3.5 Protein interaction analysis
Based on the analysis results of the predicted protein (Figure 5) and phylogenetic tree (Supplements Figure S2), we finally selected some genes (CSP1, CSP5, CSP10, and OBP17) of the yellow peach moth for protein expression. Subsequently, the expressed proteins were tested for interaction with GOBP1 and GOBP2 proteins using SPR and ITC. The SPR results showed GOBP1 could bind with CSP5 and CSP10; GOBP2 can bind CSP5, CSP10, and OBP17 (Supplements Figure S3). The two proteins cannot bind to each other, and neither can bind to CSP1 (Supplements Figure S3). Multi-concentration SPR determination more accurately showed GOBPs and ligands’ binding ability (Table 2 and Figure 6). The KD of GOBP1 reaction with CSP5 (0.62 μM) and CSP10 (0.26 μM) are more than GOBP2 (0.34 μM and 0.08 μM), respectively, which indicating that GOBP2 has the more vital binding ability with ligands.
The results of ITC are shown in Figure 7. Each peak in the upper part represents the heat generated by a single titration of the same volume of small molecules into the protein solution, and the lower part is the protein binding nonlinear fitting graph. After the first titration, the peak heat value reaches the lowest and then gradually rises. After calculation, the KD of GOBP1 reaction with CSP5 (1.70 μM) and CSP10 (2.04 μM) are more than GOBP2 (1.39 μM and 1.87 μM), respectively. Those results are consistent with the results of SPR that GOBP1 can directly interact with CSP5 and CSP10, and GOBP2 can directly interact with CSP5, CSP10, and OPB17. However, GOBP1 and GOBP2 cannot directly interact. In addition, all the reaction ΔG < 0 and |△H| > T|△S| are spontaneous chemical reactions and this reaction by enthalpy-driving (Table 2).