Fig. 5
From: A strategy to characterize chlorophyll protein interaction in LIL3
LIL3 mutants resolve Chl a binding to N174. Lil3 mutants E171A (a), R176A (b), and N174A (c) were solubilized in DDM micelles (6 mM) at increasing protein concentrations 0.61 nM–2.5 µM in the presence of a constant concentration (120 nM) of Chl a. The fluorescence difference from three MST measurements was plotted against the LIL3 mutant concentrations. Recombinant LIL3 WT and mutants were reconstituted with Chl a and assay components were isolated using native 3–12% LN-PAGE. The mobility of Chl a in native gels was determined by laser excitation/emission scanning at 680/700 nm (d). The mobility of LIL3 WT and mutants in the gels were determined by in-gel staining using colloidal Coomassie (d). Lane numbers refer to native mark (1), and reconstitution assays containing LIL3 (30 µM), Chl a (6 µM). Lanes: Native Mark (1), Chl a (2), LIL3.2 WT (3), LIL3.2 WT reconstituted (4), LIL3.2 E171A (5), LIL3.2 E171A reconstituted (6), LIL3.2 N174A (7), LIL3.2 N174A reconstituted (8), LIL3.2 R176A (9), LIL3.2 R176A reconstituted (10)