Conformational study of a new SGTx1 neurotoxin from the spider Scodra griseipes using mass spectrometry

Abstract

SGTx1 is a new neurotoxin from the venom of Scodra griseipes. Because of the small quantity of this natural peptide available, mass spectrometry was used to obtain information on its higher-order structure. The kinetics of reduction by 1,4-dithiothreitol (DTT) was monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and showed that one of the three disulfide bridges was appreciably more accessible to the DTT. Studies based on the charge state distribution (CSD) and H/D exchange of the non-reduced peptide, under neutral and acidic conditions, were performed using electrospray mass spectrometry (ES-MS). In neutral solution, SGTx1 showed a maximum charge state of four compared with seven potentially protonated basic residues, and all labile hydrogens were exchanged. However, under acidic conditions, a maximum charge state of only five was observed, and four of the labile hydrogens could not be deuterated. These observations are interpreted in terms of a rigid structure maintained by the disulfide bridges, which can be temporarily relaxed by disulfide bridge scrambling only at higher pH values.

Conformational Properties of α-Dendrotoxin Using Electrospray Mass Spectrometry

“…Thus, we have shown here a dramatic difference in CSD between disulfideintact and disulfide-reduced peptides. Such differences have already been shown for ω-conotoxins 36 and SGTx1 toxin, 37 which contain only 26 and 34 amino acids, respectively. An alternative explanation could involve Coulombic forces at acidic pH in solution.…”

“…Optimization of the parameters was obtained for water + methanol (80 / 20,v : v) at pH 7. 36,37 The ES ionization source temperature was also optimised at 100°C. In fact, at 80°C the spectrum was much too noisy and there was no difference in the CSD, centered at 8+, between these two temperatures.…”

“…The extraction cone voltage was chosen at a middle value of 150 V. A high value of this voltage (250 V) shifts the envelope of the multiply-charged ions towards a low CSD (5+), with a much better transmission, in contrast to a low value of extraction cone voltage (50 V), which shifts the ions to a higher CSD (7+). 36,37…”

“…[4][5][6][7][8][9] So, examination of CSD in positive-ion ES might be a good way to measure the number of charges corresponding to the number of ionized basic residues. Nevertheless, it has been shown that the observation of CSD might be very different 36,37 when the structure presents multiple disulfide bridges and a large number of basic amino acids. α-Dendrotoxin is a 59-amino-acid, three-disulfide-bridged, peptide (calculated average mass 7048.1 Da), including 15 basic residues (eight Arg, six Lys and one His).…”

“…7,33 H/D exchanges are slow when the hydrogen of an amino acid residue is either buried in an hydrophobic core or involved in hydrogen bonding, such as α-helices or β-sheets, or the formation of ionic linkages. Such H/D exchange studies have not been realized with multiple disulfide-bridged proteins, except for proinsulin, 34 pancreatic trypsin inhibitor, 34 ribonuclease, 31,32 lysozyme, 35 conotoxin 36 and a new SGTx1 toxin of the venom of Scodra griseipes tarantula 37 (three and four disulfide bridges).…”