Cyclization of Short Peptides Designed from Late Embryogenesis Abundant Protein to Improve Stability and Functionality

Abstract

LEA peptides, which are designed based on late embryogenic abundant (LEA) protein sequences, have demonstrated chaperone-like functions, such as improving drought stress tolerance of Escherichia coli (E. coli). Previous studies have focused on the biological functions of linear LEA peptides. However, the function of cyclic LEA peptide still unknown. This study aimed to explore the cyclic LEA peptides' bio function like enhance the drought stress tolerance of E. coli by cyclizing the LEA peptide using SICLOPPS (Split Intein Circular Ligation of Peptides and Proteins). The results indicated that cyclization significantly improved the function and extended the potential applications. At the same time, we found that peptides containing numerous lysine residues exhibited reduced performance, which may be due to the exteins' residues affecting the SICLOPPS efficiency.

Keywords: LEA peptide; SICLOPPS; cyclization; drought stress.

Figure 1

Cyclizing LEA peptides by SICLOPPS. The linear and cyclic LEA peptides’ capacity to improve cell desiccation tolerance was comparatively analysed. Large cyclic LEA peptides were also produced by extending the LEA model to compare the small and large cyclic LEA peptides′ functions.

Figure 2

Desiccation resistance test results. (a) Drop spotting analysis of C−K desiccation resistance under different Ara concentrations; X is the control sample, transformed with empty pBAD and induced by 0.2 % Ara. (b) Drop spotting analysis of C‐II desiccation resistance under different Ara concentrations. (c) Growth curve of X, L−K, C−K, L‐II, and C‐II under 0.002 % Ara induction (n=3). (d) and (e) Drop spotting images of samples before and after desiccation when induced by 0.002 % Ara.

Figure 3

Results of desiccation resistance test. (a) The desiccation survival rate of linear versus cyclic LEA peptide samples. Data are mean±SD (n=5~8). (b) The desiccation survival rate of larger cyclic LEA peptide samples. Data are mean±SD (n=5). *, significance at P <0.05; **, significance at P<0.01; ns, not significant.

Figure 4

SICLOPPS cleavage and cyclization processes. A schematic representation of SICLOPPS’ splitting and cyclization mechanism. B the predictive structure of the C‐II precursor, the split‐intein is dawn as ribbon (IN: yellow; IC: red) and the C‐II peptide is green. C, the predictive structure of the C−K precursor, the split‐intein is dawn as ribbon (IN: yellow; IC: red) and the C−K peptide is blue. (a, b, c, d) The side view of active groups of the C‐II splitting and ligation process, the relative active groups are shown in red letters on the above scheme. (f, g, h, i) The side view of active groups of C−K splitting and ligation process, the presenting order same as C‐II. (e) The structure of cyclic LEA II. (j) The structure of cyclic LEA K. All screenshots are 0 ns of conformations’ molecular dynamic simulation; red solid line, the distance of active groups, red dot line, strong interaction.

Figure 5

Molecular dynamic simulation of the branched intermediate step. (a) Overview of C‐II structure. (b), (c), and (d) are the site views of C‐II conformations at 0, 100, and 350 ns, respectively. (e) Overview of C‐K structure. (f), (g), and (h) are the site views of C‐K conformations at 0, 100, and 350 ns, respectively. Blue solid line, the distance of active groups; red dot line, strong interaction.