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Page 1: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

The role of disulfide bonds on tThe role of disulfide bonds on the activity, stability and he activity, stability and foldingfolding of bovine-angiogeninof bovine-angiogenin

Eun-Hye KoDept. of Bioinformatics & Life Science,and Protein engineering & Design Lab.

Soongsil University

Page 2: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Angiogenin (ANG)Angiogenin (ANG)

A member of ribonuclease A superfamily (with RNase A activity and sequence homology to RNase A)

Has minuscule RNase activity (10-4 ~ 10-6), but critical to the biological function

Inducer of angiogenesis, a process of new blood vessel formation (neovacularization)

Page 3: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Angiogenin (ANG)Angiogenin (ANG)

Angiogenesis is involved in normal physiological processes as well as pathological conditions

Understanding of angiogenin will contribute to

1. the folding patterns of RNase A superfamily

(RNase A, Onconase, Angiogenin)

2. the development of therapeutics for the treatment of pathological neovascularization

Page 4: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

ANG has 125 amino acid residues with three disulfide bonds (Cys27-Cys82, Cys40-Cys93 and Cys58-Cys108)

What is the role of disulfide bonds on the structure, activity and stability of ANG?

Page 5: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Role of disulfide bonds on the Role of disulfide bonds on the structure, stability & functionstructure, stability & function

Page 6: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

des mutants of b-ANGdes mutants of b-ANG

[C27S, C82S], [C40S, C93S], and [C58S, C108S], respectively, were produced in E. coliDenaturation & RefoldingPurification: Cation-exchange chromatography

Biophysical analysis : Circular dichroism, Stopped-flow

Biological (Angiogenesis) assays: CAM assay, Wound healing migration

Page 7: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Expression & purification of des mutantsExpression & purification of des mutants

SDS-PAGE of des mutants of b-ANG

97.4 kD

66.2 kD

45 kD

31 kD

21.5 kD

14.4 kD

1 2 3 4 5 6 7

1 2 3 4 5 6 7

1 2 3 4 5 6 7

[C27S, C82S] [C40S, C93S] [C58S, C108S]

Expression level : 35 %

Page 8: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

CD analysisCD analysis

- 55

- 45

- 35

- 25

- 15

- 5

5

190 200 210 220 230 240 250

wavelength (nm)

CD

(m

deg)

w/t (●), [C27S, C82S] (●), [C40S, C93S] (●), and [C58S, C108S] (●)

[C27S, C82S] & [C58S, C108S]

: much less structured

[C40S, C93S]

: native-like, similar to the wild type

Page 9: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

TTmm analysis analysis

- 40

- 30

- 20

0 10 20 30 40 50 60 70 80 90

Temperature (C)

CD

(m

deg)

wild type

[C27S,C82S] [C40S,

C93S]

[C58S,C108S]

Page 10: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Bovine ANG Tm ( )℃ pH Purity (%)

Wild-type 65 7.4 > 95

[C27S, C82S] <10 7.4 > 90

[C40S, C93S] 45 7.4 > 90

[C58S, C108S] <10 7.4 > 90

The melting temperature and purity for The melting temperature and purity for w/t and des mutants of b-ANGw/t and des mutants of b-ANG

Page 11: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Biological assaysBiological assays

Angiogenic activities of w/t and des mutants of b-ANG

(CAM assay & wound healing migration)

Wound healing Wound healing migrationmigration

CAM assayCAM assay

Page 12: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Role of disulfide bondsRole of disulfide bonds

critical for the stability & structure

critical for the biological function

27-82

58-108

40-93

no effect on the biological function

medium effect for the stability & biological function

no or slight effect on the structure

Page 13: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Under investigation:Under investigation:double des mutantsdouble des mutants

45 kD

97.4 kD

66.2 kD

1 2 3 4 5 2 3 4 5 2 3 4 5

[C27-C82] [C40-C93] [C58-C108]

SDS-PAGE of double des mutants of b-ANG

Using PDI fusion system for soluble expression

Expression level : 35~40 %

Page 14: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Kinetic studies of ANG & des Kinetic studies of ANG & des mutants : effect of disulfide mutants : effect of disulfide bonds on the folding behaviorbonds on the folding behavior

Page 15: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Stopped-flowStopped-flow

Various spectroscopic methods coupled with stopped-flow methods:

- NMR spectroscopy, CD spectroscopy, Fluorescence spectroscopy

- FT-IR, light scattering, protein engineering-coupled with

spectroscopy, etc.

