This document summarizes an experiment that used multiple spectroscopic techniques simultaneously to observe the unfolding of the protein cytochrome c. The experiment titrated cytochrome c with increasing concentrations of guanidine hydrochloride to induce unfolding. Circular dichroism, absorbance, and fluorescence were measured in each scan to monitor secondary structure, heme environment, and proximity of tryptophan to heme, respectively. Data from wild-type cytochrome c and two mutants were analyzed to determine thermodynamic parameters of unfolding and compare to previous literature values. Results from the different techniques were consistent and in agreement with published data, validating the simultaneous multi-technique approach.

1. Employing Multiple Spectroscopic
Techniques Simultaneously to Observe
Protein Unfolding
Ben Kelty and Brennan Cull
Advisor: Dr. Justin J Link
Biophysics Program
Department of Physics

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

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

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

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

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

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

8. Our Contribution
• Reproduce past experiments
to determine precision of
parameters and conditions
• Troubleshoot technical issues
• Successfully ran a scan
containing all three techniques
• Tested two mutants and
compared scans to Wild-Type

9. 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

10. 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)
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

11. Data Analysis (Wild-Type)
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).

12. 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).

13. 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
Data Analysis Comparison

14. Conclusions/
Word of Advice
• When in doubt buy two
replacement parts
• If you are not having fun you are
doing something wrong
• Do not be afraid to ask questions

15. Acknowledgements
• Dr. Justin J Link
• Brennan Cull and
Michael Crowe
• Previous Research Students
• Xavier University Physics
Department

16. Questions?