APS Protein Unfolding

Employing Multiple Spectroscopic Techniques Simultaneously to Observe

Protein Unfolding

Brennan CullAdvisor: Dr. Justin J Link

Biophysics ProgramXavier UniversityCincinnati, OH

• 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 InformationEmail: [email protected]

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

Email: [email protected]

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

W66

W77W72

W82

W36

W23

W102W45

W15

W51

Location of the Tryptophan within Wild-Type Cytochrome c

W59


Spectroscopic Techniques

Fluorescence monitors proximity of hemerelative to tryptophan

Zang C., et al. (2009) J Am ChemSoc 131(8):2846–2852

Absorbance monitors the local environment of the heme

Circular dichroism determines the components of secondary structure

W59


Data Analysis (Wild-Type)

320 340 360 380 400 420

0.0

0.1

0.2

0.3

0.4

GdnH

Cl (

M)

Wavelength (nm)

Flu

or.

(A

U)

220 225 230 235 240

-100

-80

-60

-40

-20

0

GdnH

Cl (

M)

Wavelength (nm) C

D (

md

eg

)

380 400 420 440

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

GdnH

Cl (

M)

Wavelength (nm)

Ab

s.

(OD

)

222 nm403.1nm

350nm

0 1 2 3 4 5-0.1

0.0

0.1

0.2

0.3

0.4

Flu

or.

at

35

0n

m

GdnHCl (M)


0 1 2 3 4 5-0.1

0.0

0.1

0.2

0.3

0.4

Flu

or.

at

35

0n

m

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

Flu

or.

at

35

0n

m

GdnHCl (M)

0 1 2 3 4 5

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

Ab

s.

at

40

3.1

nm

GdnHCl (M)

0 1 2 3 4 5

-100

-80

-60

-40

-20

0

CD

at

22

2n

m

GdnHCl (M)

0 1 2 3 4 5

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

Ab

s.

at

40

3.1

nm

GdnHCl (M)0 1 2 3 4 5

-100

-80

-60

-40

-20

0

CD

at

22

2n

m

GdnHCl (M)

𝑆𝑜𝑏𝑠

=𝐶𝑓 +𝑚𝑓 𝐺𝑑𝑛𝐻𝐶𝑙 + 𝐶𝑢 +𝑚𝑢 𝐺𝑑𝑛𝐻𝐶𝑙 𝑒

−Δ𝐺+𝑚𝑔[𝐺𝑑𝑛𝐻𝐶𝑙]

𝑅𝑇

1 + 𝑒−Δ𝐺+𝑚𝑔[𝐺𝑑𝑛𝐻𝐶𝑙]

𝑅𝑇


0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

Abs. at 403.1nm (OD)

Fra

cti

on

Un

fold

ed

GdnHCl (M)0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

CD at 222nm (mdeg)

Fra

cti

on

Un

fold

ed

GndHCl (M)

0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

Fluor. at 350nm (AU)

Fra

cti

on

Un

fold

ed

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)


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

Fra

cti

on

Un

fold

ed

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

Fra

cti

on

Un

fold

ed

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

Fra

cti

on

Un

fold

ed

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

Fra

cti

on

Un

fold

ed

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

Fra

cti

on

Un

fold

ed

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

Fra

cti

on

Un

fold

ed

GdnHCl (M)

F82W

pWT

Wild-Type

Wild TypepWT

F82W

Wild TypepWT

F82W

Wild TypepWT

F82W

Wild TypepWT

F82W

Comparison of WT and Mutants


Acknowledgements

• Dr. Justin J Link

• Ben Kelty

• Previous Research Students

• Xavier University Physics Department

• John Hauck Foundation


0 1 2 3 4 5

0.0

0.2

0.4

0.6

0.8

1.0

Circular Dichroism

Fra

cti

on

Un

fold

ed

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

Fra

cti

on

Un

fold

ed

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

Fra

cti

on

Un

fold

ed

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

Fra

cti

on

Un

fold

ed

GdnHCl (M)

F82W

pWT

Wild-Type

Comparison of WT and Mutants

Wild TypepWT

F82W

Wild TypepWT

F82W

Wild TypepWT

F82W

Wild TypepWT

F82W


APS Protein Unfolding

Documents