CHARACTERIZATION OF THE DIMERIZATION PROCESS OF HIV-1 REVERSE-TRANSCRIPTASE HETERODIMER USING INTRINSIC PROTEIN FLUORESCENCE

Intrinsic protein fluorescence has been used to study dimerization of the HIV-1 reverse transcriptase (RT). We observed a 25% increase of th e tryptophan fluorescence of the enzyme during dissociation of the sub units induced by the addition of acetonitrile. Upon reassociation of t he separated subunits, the original fluorescence emission of the heter odimer is restored. A two-state transition model for the RT dimerizati on process in which the dimers are in equilibrium with folded monomers is proposed. The free energy of dissociation was determined to be 12. 2 (+/- 0.2) kcal/mol. In the absence of Mg2+ ions a decrease of this v alue was observed, whereas the addition of a synthetic primer/template (18/36mer) results in an increase of dimer stability. Analyzing the e ffect of Mg2+ on the establishment of the binding equilibrium, a drama tic effect with a 100-fold acceleration of the association by the diva lent ion was observed.