The secondary structure of echistatin from 1H‐NMR, circular‐dichroism and Raman spectroscopy

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Abstract
Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20°C, echistatin contains no α-helix but contains mostly β-turns and β-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40°C and 72°C being irreversible. Raman spectra also indicate considerable β-turn and β-sheet (20%) structure and an absence of α-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27°C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either α-helical or β-sheet conformation. A number of turns could be characterised. An irregular β-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.