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

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).

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

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  1. 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

  2. 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)

  3. 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

  4. 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?

  5. Role of disulfide bonds on the structure, stability & function

  6. des mutants of b-ANG • [C27S, C82S],[C40S, C93S],and [C58S, C108S], respectively, were produced in E. coli • Denaturation & Refolding • Purification : Cation-exchange chromatography • Biophysical analysis : Circular dichroism, Stopped-flow • Biological (Angiogenesis) assays : CAM assay, Wound healing migration

  7. 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 97.4 kD 66.2 kD 45 kD 31 kD 21.5 kD 14.4 kD [C27S, C82S] [C40S, C93S] [C58S, C108S] Expression & purification of des mutants SDS-PAGE of des mutants of b-ANG Expression level : 35 %

  8. CD analysis • [C27S, C82S] & [C58S, C108S] : much less structured • [C40S, C93S] : native-like, similar to the wild type w/t (●), [C27S, C82S] (●), [C40S, C93S] (●), and [C58S, C108S] (●)

  9. [C58S, C108S] wild type [C27S, C82S] [C40S, C93S] Tm analysis

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

  11. Biological assays CAM assay Wound healing migration Angiogenic activities of w/t and des mutants of b-ANG (CAM assay & wound healing migration)

  12. Role of disulfide bonds no or slight effect on the structure medium effect for the stability & biological function 40-93 critical for the stability & structure 58-108 27-82 critical for the biological function no effect on the biological function

  13. 1 2 3 4 5 2 3 4 5 2 3 4 5 97.4 kD 66.2 kD 45 kD [C40-C93] [C58-C108] [C27-C82] Under investigation:double des mutants Using PDI fusion system for soluble expression Expression level : 35~40 % SDS-PAGE of double des mutants of b-ANG

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

  15. Stopped-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.

  16. Representative data curves of b-ANG

  17. at pH 8, 15 ℃ and 0.55 M GdnHCl at pH 8, r.t. and 0.4 M GdnHCl The parameters for conformational folding kinetics Eun-Hye Ko, Harold A. Scheraga and Hang-Cheol Shin Lovy Pradeep, Hang-Cheol Shin and Harold A. Scheraga

  18. Comparison ofthe structural stability & folding behavior by a difference of sequence

  19. 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

  20. M 1 2 3 4 5 6 97.4 kD 66.2 kD 45 kD 31 kD 21.5 kD 14.4 kD Expression, purification and CD analysis of rat-ANG SDS-PAGE of rat-ANG Expression level : < 10 % b-ANG (●, Tm = 65 ℃) and rat-ANG (●, Tm = 67 ℃)

  21. 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 Acknowledgements

  22. http://bioinfo.ssu.ac.kr/~proshin Protein engineering & design Lab. • Prof. Hang-Chol Shin • Seung-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.

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