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 UniversityIndianapolis, IN 46208

OutlineI. Introduction to Cytochrome c

II. Types of Experiments

a. Solution Spectroscopy

b. Surface Spectroscopy

III. Two types of cytochrome c

IV. Experiments

a. Horse cytochrome c

b. Yeast cytochrome c

V. Conclusions


• Structure• Function

II. Types of Experimentsa. Solution Spectroscopyb. Surface Spectroscopy

III. Two types of cytochrome cIV. Experiments

a. Horse cytochrome cb. Yeast cytochrome c

V. Conclusions






V. Conclusions

Cytochrome c

• Water soluble peripheral protein

• Resides in intermembrane space of mitochondria

• Located near negatively charged phospholipid bilayer surface

• Positively charged at pH 7.00 pI = 10.7

Cytochrome c

N N

NN

Fe

CH3H3C

H3C CH3

HO2C CO2H

CH3H3CS S

Cys Cys

Heme






V. Conclusions

General Function

Voet, Donald; Voet, Judith; Pratt, Charlotte. Fundamentals of Biochemistry Upgrade Ed. 2001:501






IV. Experiments



V. Conclusions






IV. Experiments



V. Conclusions

Solution SpectroscopySoret Band

• Soret peak at 408 nm

• Used to measure unfolding

•Soret band shifts left

300 400 500 600-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

Ab

sorb

an

ce

Wavelength (nm)

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

Heme

• Prosthetic Group

• Iron complex in porphyrin ring

• Liganding interactions with– Methionine 80– Histidine 18

Graphic derived from PDB file 1AKK

Banci et al., Biochemistry, v 36, pp 98679877, 1997.

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






IV. Experiments



V. Conclusions

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) above pH ~ 3.00

ExampleSurface Spectra

300 400 500 600-0.002

0.000

0.002

0.004

0.006

0.008

0.010

A

bso

rba

nce

Wavelength (nm)


Solution and Surface

300 400 500 600-0.002

0.000

0.002

0.004

0.006

0.008

0.010

Abs

orba

nce

Wavelength (nm)


300 400 500 600-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

Ab

sorb

anc

e

Wavelength (nm)

Solution Surface

OutlineI. Introduction to Cytochrome cII. Types of Experiments

a. Solution Spectroscopyb. Surface Spectroscopy

III. Two types of cytochrome c• Horse Heart Cytochrome c (HCC)• Yeast Cytochrome c (YCC)

IV. Experimentsa. Horse cytochrome c b. Yeast cytochrome c

V. Conclusions

Amino acid sequence:

HCC: GDVEKGKKIFVQKCAQCHTVEKG

YCC: TEFKAGSAKKGATLFKTRCLQCHTVEKG GKHKTGPNLHGLFGRKTGQAPGFTYTDAN GPHKVGPNLHGIFGRHSGQAQGYSYTDAN

KNKGITWEETLMEYLENPKKYIPGTKMI IKKNVLWDENNMSEYLTNPXKYIPGTKM

FAGIKKKTEREIDLIAYLKKATNE AFGGLKKEKDRNDLITYLKKACE

102






IV. Experiments



V. Conclusions






IV. Experiments

a. Horse cytochrome c ~Surface Coverage studies


V. Conclusions

Studies on Horse Cytochrome c

Surface Coverage Study

1. Denaturation with [alcohol] in solution

2. Denaturation with [alcohol] on surface

3. Comparison between the denatured proteins in solution and on the surface






Alcohol Study in Solution

400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Solution Absorption

A

bsor

banc

e

Wavelength (nm)• [Succinate Buffer] = 10 mM • [NaCl] = 150 mM• pH = 4.7

20µM [HCC], 0% n-propanol (native state)

~409 nm


400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Solution Absorption

A

bsor

banc

e


20µM [HCC], 60% n-propanol (denatured state)

~400nm


400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Solution Absorption

A

bsor

banc

e


20µM [HCC], 0% n-propanol20µM [HCC], 60% n-propanol






Alcohol Study on Surface

• [Succinate Buffer] = 10 mM • [NaCl] = 150 mM• pH = 4.7

20µM [HCC], 0% n-propanol

~409 nm

400 450-0.002

0.000

0.002

0.004

0.006

0.008

Abs

orb

ance

Wavelength (nm)



20µM [cyt c], 60% n-propanol

~405 nm

400 450-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

A

bso

rban

ce

Wavelength (nm)

Two types of Interactions:

1. Surface and protein

2. Protein and protein

Adsorption Isotherm

The adsorption isotherm shows that the surface coverage of cyt c reaches a saturation level at 15-20 µM bulk concentration

Choose 0.1 uM [HCC]0 20 40 60

0.002

0.004

0.006

0.008

0.010

0.012

0.014

Adsorption Isotherm

Abs

orba

nce

[cyt c]


