Employing Multiple Spectroscopic Techniques Simultaneously to Observe

Protein UnfoldingBen Kelty and Brennan Cull

Advisor: Dr. Justin J LinkBiophysics Program

Department of Physics

• 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

PurposeDevelop 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

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

Equine Cytochrome c

• Foldons

Maity H., et al. (2005) PNAS 102: 4741-6

Mutants

W89

W66W77 W72

W82

W36

W23

W102W45W15

W51

Location of the Tryptophan within Wild-Type Cytochrome c

W59

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

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

Data Analysis (Wild-Type)

320 340 360 380 400 420

0.00.10.20.30.4

GdnHCl

(M)

Wavelength (nm)

Flu

or. (

AU)

220 225 230 235 240

-100-80-60-40-200

GdnHCl (M

)Wavelength (nm)

CD

(mde

g)

380 400 420 440

0.00.20.40.60.81.01.21.4

GdnHC

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

Flu

or. a

t 350

nm

GdnHCl (M)

Data Analysis (Wild-Type)

0 1 2 3 4 5-0.1

0.0

0.1

0.2

0.3

0.4

Flu

or. a

t 350

nm

GdnHCl (M)

0 1 2 3 4 50.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

Abs.

at 4

03.1

nm

GdnHCl (M)0 1 2 3 4 5

-100

-80

-60

-40

-20

0

CD a

t 222

nm

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.

at 4

03.1

nm

GdnHCl (M)0 1 2 3 4 5

-100

-80

-60

-40

-20

0

CD a

t 222

nm

GdnHCl (M)

𝑆𝑜𝑏𝑠=(𝐶 𝑓 +𝑚 𝑓 [𝐺𝑑𝑛𝐻𝐶𝑙 ])+(𝐶𝑢+𝑚𝑢 [𝐺𝑑𝑛𝐻𝐶𝑙 ])𝑒

−Δ𝐺+𝑚𝑔[𝐺𝑑𝑛𝐻𝐶𝑙 ]𝑅𝑇

1+𝑒−Δ𝐺+𝑚𝑔[𝐺𝑑𝑛𝐻𝐶𝑙 ]

𝑅𝑇

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)

Frac

tion

Unfo

lded

GdnHCl (M)0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

CD at 222nm (mdeg)

Frac

tion

Unfo

lded

GndHCl (M)

0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

Fluor. at 350nm (AU)

Frac

tion

Unfo

lded

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/AKnapp, JA and Pace CN. Biochemistry 13, 1289-94. (1974).Maity, H et al. J Mol Biol 343, 223-33. (2004).

Fraction Unfolded (Wild-Type)

Technique ΔG (kcal/mol) Cm (M)CD 7.83 2.30Absorbance 7.12 2.25Fluorescence 7.71 2.34Published (CD) 7.27 2.42Published (HX) 7.40 N/AGlobal 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

Frac

tion

Unfo

lded

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 AbsorbanceFluorescence

Frac

tion

Unfo

lded

GdnHCl (M)

Knapp, JA and Pace CN. Biochemistry 13, 1289-94. (1974).Maity, H et al. J Mol Biol 343, 223-33. (2004).

0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

Circular Dichroism

Frac

tion

Unfo

lded

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

Frac

tion

Unfo

lded

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

Frac

tion

Unfo

lded

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

Frac

tion

Unfo

lded

GdnHCl (M)

F82W pWT Wild-Type

Data Analysis Comparison

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

Acknowledgements

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

Department

Questions?