1. 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)
Introduction
Proteins are long chains of amino acids that are essential
to life because they have many roles in the body including
structure, reaction catalysis, and binding. A protein’s
native state is a folded state, and for a protein to function
properly, it must be able to fold and unfold correctly.1 Our
goal was to study protein unfolding using the protein
horse heart cytochrome c (hhcyt c) as our model protein.
It is our hope that others could use our protocol to study
the unfolding of more complex proteins in the future.
hhcyt c was chosen for several reasons. This protein has
been well studied, is relatively small (104 AA), has a single
tryptophan amino acid, and a heme cofactor.2 Having the
single tryptophan amino acid was critical to our study
since we could use it as a site-specific probe for our
spectroscopic techniques and create mutants where the
tryptophan amino acid is moved to other locations on our
protein, allowing us to study the unfolding of different
regions of our protein.
Procedure
Results
Acknowledgements
Conclusions
My research built off of research done by previous
students who worked with Dr. Link. While previous
students had been able to study the protein using CD,
Fluorescence, and Absorbance, this was done in separate
scans on separate days. My research group was the first to
develop and use a protocol where all three techniques
were used during one automated scan. This helps increase
the accuracy of our experiment by reducing the possibility
of error sources such as different protein concentrations,
different orientation of the cuvette, and impurities being
introduced to our sample. After analyzing the data from
each scan, the stability of the protein was determined
using the change in Gibbs free energy between the native,
folded state and the unfolded protein. The goal of this
research is to use the results from the mutant proteins as
well as the wild type protein and compare the unfolding
curves of each protein. Ideally, this will result in a better
understanding of how the different regions of the protein
unfold as a whole and would give us a clearer
understanding of how the overall protein unfolds.
However, only two of the thirteen mutant proteins were
able to be studied so far. This gives future research
students the opportunity to finish this project by studying
the remaining mutant proteins.
References
Employing Multiple Spectroscopic Techniques
Simultaneously to Observe Protein Unfolding
Ben Kelty (Biophysics Major)
Advisor: Dr. Justin Link
XAVIER UNIVERSITY Physics Department
I would like to thank Dr. Justin Link for advising me in my
research for the past two summers. I would also like to
thank Michael Crowe and Brennan Cull for working on this
project with me. Finally, thank you to the John Hauck
foundation and the Borcer fund for funding my research
and to the Xavier University Physics Department for giving
me this great opportunity.
Figure 1: A. Illustration of horse heart cytochrome c (PDB: 1HRC) displaying the
ligands connection to the heme group (fuchsia) and the location of the single
tryptophan amino acid (yellow). B. Overlay of the different tryptophan locations
(yellow spheres) for mutant versions of hhcyt c. Image created using PyMOL.
A. B.
A 33-step titration protocol was used to unfold the protein
hhcyt c using the denaturant guanidine hydrochloride
(GdnHCl) in 25mM NaPi pH=7.6 buffer. At each step, the
protein was monitored using the spectroscopic
techniques of Circular Dichroism (CD), Fluorescence, and
Absorbance using a J-810 spectropolarimeter. By using the
three spectroscopic techniques at each step, we were able
to monitor the unfolding of the protein based on the
changes in signal intensity. This protocol was used on wild
type hhcyt c, as well as two mutants where the single
tryptophan (trp) amino acid was moved to different
locations on the protein in order to monitor the different
regions of the protein as the protein is unfolded. In future
research, the 33-step titration protocol will be used on
eleven more mutant versions of hhcyt c . After all thirteen
mutants have been studied, the data will be compared in
order to understand how the different regions of
cytochrome c unfold as a whole.
𝑺 𝒐𝒃𝒔 =
𝑪 𝒇 + 𝒎 𝒇 𝑫 + 𝑪 𝒖 + 𝒎 𝒖 𝑫 𝒆
−𝜟𝑮+𝒎 𝒈 𝑫
.
𝑹𝑻
𝟏 + 𝒆
−𝜟𝑮+𝒎 𝒈[𝑫]
.
𝑹𝑻
Figure 2: Three dimensional plot of data for wild type cytochrome c. Scans were taken at each step of the 33-step titration where the concentration of GdnHCl was increased at each step. A. Data
measuring the Circular Dichroism (CD) of hhcyt c as it unfolds. B. Data measuring the Fluorescence of hhcyt c as it unfolds. C. Data measuring the Absorbance of hhcyt c as it unfolds.
A. B. C.
A. B. C.
Equation 1: The two state unfolding equation that was used to create two-state unfolding
curves for the data. 𝑺 𝒐𝒃𝒔 is the observed signal at a specified wavelength (222nm for CD,
350nm for Fluorescence, and 403.1 nm for Absorbance), 𝑪 𝒇 and 𝑪 𝒖 are the y-intercepts for
the folded and unfolded regions respectively while 𝒎 𝒇 and 𝒎 𝒖 are the slopes of the folded
and unfolded regions respectively. 𝒎 𝒈 is the equilibrium m value and is a measure of the
change in surface area of the protein as it unfolds. [D] is the concentration of the denaturant,
guanidine hydrochloride, 𝜟𝑮 is Gibbs Free Energy, R is the ideal gas constant, and T is the
temperature of the sample in Kelvin. 3
Figure 3: Plots of signal intensity versus the concentration GdnHCl in the sample. The data was then fit to a two-state unfolding model which yielded values to be used in equation 1 to then create
data to make a fraction of the protein that is unfolded versus the concentration of GdnHCl plot (figure 4). A. A plot of CD versus concentration of GdnHCl. B. A plot of Fluorescence versus
concentration of GdnHCl. C. A plot of Absorbance versus concentration of GdnHCl.
Table 1: This table compares the Gibbs Free Energy and Cm (the concentration of
GdnHCl when half of the protein is unfolded) obtained from the plots of each of the
three techniques, published literature, and the global fit.
Figure 4: Plot of the fraction of protein in the sample that is unfolded versus the concentration
of GdnHCl in the sample. This data was obtained by using the values obtained by fitting the
plot in figure 3 to equation 1. The fraction unfolded data points for CD, Fluorescence, and
Absorbance were then all fitted to a single two-state unfolding curve, a technique called
global fitting.
Figure 5: The global fit curves from the wild type hhcyt c and the mutants F82W and
pseudo wild-type (pWT) are overlaid onto one plot.
1. Serdyuk, Igor N., Nathan R. Zaccai, and G. Zaccai. 2007. Methods
in Molecular Biophysics: Structure, Dynamics, Function.
Cambridge: Cambridge UP. 61-67
2. Goldbeck R, Chen E, Kliger D. 2009. Early events, kinetic
intermediates and the mechanism of protein folding in cytochrome
c. Int. J. Mol. Sci. 10: 1476-1499.
3. Bhuyan A., Udgaonkar J. 2001. Folding of horse cytochrome c in
the reduced state. J. Mol. Biol. 312: 1135-1160.
4. Knapp, J. A., and C. N. Pace. 1974. Guanidine Hydrochloride and
Acid Denaturation of Horse, Cow, and Candida Krusei Cytochromes
C. Biochemistry 13: 1289-294.
5. Maity, Haripada, Mita Maity, and Walter S. Englander. 2004. How
Cytochrome C Folds, and Why: Submolecular Foldon Units and
Their Stepwise Sequential Stabilization. J. Mol. Biol. 343: 223-33.
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)
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Global FitFractionUnfolded
GdnHCl (M)
F82W
pWT
Wild-Type