Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Abstract

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine-rich snakin-1 and -2 antimicrobial peptides by using a combination of solid-phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40-50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin-2 compared to natural snakin-2, we demonstrated that synthetic snakin-2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin-2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

“…6, F. oxysporum was highly susceptible to recombinant SN-1 and spore germination was completely inhibited with 60 μM of SN-1. This result was in accordance with the findings described in previous reports for the potato native SN-1 and chemically synthetic SN-1 [19,24].…”

“…Numerous reports have documented the strong antimicrobial activities of snakin peptides against a wide range of microorganisms [11,19,21,24,26,27]. Enhanced resistance to pathogen infections has also been reported in plants that express snakin genes [28,29].…”

“…All snakin peptides are characterized by twelve conserved cysteine residues that are involved in the formation of six disulfide bonds [19]. These disulfide bonds are thought to be important for maintaining their tertiary structure and also responsible for their biological activity [13,24,25]. In previous studies, snakin peptides from potato (Solanum tuberosum) were found to exert strong antimicrobial activity against phytopathogens [11,19,24,26] and animal pathogens [27].…”

“…Similarly, chemical synthesis has been used to produce disulfide-rich SN-1 and SN-2 peptides [24] in an inactive, unfolded state that required laborious oxidative refolding and purification techniques to recover the functionally active peptides. Recently, the SN-2 peptide from tomato (Solanum lycopersicum) was successfully expressed in E.coli as a thioredoxin fusion protein in the active form, although the yield was approximately 1 mg/L [21].…”

One new kind of phytohormonal signaling integrator: Up-and-coming GASA family genes

“…37 The disulfide pattern of the synthetic Snakin-2 protein, in which 3 pairs of disulfide bonds has been determined as CysIII-CysIV, CysVIII-CysXI and CysIX-CysX, is different slightly with that of the Snakin-1 protein. 38 The previous researches have shown that cysteine disulfide bond formation mode affects the spatial protein structure and function directly. 39 Normally, GASA proteins form 5 or 6 disulfide bonds and these disulfide bonds may be necessary for the senior structural of GASA and be critical for the GASA protein interactions with other proteins.…”