The lane headings Proteases inhibitor showed the time post-infection in hours. Co-localization of host AST, GroEL and viral VP371 proteins during bacteriophage infection To characterize the VP371-GroEL-AST interactions during GVE2 infection, these three proteins were labeled and examined using immunofluorescence microscopy. The results indicated that the host AST, GroEL, and viral VP371 proteins were co-localized
in the GVE2-infected Geobacillus sp. E263 (Figure 3A). In the virus-free Geobacillus sp. E263, however, the AST and GroEL were bound to each other (Figure 3A), while no signal was observed in the GST control and no obvious co-localization was found between the GST-MreB control and GroEL proteins (Figures 3B and 3C). Considering Dibutyryl-cAMP mouse the importance of the VP317 and AST proteins in the GVE2 infection [5, 25], the immunofluorescence microscopy results suggested that the VP371-
GroEL-AST complex might be involved in the bacteriophage infection in high temperature environment. Figure 3 Co-localization of host aspartate aminotransferase (AST), GroEL, and viral VP371 in Geobacillus LY2874455 purchase sp. E263. The host bacteria were challenged with GVE2. At different time post-infection, the GVE2-infected Geobacillus sp. E263 was labeled with the antibodies against the AST, GroEL, or VP371 (A). The GST (B) and the GST-MreB (C) were used as controls to detect the nonspecific co-localization with GroEL at 2 h post-infection. The bacteria were examined under a fluorescence microscope. The lane headings indicated the labeled proteins. The numbers showed the time post-infection in hours. Thermodynamic characterization of the VP371-GroEL-AST interactions The binding properties of the interactions in the VP371-GroEL-AST linear complex were characterized by ITC. Figure 4 showed a thermogram for all 3 kinds of protein–protein combinations and binding isotherms only for the valuable interaction (AST-GroEL or VP371-GroEL).
Figure 4 Thermodynamic characterization of the VP371-GroEL-aspartate aminotransferase (AST) interactions. The purified proteins of VP371-GroEL-AST linear complex and GST as control group were combined for isothermal titration calorimetry to measurements. The experiment was performed at 25°C in phosphate buffered saline (pH 7.4) with 10-μL injections. (A) Thermogram (left) and binding isotherm (right) for the interaction between AST and GroEL. Concentrations of AST and GroEL were 44.5 and 8.5 μM, respectively. (B) Thermogram (left) and binding isotherm (right) for the interaction between VP371 and GroEL. Concentrations of VP371 and GroEL were 38.5 and 6.5 μM, respectively. (C) Thermogram for the titrations of 38.5 μM VP371 to 7 μM AST, 44.5 μM AST to 8.5 μM GST, 38.5 μM VP371 to 6.5 μM GST, and 44.5 μM GST to8.5 μM GroEL. (D) Thermodynamic parameters for binding of aspartate aminotransferase-GroEL and VP371-GroEL at different temperatures. All experiments were performed in phosphate buffered saline (pH 7.4) using isothermal titration calorimetry.