Structural-Functional Evaluation of Synthetic Tityus stigmurus Toxins Lacking Native Disulfide Bonds

Introduction: Voltage-gated sodium channels (Nav) are proteins that regulate the flow of Na+ in cells, being essential for depolarization and the initiation of the action potential. Scorpion venom, which causes millions of accidents annually, contains peptides that interact with ion channels, including Nav channels. These peptides are classified as NDBPs and DBPs, with α-toxins and β-toxins influencing the activity of sodium channels. This study aims to synthesize, purify and characterize the toxins Tst1 and Tst3, from the scorpion Tityus stigmurus, with a reduced number of disulfide bonds (from 4 to 1).

Objectives: To perform solid-phase chemical synthesis of Tst1 and Tst3 with reduced disulfide bonds, purify the synthetic peptides, verify their secondary structure via circular dichroism, and characterize their electrophysiological activity on Nav1.4 channels.

Methodology: The modified toxins were produced using standard solid-phase peptide synthesis (SPPS). The crude peptides were subsequently purified using reverse-phase high-performance liquid chromatography (RP-HPLC) to isolate the target molecules. Molecular masses were confirmed by MALDI-TOF mass spectrometry. Secondary structure conformational analysis was performed using Circular Dichroism (CD) spectroscopy. Finally, biological activity was assessed using patch-clamp electrophysiology on cells expressing Nav1.4 channels.

Results: The modified toxins were successfully purified in two RP-HPLC steps. MALDI-TOF analysis confirmed their identities: Tst1 presented masses of [M+H]+ = 6934.29 Da and [M+2H]2+ = 3469.60 Da, while Tst3 presented masses of [M+H]+ = 7308.40 Da and [M+2H]2+ = 3652.45 Da. Both synthetic toxins were tested at a concentration of 10 μM on Nav1.4 channels to verify their activities on a subtype affected by the natural toxins; however, they did not present any effect on these channels.

Conclusion: The successful synthesis of Tst1 and Tst3 analogs with only one disulfide bond enabled a clear structural-functional evaluation. The complete loss of activity on Nav1.4 channels indicates that removing the three native disulfide bonds disrupts the three-dimensional conformation needed for target interaction. This highlights the crucial role of the intact multiple disulfide bridge framework in maintaining the stability and biological activity of these scorpion toxins.