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Stability of protein‐bound conformations of bioactive peptides: The folded conformation of an epidermal growth factor‐like thrombomodulin fragment is similar to that recognized by thrombin
Author(s) -
Song Jianxing,
Xu Ping,
Koutychenko Anatol,
Ni Feng
Publication year - 2002
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.10253
Subject(s) - chemistry , antiparallel (mathematics) , thrombomodulin , peptide , thrombin , stereochemistry , peptide conformation , turn (biochemistry) , crystallography , protein structure , epidermal growth factor , side chain , biophysics , biochemistry , receptor , platelet , physics , organic chemistry , quantum mechanics , biology , magnetic field , immunology , polymer
The relationship between the free and bound conformations of bioactive peptides is explored using the epidermal growth factor (EGF)‐like thrombomodulin fragment hTM409–426 as a model system. The hTM409–426 peptide has a sequence of C 409 PEGYILDDGFIC 421 TDIDE (with a disulfide bond between Cys409 and Cys421) and is a selective inhibitor of thrombin. Upon binding to thrombin, hTM409–426 adopts a well‐defined conformation—namely, a β‐turn followed by an antiparallel β‐sheet, similar to those found in all other EGF‐like protein repeats (Hrabal et al., Protein Science , 1996, Vol. 5, 195–203). Here we demonstrate that, at pH 6.8 and at 25°C, the hTM409–426 peptide in the free state is very flexible, but still populates a type II β‐turn over residues Pro410–Glu411–Gly412–Tyr413 and the clustering of some hydrophobic side chains, both of which are present in the thrombin‐bound conformation. At a lower temperature of 5 °C, significant conformational shifts of the CαH proton resonances and extensive medium‐ and long‐range NOEs are observed, indicating the presence of folded conformations with unique backbone–backbone and side‐chain interactions. A comparison of the NOE patterns in the free state with transferred NOEs shows that the free‐state folded and the thrombin‐bound conformations of the hTM409–426 peptide are very similar, particularly over residues Pro410–Ile424. The folded conformation of hTM409–426 appears to be stabilized by two hydrophobic clusters, one formed by the side chains of residues Pro410, Tyr413, Leu415, and Phe419 and the Cys409–Cys421 disulfide bond, the second involving residues Ile414 and Ile424. These results indicate that the overall topology of the thrombin‐bound conformation of the hTM409–426 peptide is prefolded in the free state and the primary sequence (including the disulfide bond) may be selective for an ensemble of conformations similar to that recognized by thrombin. Published 2002 Wiley Periodicals, Inc. Biopolymers 65: 373–386, 2002