Proton magnetic resonance characterization of phoratoxins and homologous proteins related to crambin.
The mistletoe protein toxins ligatoxin, phoratoxins A and B, and viscotoxins A3 and B have been investigated by 1H NMR spectroscopy at 300 and 600 MHz. The five polypeptides define a set of closely related homologues, containing 46 amino acid residues each, in a structure constrained by three cystine bridges. Their methyl and aromatic spectra were analyzed and a number of signals identified and assigned via comparative criteria, two-dimensional chemical-shift correlated spectroscopy, acid-base titration, and proton Overhauser experiments in 1H2O. The spectra indicate a compact globular conformation and a common folding pattern for the toxins. In particular, use was made of well-resolved aliphatic and aromatic resonances in order to compare the mistletoe proteins with the thionins, a set of homologous toxins from gramineae, and with crambin, a closely related polypeptide from a crucifer, which we have previously studied by NMR. We observe that while all the investigated proteins have very similar secondary and tertiary structures, they differ widely in their dynamic characteristics as probed 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 groups showing exchange half-lives that extend up to several days at pH* 5.8 or that are too long to be measurable at ambient temperature. The temperature dependence of the 1H NMR spectrum also indicates that the toxins are endowed with a thermally very stable native (ground-state) structure, with little evidence of large amplitude structural breathings up to approximately 370 K, although irreversible chemical degradation (denaturation) becomes evident at temperatures greater than or equal to 350 K. It is concluded that the mistletoe toxins afford valuable rigid structures for NMR conformational studies.
D000595:Amino Acid Sequence; D004220:Disulfides / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D014784:Mistletoe; D028321:Plant Preparations; D010940:Plant Proteins; D010946:Plants, Medicinal; D011487:Protein Conformation; D054790:Ribosome Inactivating Proteins, Type 2; D013329:Structure-Activity Relationship; D014118:Toxins, Biological
D004220:Disulfides; D028321:Plant Preparations; D010940:Plant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C009674:ribosome inactivating protein, Viscum; C008873:phoratoxin protein, Phoradendron tomentosum; C024934:crambin protein, Crambe abyssinica; C488038:viscotoxin