400 450-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

A

bsor

banc

e

Wavelength (nm)


0.1µM [cyt c], 60% n-propanol

~ 400 nm

*Note the spectrum for 0.1µM is enlarged


*Note the spectrum for 0.1µM is enlarged


20µM [cyt c], 60% n-propanol0.1µM [cyt c], 60% n-propanol

400 450-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

Ab

sorb

anc

e

Wavelength (nm)






Surface vs. Solution:

400 450

0.0

0.2

0.4

0.6

0.8

1.0

1.2

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012Solution:

20 0% n-propanol 20 60% n-propanol

Abs

orba

nce

Wavelength (nm)

Surface: 20 , 60% n-propanol

Solution and Surface spectra

0.1 , 60% n-propanol

400 450-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

Abs

orba

nce

Wavelength (nm)

[Succinate Buffer] = 10 mM pH = 4.7[NaCl] = 150 mM

*Note the spectrum for 0.1 µM is enlarged

Surface Adsorption

400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Solution Absorption

Abs

orba

nce

Wavelength (nm)


[Succinate Buffer] = 10 mM pH = 4.7[NaCl] = 150 mM

*Note the spectrum for 0.1 µM is enlarged

•At lower bulk [HCC], surface adsorbed proteins are more denatured than at higher [HCC]

•Denatured proteins in the solution are renatured at the surface

400 450

0.0

0.2

0.4

0.6

0.8

1.0

1.2

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012Solution:

20 0% n-propanol 20 60% n-propanol

Abs

orba

nce

Wavelength (nm)

Surface: 20 , 60% n-propanol

Solution and Surface spectra

0.1 , 60% n-propanol


0 5 10 15 20 25 30 35 40 45 50 55 60399

400

401

402

403

404

405

406

Surface Solution

La

md

a M

ax

(nm

)

[ Cyt c ] (M)

Solution and Surface Absorbance

[Succinate Buffer] = 10 mM 60% n-propanol[NaCl] = 150 mM pH = 4.7






IV. Experiments



V. Conclusions

Experiments:

1. YCC free in solution» Proteins denature at higher [alcohol] and at lower pH

3. YCC covalently attached » YCC on surface takes longer to unfold than the solution» YCC on surface denatures partially

1. YCC electrostatically attached Alcohol Study:

- Solution- Surface

YCC Electrostatically attached

Yeast Cytochrome c

-disulfide linkage.

-Dimerization of YCC

dimerMonomer

Significance of Sulfur

Method to retain monomer

1. Treatment with iodoacetate:

Reaction:

I

OH

O

+

IodoacetateYCC

OH

O

+

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:

• Separate molecules of different sizes

• Heavy molecules elute rapidly

• Dimer (2 x 12,588 g/mol)• Monomer (12,588 g/mol)

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 μg

YCC uncapped (6 mths) 1 μg

YCC uncapped (4 mths) 1 μg

YCC capped /purified 1 μg

Procedure to get the data:

1. Make samplesa. Bufferb. Waterc. Proteinsd. Denaturants (alcohol)e. pH

2. Surface washing 3. Kinetic study4. Scans 5. Data analysis

Encountered Problems at Step 2

Intensity proportional to Number of proteins on surface

@ 409 nm

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.

Solution: Tried using diluted soap for rinsing the surface

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

Abs

orb

ance

Time (min)

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

• Proteins stick to the surface longer

• Take the data when see the monolayer

For every sample: 1. Kinetic run 2. Take scan

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 E7

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]

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

Ab

s

~0.0054

Covalently attached studies done at ~1.00 uM [YCC]

Electrostatically adsorbedSurface Adsorption Isotherm

Denaturation Study

Variation in [n-propanol] alcohol:

- On Surface

- In Solution


0 20 40 60 80

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 = 1.00 uM


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

Surface and Solution

0 20 40 60 80399

400

401

402

403

404

405

406

407

408

409

410

La

md

a M

ax

(nm

)

% Alcohol

Surface Solution

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

Conclusions:Horse Cytochrome c:

Under denaturing conditions (60% alcohol):

• At low [cyt c] the proteins adsorb to the surface with little change to their state of denaturation

• As the [cyt c] increases the proteins are renaturing on the surface due to increasing protein - protein interactions

• When the surface is saturated (>15-20 µM) the protein-protein interactions remain constant and protein renaturation reaches a limit

Yeast Cytochrome c:

• Stabilization in monomeric form by treating with iodoacetate

• Shows distinctly different unfolding behavior than horse– Spectroscopic phenomenon – Reverse soret band behavior

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 University Department of Chemistry

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

Date post:	03-Jul-2015
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Study of the Folding and Unfolding of Proteins Adsorbed to a Fused Silica Surface

Technology