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

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)

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

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?

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

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

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 %

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

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]

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

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

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

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 %

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

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.

Representative Representative data curves data curves

of b-ANGof b-ANG

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

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

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

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

SDS-PAGE of rat-ANG

- 55

- 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 %

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

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