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. 2008 Aug 22;190(21):7302–7307. doi: 10.1128/JB.00593-08

FIG. 4.

FIG. 4.

The monomer-dimer equilibrium of SecAΔ11, SecAΔ11/N95, and SecAΔ8 proteins is altered, and these proteins have defects in their SecA-dependent translocation ATPase activities. SecA proteins were overproduced and purified using either His-bind resin (Novagen) or SP Sepharose (Sigma) as previously described (17). (A and B) Light scattering data were collected using a Superose 6 10/30 HR size exclusion chromatography column (GE Healthcare) connected to a high-performance liquid chromatography system (Alliance 2965; Waters Corp.) equipped with an autosampler. The eluate from the size exclusion chromatography column was monitored by using a photodiode array UV/Vis detector (996 PDA; Waters Corp.), a differential refractometer (OPTI-Lab or OPTI-rEx; Wyatt Corp.), and a static, multiangle laser light scattering detector (DAWN-EOS; Wyatt Corp.). The system was equilibrated with a solution containing 10 mM Tris-HCl (pH 7.5), 100 mM or 300 mM KCl, 10 mM magnesium acetate, 1 mM dithiothreitol, and 1× protease inhibitor cocktail at a flow rate of 0.3 ml/min. The weight average molecular mass (MW) for SecA protein was determined for the concentration ranges shown with either (A) 100 mM KCl or (B) 300 mM KCl. Two software packages were used for data collection and analysis; the Millennium software (Waters Corp.) controlled the high-performance liquid chromatograph operation and data collection from the multiwavelength UV/Vis detector, while the ASTRA software (Wyatt Corp.) collected data from the refractive index detector and the light scattering detector and recorded the UV trace at 280 nm sent from the 996 PDA detector. The lines indicate the nonlinear least-square fits of weight average molecular mass determined for the apex of each eluting peak at various concentrations for a monomer-dimer association model; the error bars indicate 5% uncertainties for molecular mass determinations expected from measurement of light scattering. Symbols: filled circles, SecA-his; open squares, SecAΔ8his; filled triangles, SecAΔ11his; open triangles, SecAΔ11/N95. (C) Endogenous, membrane, and translocation ATPase activities of the indicated SecA mutant proteins, determined as described previously (17).