The conformational properties of the pentapeptide Ser-Phe-Leu-Leu-Arg (P5), a human thrombin receptor-derived sequence forming part of a tethered ligand which activates the thrombin receptor, and its more active amide derivative Ser-Phe-Leu-Leu-Arg-NH
2 (P5-NH
2), have been studied by proton NMR spectroscopy in dimethylsulfoxide. Measurements of nuclear Overhauser effects, performed using two-dimensional rotating frame nuclear Overhauser (ROESY) and one-dimensional nuclear Overhauser enhancement (NOE) spectroscopy, revealed that P5 exists mainly in an extended conformation. However, proton–proton 1D-NOEs between Phe C
αH and Ser C
αH, Leu
3 C
αH and Leu
3 NH, and Leu
4 C
αH and Leu
4 NH, as well as between the Ser and Arg sidechains, also implicated a minor conformer for P5 having a curved backbone and a near-cyclic structure. In contrast to P5, measurements of NOEs and ROEs for P5-NH
2 revealed a more stabilized cyclic structure which may account for its higher biological potency. Thus strong interresidue sequential NH (
i)–NH (
i + 1) interactions, as well as C-terminal carboxamide to N-terminal side-chain interactions, i.e., Arg CONH
2 to Phe ring and Arg CONH
2 to Ser
$C_\alpha /C_{\beta \beta '} $ , observed at lower levels of the ROESY spectrum, supported a curved backbone structure for SFLLR-NH
2. Since the higher potaency P5-NH
2 analogue adopts predominantly a cyclic structure, a cyclic bioactive conformation for thrombin receptor agonist peptides is suggested.
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