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. 2017 Feb 21;292(14):5871–5883. doi: 10.1074/jbc.M116.761809

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© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 5. — GDP and GTP binding and dissociation kinetics of ObgE_FL (a, c, and e) and ObgE_340 (b, d, and f) determined via stopped flow fluorescence analysis. a and b, transients obtained by following mGDP (0.2/0.1 μm, before/after mixing) fluorescence upon rapid mixing with different concentrations of ObgE_FL (a) and ObgE_340 (b). Concentration values given on the graph represent ObgE concentrations before and after mixing. The lower panels show the concentration dependence of the observed rate constant (k_obs). The slope of the linear fit yields k_on. c and d, transients obtained by following mGTP (0.2/0.1 μm, before/after mixing) fluorescence upon rapid mixing with different concentrations of ObgE_FL (c) and ObgE_340 (d). The same concentrations as in a and b are used. The lower panels show the concentration dependence of the observed rate constant (k_obs). The slope and intercept of the linear fit yield k_on and k_off, respectively. e and f, direct determination of k_off by following the release of mGDP from ObgE_FL (e) and ObgE_340 (f) upon mixing 200 μm unlabeled GDP with a mixture of 0.4 μm ObgE and 1.5 μm mGDP. g, summary of nucleotide binding/dissociation kinetics and the deduced K_D values of ObgE_FL and ObgE_340.