denaturation : Temperature change pH change Chemical change - Urea - Alcohol Tertiary Primary

Three dimensional structure

Primary, Secondary, and Tertiary

Helices maximize hydrogen bonds

Conformation is considered “native” in solution under physiological conditions (pH≈7)

Process of Denaturation :

Temperature change

pH change

Chemical change

- Urea

- Alcohol

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 ATR (Attenuated Total internal Reflection) Only detects proteins on surface Detector Light Source Quartz prism Hydrophilic surface Negatively charged (similar to phospholipid bilayer) Prism θ Glass plate O-ring Sample solution To detector

ATR (Attenuated Total internal

Reflection)

Only detects proteins on surface

Quartz prism

Hydrophilic surface

Negatively charged (similar

to phospholipid bilayer)

Experiments: YCC free in solution YCC covalently attached 3. YCC electrostatically attached

YCC free in solution

YCC covalently attached

3. YCC electrostatically attached

YCC Free in Solution

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

YCC Covalently Attached

How is YCC covalently tethered on silica? (3-aminopropyl)-trimethoxylsilane + N[ γ -maleimidobutyryloxy]sulfosuccinimide ester + SiO 2 SiO 2 SiO 2 YCC sGMBS

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

Surface Alcohol Denaturing Shifts of the Soret band maximum: Surface bound YCC Free YCC Methanol 1-propanol Conditions: 7mM phosphate buffer, pH 4.00 , , YCC in solution denatures with respect to change in [alcohol] YCC on surface denatures partially

Shifts of the Soret band maximum:

Surface bound YCC

Free YCC

YCC in solution denatures with

respect to change in [alcohol]

YCC on surface denatures

partially

YCC Electrostatically attached

Yeast Cytochrome c -disulfide linkage. -Dimerization of YCC dimer Monomer Significance of Sulfur

-disulfide linkage.

-Dimerization of YCC

Why work with monomer? Dimer has different functionality Want to compare with the covalently tethered proteins No chances for these proteins to dimerize as the sulfur is covalently bound to the surface

Dimer has different functionality

Want to compare with the covalently tethered proteins

No chances for these proteins to dimerize as the sulfur is covalently bound to the surface

Method to retain monomer 1. Treatment with iodoacetate: Reaction: + Iodoacetate YCC

1. Treatment with iodoacetate:

Reaction:

2. Size Exclusion Chromatography: Separate molecules of different sizes Heavy molecules elute rapidly Sephadex G-50 Dimer (2 x 12,588 g/mol) Monomer (12,588 g/mol) Method to retain monomer

Separate molecules of different sizes

Heavy molecules elute rapidly

Sephadex G-50

Dimer (2 x 12,588 g/mol)

Monomer (12,588 g/mol)

3. Gel Electrophoresis: Molecular HCC YCC Weight 1μg 1μg Marker 15μL 15μL Method to retain monomer Dimer ~24,000g/mol Monomer~12,000g/mol

Procedure to get the data: 1. Make samples 2. Kinetic study 3. Surface washing Scans 5. Data analysis

1. Make samples

2. Kinetic study

3. Surface washing

Scans

5. Data analysis

Encountered Problems at Step 2 Right after Taiwan Proteins did not stick to the Surface…. Does not look like a Kinetic scan Intensity proportional to number of proteins on surface

Right after Taiwan

Several Factors could play a role [YCC] [Buffer] [alcohol] [NaCl] pH Result: Found that by using the base bath, the surface was getting too basic and was not allowing proteins to stick to the surface. Tried using diluted soap by rinsing the surface several times and turned Out to be a success.

[YCC]

[Buffer]

[alcohol]

[NaCl]

pH

Kinetic Study Proteins stick to the surface longer See the monolayer Take the data when see the monolayer For every sample: 1. Kinetic scan 2. Record the time (monolayer) 3. Take data

Proteins stick to the surface

longer

See the monolayer

Take the data when

see the monolayer

Adsorption Isotherm pH 4.0, 7mM Succinate Buffer purpose: know the concentration at which the covalently anchored studies were done Surface saturation around 10 µM YCC concentration K ad YCC = 1.3 E 6 K ad HCC = 1.3 E 6

purpose: know the concentration

at which the covalently anchored

studies were done

Surface saturation around

10 µM YCC concentration

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 Electrostatically adsorbed Surface Adsorption Isotherm Covalently attached studies done at ~1.00 uM [YCC]

Future Direction Denaturation studies: Variation in pH Variation in alcohol Methanol 1-propanol Methods: Solution Absorption ATR Spectroscopy For Electrostatic Attachment :

Denaturation studies:

Variation in pH

Variation in alcohol

Methanol

1-propanol

Methods:

Solution Absorption

ATR Spectroscopy

Special Thanks To Dr. Geoffrey C. Hoops Dr. Todd A. Hopkins Dr. Meng-Chih Su Victoria Fahrenbach Tara Benz Greg Campanello Carrie Ann Hedge Ken Clevenger Butler Summer Institute Holcomb Undergraduate Grants Lilly Endowment Butler University Department of Chemistry Collaborators: Y.-Y. Cheng, S. H. Lin, and H.-C. Chang Institute of Atomic and Molecular Sciences, Academia Sinica

Dr. Geoffrey C. Hoops

Dr. Todd A. Hopkins

Dr. Meng-Chih Su

Victoria Fahrenbach

Tara Benz

Greg Campanello

Carrie Ann Hedge

Ken Clevenger

Butler Summer Institute

Holcomb Undergraduate Grants

Lilly Endowment

Butler University

Department of Chemistry

Collaborators:

Y.-Y. Cheng, S. H. Lin, and H.-C. Chang

Institute of Atomic and Molecular Sciences,

Academia Sinica

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

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