Figure 4

Apparent dNTP binding affinity of mutant HIV-1 RT carrying insertion in the β7–β8 loop of either or both the subunits. The 33-mer DNA primed with 5'-³²P labeled dideoxy (ddC) terminated 21-mer DNA primer was incubated with the individual enzyme at 4°C for 10 min. The binding affinity [K_{d (dNTP)}] of the wild type enzyme and its mutant derivatives in the ternary complex was determined by incubating the preformed E-TP binary complex of the individual enzyme species at different concentrations (0.26–800 μM) of the next correct dNTP (dGTP). Following incubation with dGTP, 300 fold molar excess of the unlabeled TP was used as trap to remove the readily dissociable binary complexes from the ternary complex population. The extent of stable ternary complexes formed were resolved on 6% native polyacrylamide gel and analyzed by phosphorImaging. The [K_{d [dNTP]}], in the ternary complex for each enzyme was determined by quantifying the E-TP-dNTP ternary complexes as a function of increasing concentrations of dGTP and fitting the data to the equation for single-site ligand binding using Sigma-Plot. Lanes 1–6 represent the ternary complex formed in the presence of dGTP at 0.2, 1.28, 6.4, 32, 160 and 800 μM, concentrations.