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
