TY - JOUR
T1 - Structural analysis of a biologically active echistatin analogue des(46-49)-[Ala8,37]-echistatin γ with three disulfide bonds by 2D-NMR and computer graphics
AU - Chuang, Li Chin
AU - Chen, Pei Yeh
AU - Chen, Chinpan
AU - Huang, Tai Huang
AU - Wang, Kung Tsung
AU - Chiou, Shyh Horng
AU - Wu, Shih Hsiung
N1 - Funding Information:
This work was supported by Academia Sinica and the National Science Council, Taipei, Taiwan. We are grateful to Dr. Ming-Fai Tam at the Institute of Molecular Biology, Academia Sinica, for performing the mass spectrometry.
PY - 1996/3/18
Y1 - 1996/3/18
N2 - An echistatin analogue, designated as des(46-49)-[Ala8,37]-echistatin γ, was synthesized chemically by solid-phase peptide synthesis. The analogue was made by replacing Cys8 and Cys37 residues with two alanines and the deletion of C-terminal peptide 46-49 of echistatin γ, resulting in an artificial polypeptide of 45 amino acids with three disulfide bonds. In the platelet aggregation assay, the analogue exhibits almost the same activity as echistatin γ, indicating that the linear sequence of des(46-49)-[Ala8,37]-echistatin γ contains all of the primary-structure information that is required for proper folding of this synthetic polypeptide. The tertiary structure of the analogue, as determined from high-resolution nuclear magnetic resonance (NMR) coupled with dynamic simulated annealing, is very similar to that of echistatin α1 which differs from echistatin γ by 8 residues. In particular the two important sites of the Arg-Gly-Asp (RGD) loop and the C-terminal Lys45, both of which show some degree of disorder, are maintained in similar spatial orientation and proximity as those in echistatin al even without the constraint provided by the disulfide bond of the (Cys8-Cys37) pair. These results provide new insights in further defining distinct structural features of echistatin γ, which are involved in supporting the active polypeptide conformation to achieve biological activity in the absence of one pair of disulfide bonds.
AB - An echistatin analogue, designated as des(46-49)-[Ala8,37]-echistatin γ, was synthesized chemically by solid-phase peptide synthesis. The analogue was made by replacing Cys8 and Cys37 residues with two alanines and the deletion of C-terminal peptide 46-49 of echistatin γ, resulting in an artificial polypeptide of 45 amino acids with three disulfide bonds. In the platelet aggregation assay, the analogue exhibits almost the same activity as echistatin γ, indicating that the linear sequence of des(46-49)-[Ala8,37]-echistatin γ contains all of the primary-structure information that is required for proper folding of this synthetic polypeptide. The tertiary structure of the analogue, as determined from high-resolution nuclear magnetic resonance (NMR) coupled with dynamic simulated annealing, is very similar to that of echistatin α1 which differs from echistatin γ by 8 residues. In particular the two important sites of the Arg-Gly-Asp (RGD) loop and the C-terminal Lys45, both of which show some degree of disorder, are maintained in similar spatial orientation and proximity as those in echistatin al even without the constraint provided by the disulfide bond of the (Cys8-Cys37) pair. These results provide new insights in further defining distinct structural features of echistatin γ, which are involved in supporting the active polypeptide conformation to achieve biological activity in the absence of one pair of disulfide bonds.
UR - http://www.scopus.com/inward/record.url?scp=0029881175&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029881175&partnerID=8YFLogxK
U2 - 10.1006/bbrc.1996.0422
DO - 10.1006/bbrc.1996.0422
M3 - Article
C2 - 8645291
AN - SCOPUS:0029881175
SN - 0006-291X
VL - 220
SP - 246
EP - 254
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -