1H NMR Study of the Solution Properties of the Polypeptide Neurotoxin I from the Sea Anemone Stichodactyla helianthus

Raymond S. Norton, Anne I. Cossins, William R. Kem

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Abstract

The solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I) have been investigated by high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz. The pH dependence of the spectra has been examined over the range 1.1–12.2 at 27 °C. Individual pKa values have been obtained for the α-ammonium group of Ala-1 (8.6) and the side chains of Glu-8 (3.7), Tyr-36 (10.9), and Tyr-37 (10.8). For the remaining seven carboxyl groups in the molecule (from five Asp, Glu-31, and the C-terminus), four pKa values, viz., 2.8, 3.5, 4.1 and 6.4, can be clearly identified. The five Lys residues titrate in the range 10.5–11, but individual pKa values could not be obtained because of peak overlap. Conformational changes associated with the protonation of carboxylates occur below pH 4, while in the alkaline pH range major unfolding occurs above pH 10. The molecule also unfolds at elevated temperatures, having a transition temperature of ca. 55 °C at pH 5.25. Exchange of the backbone amide protons has been monitored at various values of pH and temperature in the ranges pH 4–5 and 12–27 °C. Up to 18 slowly exchanging amides are observed, consistent with the existence of a core of hydrogen-bonded secondary structure, most probably β-sheet. Comparison of these properties of Sh I in solution with those of the related polypeptides anthopleurin A and Anemonia sulcata toxins I and II indicates that Sh I is less stable thermally and that there are some significant differences in the ionic interactions that maintain the tertiary structure. The solvent accessibility of aromatic residues has been probed with photochemically induced dynamic nuclear polarization NMR at 360 MHz. The aromatic rings of Trp-30 and both Tyr residues are accessible to flavin dye, but Tyr-37 shows a weaker response then Tyr-36, suggesting that Tyr-37 is partially shielded.

Original languageEnglish
Pages (from-to)1820-1826
Number of pages7
JournalBiochemistry
Volume28
Issue number4
DOIs
Publication statusPublished - 1989
Externally publishedYes

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