Surface Coverage Dependent Renaturation of Oxidized

Cytochrome c Adsorbed to a Fused Silica Surface

S. A. Mistry, S. A. Hocker, V. S. Fahrenbach, T. L. Benz, G. C. Campanello

G. C. Hoops, T. A. Hopkins, M.-C. Su

Department of Chemistry, Butler University, Indianapolis, IN 46208

Cytochrome c• In most 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 consists of Iron

• Two states: oxidized Fe(III) and reduced Fe(II)

Heme Tryptophan Tyrosine

Cyt c Function

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

Plays a major role in the electron transport chain in the inner membrane of mitochondria

Shuttles electrons between complexes III & IV

Solution Absorption

20 µM [Cyt c] in 10mM pH 4.7 Succinate Buffer

Cox, M., Nelson, D. Principles of Biochemistry 2000:194

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

• Conformation is considered

“native” in solution under

physiological conditions (pH≈7)

Solution Alcohol Denatured

•At 20 μM, native cyt c has soret location at 408nm

• n-propanol denatured cyt c at 400nm

•Protein degree of denaturation is not dependent on [cyt c]400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Solution Absorption

Abs

orba

nce

Wavelength (nm)

20µM [cyt c], 0% n-propanol

20µM [cyt c], 60% n-propanol

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

ATR spectroscopy of cyt c on fused silica surface

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)

Alcohol denatured Isotherm

The adsorption isotherm shows

that the surface coverage of

cyt c reaches a saturation level

at 15-20 µM bulk concentration0 20 40 60

0.002

0.004

0.006

0.008

0.010

0.012

0.014

Adsorption Isotherm

Ab

sorb

anc

e

[cyt c]

Surface Spectra:

• Denatured 20 μM cyt c has soret location at 405 nm

• 0.1 μM cyt c at 401 nm

• Degree of denaturation of proteins is dependent on [cyt c]

*Note the spectrum for 0.1µM is enlarged

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

0.012

Abs

orba

nce

Wavelength (nm)

20µM [cyt c], 60% n-propanol

0.1µM [cyt c], 60% n-propanol

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

Surface adsorbed vs. Solution:

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

*Note the spectrum for 0.1 µM is enlarged

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

•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

Surface adsorbed vs. Solution:

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

Concentration dependence:

•In solution cyt c proteins are “completely” denatured

•At all [cyt c] the surface adsorbed proteins are less denatured than in solution

•The degree of denaturation of surface adsorbed proteins depends on concentration

Protein-Protein Interactions:

• 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

ConclusionProtein-protein interactions are an important

factor in the conformation of cyt c

adsorbed to fused silica surface

Future Direction• Increase signals of low concentration spectra via multiple

reflection ATR absorbance spectroscopy using a multi pass cell

• Surface Fluorescence to indicate conformational changes

Special Thanks To:

Dr. Todd A. Hopkins

Dr. Geoffrey C. Hoops

Dr. Meng-Chih Su

Scott A. Hocker

Victoria Fahrenbach

Tara Benz

Greg Campanello

Butler University Department of Chemistry

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