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  • Title: Conformational studies of the 'RGD' containing peptide echistatin and close analogs by circular dichroism and fluorescence.
    Author: Brockel C, Cowley DJ, Pelton JT.
    Journal: Biochim Biophys Acta; 1992 Jul 31; 1122(2):196-202. PubMed ID: 1643093.
    Abstract:
    Echistatin, one of the smallest and most active natural disintegrins, and its [Trp13]echistatin, [Trp27]echistatin, [Phe13,Trp31]echistatin analogs have been investigated by far-UV circular dichroism spectroscopy and fluorescence spectroscopy. All analogs inhibited ADP-stimulated platelet aggregation with EC50 values between 30 and 50 nM. The analogs were related closely, both in the CD spectral properties, characteristic of turn conformations, and in the location of isodichroic points connected to conformational transitions upon temperature increase. The low fluorescence quantum yield for Trp13 of 0.018, which could be enhanced 2.7-fold by DTT reduction of the peptide, is ascribed to a close proximity of this Trp13 residue to a disulfide bond. Calculation of the efficiency of fluorescence resonance energy transfer (FRET) yielded distances of 11.5 +/- 0.8 A for Tyr31-Trp27 in [Trp27]echistatin, and more than 15 A for Tyr31-Trp13 in [Trp13]echistatin, in good agreement with the structure of echistatin deduced from earlier NMR-molecular modeling studies. Both Trp13 and Trp27 in the respective analogs were quenched effectively by acrylamide with bimolecular quenching constants of 3.36 x 10(9) M-1 s-1 and 3.72 x 10(9) M-1 s-1, respectively. Iodide anion had negligible quenching effect on Trp13, despite high exposure of this residue to water, but was only 2-fold less efficient than acrylamide in quenching Trp27 fluorescence. Steady-state fluorescence anisotropy data, together with mean fluorescence lifetimes of 1.25 ns for Trp13 and 3.84 ns for Trp27 derived from full fluorescence lifetime decay analyses, yielded long rotational relaxation times of 1.39 +/- 0.18 and 1.35 +/- 0.17 ns, respectively, for these residues comparable to the expected overall rotation time of the peptides. The 'RGD'-containing loop appears to be restricted in movement on the nanosecond timescale with respect to the compact core of the peptide.
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