study of the folding and unfolding of proteins adsorbed to a fused silica surface
DESCRIPTION
Study of the folding and unfolding of proteins adsorbed to a fused silica surfaceTRANSCRIPT
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
OutlineI. Introduction to Cytochrome c
• 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
OutlineI. Introduction to Cytochrome c
• 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
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
CH3H3C S S
Cys Cys
Heme
OutlineI. Introduction to Cytochrome c
• 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
General Function
Voet, Donald; Voet, Judith; Pratt, Charlotte. Fundamentals of Biochemistry Upgrade Ed. 2001:501
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
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
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
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
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)
1 µM [YCC], 7mM Succinate Buffer, pH 4.00
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)
1 µM [YCC], 7mM Succinate Buffer, pH 4.00
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)
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: GDVEKGKKIFVQKCAQCHTVEKGYCC: TEFKAGSAKKGATLFKTRCLQCHTVEKG GKHKTGPNLHGLFGRKTGQAPGFTYTDAN GPHKVGPNLHGIFGRHSGQAQGYSYTDAN
KNKGITWEETLMEYLENPKKYIPGTKMI IKKNVLWDENNMSEYLTNPXKYIPGTKM
FAGIKKKTEREIDLIAYLKKATNE AFGGLKKEKDRNDLITYLKKACE
102
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
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 ~Surface Coverage studies
b. Yeast cytochrome c
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
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
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], 60% n-propanol (denatured state)
~400nm
Alcohol Study in Solution
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 • [NaCl] = 150 mM• pH = 4.7
20µM [HCC], 0% n-propanol20µM [HCC], 60% n-propanol
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 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
Ab
sorb
an
ce
Wavelength (nm)
Alcohol Study on Surface
• [Succinate Buffer] = 10 mM • [NaCl] = 150 mM• pH = 4.7
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
rba
nce
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]
Alcohol Study on Surface
400 450-0.002
0.000
0.002
0.004
0.006
0.008
0.010
0.012
A
bsor
banc
e
Wavelength (nm)
• [Succinate Buffer] = 10 mM • [NaCl] = 150 mM• pH = 4.7
0.1µM [cyt c], 60% n-propanol
~ 400 nm
*Note the spectrum for 0.1µM is enlarged
Alcohol Study on Surface
*Note the spectrum for 0.1µM is enlarged
• [Succinate Buffer] = 10 mM • [NaCl] = 150 mM• pH = 4.7
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
an
ce
Wavelength (nm)
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
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
Ab
sorb
an
ce
Wavelength (nm)
Surface vs. Solution:
[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
Surface 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
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
Experiments:
1. YCC free in solution» Proteins denature at higher [alcohol] and at lower pH
2. YCC covalently attached » YCC on surface takes longer to unfold than the solution» YCC on surface denatures partially
3. 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
Ab
sorb
an
ce
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
Alcohol Study on Surface
0 20 40 60 80
402
403
404
405
406
407
408
409
410
So
ret M
axi
mu
m (
nm
)
% Alcohol
Alcohol: 1 propanolpH 4.00Buffer: 7 mM Succinate[YCC] : 1.00E-6M = 1.00 uM
Alcohol Study in Solution
0 20 40 60 80398
399
400
401
402
403
404
405
406
407
408
409
410
So
ret M
axi
mu
m (
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. HopkinsDr. Meng-Chih SuVictoria FahrenbachTara BenzGreg CampanelloCarrie Ann HedgeKen Clevenger
Butler University Department of Chemistry
Collaborators:Y.-Y. Cheng, S. H. Lin, and H.-C. ChangInstitute of Atomic and Molecular Sciences,Academia Sinica