Journal: PLoS ONE
Article Title: The Terminal Immunoglobulin-Like Repeats of LigA and LigB of Leptospira Enhance Their Binding to Gelatin Binding Domain of Fibronectin and Host Cells
Figure Lengend Snippet: Determination of binding constant, kinetics and thermodynamic parameters of the LigBCen2R/GBD interaction by ELISA, SPR, and ITC. (A) Binding of serial concentrations of LigBCen2NR to immobilized GBD by ELISA. Serial concentrations of GST-LigBCen2R, GST-LigBCen2NR, GST-LigBCen2 (positive control), or GST-LigCon (negative control) were added to 1 µM of GBD or BSA coated wells (negative control, data not shown). (B) SPR analysis of LigBCen2R interacting with GBD. 1.5 µM of Recombinant Histidine-tagged LigBCen2R was immobilized on the surface of a Ni-NTA chip. GBD in Tris Buffer containing 100 µM CaCl2 at pH 7.5 flowed through the chip and the concentrations of GBD ranged from 40 to 0.625 µM (from top to bottom). The KD, kon, and koff were obtained from the average of duplicate experiments shown in Table 1 . (C) Determination of the binding affinity by ITC. The cell contained 1 ml of GBD and the syringe contained 250 µl of LigBCen2R (upper panel). Heat differences obtained from 25 injections of LigBCen2R; (lower panel). Integrated curve with experimental data (⋄) and the best fit (-). The thermodynamic parameters are shown as the average of duplicate experiments (KD = 1.88±0.09 µM, ΔH = −29.25±3.58 kcal mol-1, TΔS = −21.47±3.61 kcal mol-1 K-1, n = 0.995±0.01). (D) Fluorescence spectrum of Alexa-488 labeled LigBCen2RW1073C in the presence and absence of GBD. One µM of Alexa-488 labeled LigBCen2RW1073C in Tris buffer was excited at 485 nm. Aliquots of GBD from respective stock solutions were added. The figure shows Alexa488 fluorescence in the presence of 0, 1.62, 3.12, 6.25, 12.5, 25 µM of GBD (Inner plot). The determination of KD of Alexa488 labeled LigBCen2RW1073C and GBD by monitoring the quenching fluorescence intensity of Alexa488 labeled LigBCen2RW1073C titrated by GBD. The emission wavelength recorded in this figure was 513 nm, and KD was revealed by fitting the data point into the equation described in materials and methods (KD = 1.93.4 µM).
Article Snippet: Surface Plasmon Resonance (SPR) Association and dissociation rate constants for the interaction of Lig proteins and GBD were measured by SPR analysis performed with a Biacore 2000 instrument (GE Healthcare) at 25°C.
Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Positive Control, Negative Control, Recombinant, Chromatin Immunoprecipitation, Fluorescence, Labeling