Proton magnetic resonance characterization of phoratoxins and homologous proteins related to crambin
Journal/Book: Biochemistry 26 (4), 1187-1194. 1987;
Abstract: The mistletoe protein toxins ligatoxin, phoratoxins A and B, andviscotoxins A3 and B have been investigated by 1H NMR spectroscopy at300 and 600 MHz. The five polypeptides define a set of closely relatedhomologues, containing 46 amino acid residues each, in a structureconstrained by three cystine bridges. Their methyl and aromatic spectrawere analyzed and a number of signals identified and assigned viacomparative criteria, two-dimensional chemical-shift correlatedspectroscopy, acid-base titration, and proton Overhauser experiments in1H2O. The spectra indicate a compact globular conformation and a commonfolding pattern for the toxins. In particular, use was made ofwell-resolved aliphatic and aromatic resonances in order to compare themistletoe proteins with the thionins, a set of homologous toxins fromgramineae, and with crambin, a closely related polypeptide from acrucifer, which we have previously studied by NMR. We observe that whileall the investigated proteins have very similar secondary and tertiarystructures, they differ widely in their dynamic characteristics asprobed by the amide NH 1H-2H exchange kinetics in deuteriated solvents;thus, while crambin and the thionins exhibit very fast isotope exchange,the kinetics for the mistletoe toxins are slow, with some NH groupsshowing exchange half-lives that extend up to several days at pH* 5.8 orthat are too long to be measurable at ambient temperature. Thetemperature dependence of the 1H NMR spectrum also indicates that thetoxins are endowed with a thermally very stable native (ground-state)structure, with little evidence of large amplitude structural breathingsup to approximately 370 K, although irreversible chemical degradation(denaturation) becomes evident at temperatures greater than or equal to350 K. It is concluded that the mistletoe toxins afford valuable rigidstructures for NMR conformational studies. Author.