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. 2014 Dec 5;464(Pt 3):315–322. doi: 10.1042/BJ20140463

Figure 5. Gun4 is able to associate with and stimulate activity of ΔN159ChlH.

Figure 5

(A) FRET association experiment between Gun4 and WT ChlH (●) or ΔN159ChlH (○). Assays contained 0.1 μM ChlH, in 50 mM Tricine/NaOH, 0.3 M glycerol and 200 mM NaCl, pH 7.9, at 45°C. ChlH was labelled with Atto 488 and Gun4 labelled with Alexa Fluor 532. The quenching of emission of Atto 488 was monitored at 524 nm. A.U.–arbitrary units. The curves can be described by eqn (1) with the concentration term, [ChlH], held at 0.1 μM with characterizing parameters: WT ChlH Kd=0.009±0.003 μM and Δ159ChlH Kd=0.016±0.004 μM. (B) Assembly titrations of MgCH between Gun4. Assays contained 0.1 μM ChlD, 0.2 μM ChlI, 0.4 μM ChlH in 50 mM Mops/KOH, 0.3 M glycerol, 1 mM DTT, 10 mM free Mg2+, I=0.1 with KCl and 8 μM DIX, pH 7.7, at 45°C. The curves can be described by eqn (2). nd was held at 0.4 (i.e. one binding site on ChlH for Gun4) based on the fit in (A) with characterizing parameters WT ChlH Kapp=0.39±0.14 μM and Δ159ChlH Kapp=0.05±0.03 μM.