Page 16: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Representative Representative data curves data curves

of b-ANGof b-ANG

Page 17: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

at pH 8, 15 and 0.55 M GdnHCl ℃

Protein

Time constant τ (s) Relative amplitudes (%)

fast medium slowVery slow

fast medium slowVery slow

RNase A0.08

± 0.031.6

± 0.343 ± 5

186± 34

15 ± 714

± 0.349 ± 9 22 ± 2

ONC ·2.6

± 0.369

± 17· · 74 ± 8 26 ± 8 ·

ANG0.09

± 0.021.5

± 0.255 ± 3

334± 60

7 ± 3 17 ± 5 43 ± 533

± 10

Lovy Pradeep, Hang-Cheol Shin and Harold A. Scheraga

at pH 8, r.t. and 0.4 M GdnHCl

Protein Time constant τ (s)

τ 1 τ 2 τ 3 τ 4 τ 5

ANG 0.0825 1.40 6.76 28.1 111

des mutants

The effect of disulfide bonds on the folding rate of ANG ?

The parameters The parameters for for conformational conformational folding kinetics folding kinetics

Eun-Hye Ko, Harold A. Scheraga and Hang-Cheol Shin

Page 18: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Comparison ofComparison of the structural the structural stability stability & folding behavior & folding behavior bbyy a difference of sequence a difference of sequence

Page 19: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

rat Angiogenin (rat-ANG)rat Angiogenin (rat-ANG)

Has 122 amino acid residues with three disulfide bonds (Cys27-Cys81, Cys40-Cys92 and Cys58-Cys107)

60 % sequence identity with b-ANG

Page 20: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Expression, purification Expression, purification and CD analysis and CD analysis of rat-ANGof rat-ANG

SDS-PAGE of rat-ANG

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- 45

- 35

- 25

- 15

- 5

5

190 200 210 220 230 240 250

Wavelength (nm)

CD

(m

deg)

M 1 2 3 4 5 6

97.4 kD66.2 kD

45 kD

31 kD

21.5 kD

14.4 kD

b-ANG (●, Tm = 65 ℃) and rat-ANG (●, Tm = 67 ℃)

- 40

- 35

- 30

- 25

- 20

- 15

35 45 55 65 75 85

Temperature (℃)

CD

(m

deg)

Expression level : < 10 %

Page 21: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

AcknowledgementsAcknowledgements

Soongsil Univ.

Prof. Hang-Chol Shin

Seung-Hwan Jang, Ph.D.

Eun-Hye Ko

Hyang-Do Song

Cornell Univ.

Prof. Harold A. Scheraga

Chungbuk Natl. Univ.

Prof. Soo-Ik Chang

Dong-Ku Kang

Page 22: The role of disulfide bonds on the activity, stability and  folding  of bovine-angiogenin

Protein engineering & design Protein engineering & design LabLab.

Prof. Hang-Chol ShinSeung-Hwan Jang, Ph.D. Yeon-Hee Park, Ms.

Eung-Yoon Kim, MsC. Hyo-Jin Kim, MsC. Hyang-Do Song, MsC. Eun-Hye Ko, MsC.

Yong-Kyu Kim, MsC. Ha-A-Rin Jeon, MsC. Hye-Ran Hyun, MsC. Him-Chan Na, MsC.

http://bioinfo.ssu.ac.kr/~proshinhttp://bioinfo.ssu.ac.kr/~proshin


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