Figure 7.
Effect of an RH active site-directed ligand on the H/D exchange kinetics of RHΔNT and on the maturation behavior of the p66 homodimer. (A) 1H-15N HSQC spectra of RHΔNT in the presence of 5 mM MgCl2 and 1.5 mM HIQ after an H2O/D2O buffer change, obtained during the time periods indicated. The red spectrum in the lower right hand panel was obtained over the 760–800 min accumulation period after changing to the D2O buffer. Other conditions as in Figure 5B. (B) Time dependent changes of connection′ (Ile329 and Ile375) and RH′ domain (Ile434) Ile resonances in the [13CH3-Ile,U-2H]p66/[13CH3-Ile,U-2H]p66′ homodimer in the presence of a Mg-HIQ RH-directed ligand. The ‘E’ and ‘C’ resonance labels indicate assignments to the extended and compact polymerase conformations that are present in the p66 and p66′ subunits, respectively. Conditions favoring dimerization were introduced at t = 0, and the samples also contained 6 mM MgCl2, 1 mM HIQ in 25 mM Tris HCl-d11, pD 7.5, 100 mM KCl and 0.02% NaN3. Each spectrum corresponds to a sequential 5.5 h accumulation period. Samples were run on an Agilent 800 MHz NMR spectrometer at 35°C, under conditions identical to those reported previously without the addition of Mg-HIQ (23).