the role of disulfide bonds on the activity, stability and folding of bovine-angiogenin
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The role of disulfide bonds on the activity, stability and folding of bovine-angiogenin. Eun-Hye Ko Dept. of Bioinformatics & Life Science, and Protein engineering & Design Lab. Soongsil University. Angiogenin (ANG). - PowerPoint PPT PresentationTRANSCRIPT
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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
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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)
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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
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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?
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Role of disulfide bonds on the Role of disulfide bonds on the structure, stability & functionstructure, stability & function
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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
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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 %
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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
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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]
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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
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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
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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
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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 %
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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
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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.
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Representative Representative data curves data curves
of b-ANGof b-ANG
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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
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Comparison ofComparison of the structural the structural stability stability & folding behavior & folding behavior bbyy a difference of sequence a difference of sequence
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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
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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 %
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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
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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