Covalent Versus Electrostatic Attachment of Yeast

Cytochrome c to a Fused Silica Surface

By Sheetal Mistry

Department of Chemistry, Butler UniversityIndianapolis, 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

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)

Solution Absorption

10 µM [YCC], 7mM Succinate Buffer, pH 4.00

Soret Band

300 400 500 600

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Abs

orba

nce

Wavelength (nm)

• 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

ATR spectroscopy

Cheng, Y.-Y.; Lin, S. H.; Chang, H.-C.; Su, M.-C.: Probing Adsorption, Orientation and Conformational Changes ofCytochrome 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

DetectorPrism

θ

Glass plateO-ring

Sample solution

To detector

Light

Source

• Quartz prism

• Hydrophilic surface

• Negatively charged (similar to phospholipid bilayer)

Experiments:

1. YCC free in solution

2. YCC covalently attached

3. YCC electrostatically attached

YCC Free in Solution

Solution Absorption

Wavelength20% alcohol60% 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?

Si

H3CO

H3CO

OCH3

NH2

(3-aminopropyl)-trimethoxylsilane

+ N

O

OS

O

O-Na+O

O

N

O

O

O

N[γ-maleimidobutyryloxy]sulfosuccinimide ester

Si

H3CO

H3CO

OCH3

NH

N

O

O

O

+

Si

H3CO

H3CO

OCH3

NH

N

O

O

O

SiO2

SiO2

SiO2

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 solutiondenature as the pH is lowered

Surface Alcohol Denaturing

Shifts of the Soret band maximum:(a) Surface bound YCC(b) Free YCC

Methanol1-propanol

Conditions: 7mM phosphate buffer, pH 4.00

,

,

• 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

dimerMonomer

Significance of Sulfur

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

Method to retain monomer

1. Treatment with iodoacetate:

Reaction:

I

OH

O

+

IodoacetateYCC

OH

O

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

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

4. 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 numberof proteins on surface

Several Factors could play a role

1. [YCC]

2. [Buffer]

3. [alcohol]

4. [NaCl]

5. 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.

Kinetic Study

-2 0 2 4 6 8 10 12 14 16-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

Ab

sorb

an

ce

Time (min)

Time Scan 2 uM YCC, pH 4.00, 7mM succinate buffer• 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

Adsorption Isotherm

pH 4.0, 7mM Succinate Buffer

Abs vs. [ ]

0.0000

0.0020

0.0040

0.0060

0.0080

0.0100

0.0120

0.0140

0 2 4 6 8 10 12

[ YCC] uM

Abs

• purpose: know the concentration at which the covalently anchored studies were done

• Surface saturation around 10 µM YCC concentration

Kad YCC = 1.3 E6

Kad HCC = 1.3 E6

Determination of the [YCC]

Abs max = 0.0054

350 400 450 500 550 600

0.000

0.002

0.004

0.006

0.008

0.010

A

bs

wavelength

pH 4.00, 7mM phosphate buffer

Surface Adsorption of covalently anchored YCC

Determination of [YCC]

~0.0054

Electrostatically adsorbedSurface 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 :

Special Thanks To

Dr. Geoffrey C. Hoops Dr. Todd A. HopkinsDr. Meng-Chih SuVictoria FahrenbachTara BenzGreg CampanelloCarrie Ann HedgeKen Clevenger

Butler Summer InstituteHolcomb Undergraduate GrantsLilly Endowment

Butler University Department of Chemistry

Collaborators:Y.-Y. Cheng, S. H. Lin, and H.-C. ChangInstitute of Atomic and Molecular Sciences,Academia Sinica

Surface adsorption

0 20 40 60 80

402

403

404

405

406

407

408

409

410

S

ore

t Ma

xim

um

(n

m)

% Alcohol

Alcohol: 1 propanolpH 4.00Buffer: 7 mM Succinate[YCC] : 1.00E-6M

Solution Absorption

0 20 40 60 80398

399

400

401

402

403

404

405

406

407

408

409

410

Sor

et M

axim

um (

nm)

% Alcohol

Alcohol: 1 propanolpH 4.00Buffer: 7 mM Succinate[YCC] : 1.00E-6M