The document describes a study that used multiple spectroscopic techniques simultaneously to observe the unfolding of proteins. The purpose was to develop an efficient way to study the structure and stability of a protein as it unfolds. Equine cytochrome c and some of its mutants were used as model systems. Circular dichroism, absorbance, and fluorescence were measured in automated scans as the protein was titrated with increasing concentrations of guanidine hydrochloride. Data from the wild type protein and mutants were analyzed and compared to published literature to validate the results. The study provides a cost-effective way to examine protein unfolding using an integrated multi-technique approach.

1. Employing Multiple Spectroscopic
Techniques Simultaneously to Observe
Protein Unfolding
Brennan Cull
Advisor: Dr. Justin J Link
Biophysics Program
Xavier University
Cincinnati, OH

2. • A protein is a chain of amino acids
– Twenty different amino acids
• Proteins are essential to life
– Variety of biological functions
• Catalysis, Structure, Protection (Immune System),
and Regulating Cell Division
• Diseases caused by protein misfolding
– Alzheimer’s Disease
– Huntington’s Disease
– Atherosclerosis
– Type II Diabetes
– Many types of cancers
Petsko, G. A., and Ringe, D. Protein Structure and Function. New
Science Press, 2004. Print
Background Information
Email: cullb1@Xavier.edu

3. Purpose
Develop a cost-efficient way to study the structure
and stability of a protein as it unfolds
Overview of Procedure
• Titration that increases the concentration
guanidine hydrochloride at each step in order to
unfold the protein, cytochrome c, and a couple
of its mutants
• Measure the following in one, automated scan:
– Circular Dichroism (CD)
– Absorbance
– Fluorescence
To help support or disclaim the results of
published literature
Email: cullb1@Xavier.edu

4. Equine Cytochrome c
• Model System
• Well characterized
• Relatively small in size
• Single Tryptophan
molecule
• Cofactor: Heme
group
Zang C., et al. (2009) J Am Chem Soc 131(8):2846–2852
Email: cullb1@Xavier.edu

5. Equine Cytochrome c
• Foldons
Maity H., et al. (2005) PNAS 102: 4741-6
Email: cullb1@Xavier.edu

6. Mutants
W89
W66
W77
W72
W82
W36
W23
W102W45
W15
W51
Location of
the
Tryptophan
within Wild-
Type
Cytochrome c
W59
Email: cullb1@Xavier.edu

7. Spectroscopic Techniques
Fluorescence monitors
proximity of heme
relative to tryptophan
Zang C., et al. (2009) J Am Chem
Soc 131(8):2846–2852
Absorbance monitors
the local environment
of the heme
Circular dichroism
determines the
components of
secondary structure
W59
Email: cullb1@Xavier.edu

8. Data Analysis (Wild-Type)
320 340 360 380 400 420
0.0
0.1
0.2
0.3
0.4
G
dnH
C
l(M
)
Wavelength (nm)
Fluor.(AU)
220 225 230 235 240
-100
-80
-60
-40
-20
0
G
dnH
C
l(M
)Wavelength (nm)
CD(mdeg)
380 400 420 440
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
G
dnH
C
l(M
)
Wavelength (nm)
Abs.(OD)
222 nm 403.1nm
350nm
0 1 2 3 4 5
-0.1
0.0
0.1
0.2
0.3
0.4
Fluor.at350nm
GdnHCl (M)
Email: cullb1@Xavier.edu

9. 0 1 2 3 4 5
-0.1
0.0
0.1
0.2
0.3
0.4
Fluor.at350nm
GdnHCl (M)
Data Analysis
(Wild-Type Cross Section)
0 1 2 3 4 5
-0.1
0.0
0.1
0.2
0.3
0.4
Fluor.at350nm
GdnHCl (M)
0 1 2 3 4 5
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
Abs.at403.1nm
GdnHCl (M)
0 1 2 3 4 5
-100
-80
-60
-40
-20
0
CDat222nm
GdnHCl (M)
0 1 2 3 4 5
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
Abs.at403.1nm
GdnHCl (M)
0 1 2 3 4 5
-100
-80
-60
-40
-20
0
CDat222nm
GdnHCl (M)
𝑆 𝑜𝑏𝑠
=
𝐶𝑓 + 𝑚 𝑓 𝐺𝑑𝑛𝐻𝐶𝑙 + 𝐶 𝑢 + 𝑚 𝑢 𝐺𝑑𝑛𝐻𝐶𝑙 𝑒
−Δ𝐺+𝑚 𝑔[𝐺𝑑𝑛𝐻𝐶𝑙]
𝑅𝑇
1 + 𝑒
−Δ𝐺+𝑚 𝑔[𝐺𝑑𝑛𝐻𝐶𝑙]
𝑅𝑇
Email: cullb1@Xavier.edu

10. 0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Abs. at 403.1nm (OD)
FractionUnfolded
GdnHCl (M)
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
CD at 222nm (mdeg)
FractionUnfolded
GndHCl (M)
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Fluor. at 350nm (AU)
FractionUnfolded
GdnHCl (M)
Technique ΔG (kcal/mol) Cm (M)
CD 7.83 2.30
Absorbance 7.12 2.25
Fluorescence 7.71 2.34
Published (CD) 7.27 2.42
Published (HX) 7.40 N/A
Knapp, JA and Pace CN. Biochemistry 13, 1289-94. (1974).
Maity, H et al. J Mol Biol 343, 223-33. (2004).
Data Analysis
(Wild-Type Cross Section)
Email: cullb1@Xavier.edu

11. Fraction Unfolded
(Wild-Type)
Technique ΔG (kcal/mol) Cm (M)
CD 7.83 2.30
Absorbance 7.12 2.25
Fluorescence 7.71 2.34
Published (CD) 7.27 2.42
Published (HX) 7.40 N/A
Global Fit 7.83 2.30
0 1 2 3 4 5
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
Individual Fit
CD Fit
CD Data
Abs Fit
Abs Data
Fluor Fit
Fluor Data
FractionUnfolded
GdnHCl (M)
0 1 2 3 4 5
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
Global FIt
Global Fit
CD
Absorbance
Fluorescence
FractionUnfolded
GdnHCl (M)
Knapp, JA and Pace CN. Biochemistry 13, 1289-94. (1974).
Maity, H et al. J Mol Biol 343, 223-33. (2004).
Email: cullb1@Xavier.edu

12. 0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Circular Dichroism
FractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Global Fit
FractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Fluorescence
FractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Absorbance
FractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type
Wild Type
pWT
F82W
Wild Type
pWT
F82W
Wild Type
pWT
F82W
Wild Type
pWT
F82W
Comparison of
WT and Mutants
Email: cullb1@Xavier.edu

13. Acknowledgements
• Dr. Justin J Link
• Ben Kelty
• Previous Research Students
• Xavier University Physics
Department
• John Hauck Foundation
Email: cullb1@Xavier.edu

14. 0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Circular Dichroism
FractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Global Fit
FractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Fluorescence
FractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Absorbance
FractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type
Comparison of
WT and Mutants
Wild Type
pWT
F82W
Wild Type
pWT
F82W
Wild Type
pWT
F82W
Wild Type
pWT
F82W
Email: cullb1@Xavier.edu