H-1 NMR study of robustoxin, the lethal neurotoxin from the funnel web spider Atrax robustus

Robustoxin, the lethal neurotoxin from the Sydney funnel web spider Atr ns robustus, is a polypeptide of 42 residues cross-linked by four disulfide bo nds. This paper describes the sequence-specific assignment of resonances in the H-1 nuclear magnetic resonance spectrum of robustoxin in aqueous solut ion. Several broad backbone amide resonances were encountered in spectra re corded at 27 degrees C, making the assignments at that temperature incomple te. In spectra recorded at lower temperatures these amide resonances became sharper, but others that were sharp at 27 degrees C became broad, indicati ve of conformational averaging on the millisecond timescale for certain reg ions of the structure. Nevertheless, it was possible to establish that robu stoxin contains a small, triple -stranded, antiparallel beta-sheet and seve ral reverse turns, but no alpha-helix. These observations indicate that thi s toxin may adopt the inhibitor cystine knot structure found in polypeptide s from a diverse range of species, including a number of spiders. Analysis of the pH dependence of the spectrum yielded pK(a) values for Tyr22 and Tyr 25, one of the three carboxyl groups, and the Lys residues. (C) 1999 Elsevi er Science Ltd. All rights reserved.