Using fluorescence tagging of amino acid residues distant from the active site of HIV-1 reverse transcriptase (RT) to obtain a unique fluorescent signal for nucleotide binding during catalysis
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MDCC fluorophore labelling of HIV-1 RT has been used to monitor catalytic activity in the active site of the enzyme. Two fluorescence signals are generated in such experiments; one that results from the DNA template binding to the enzyme, and one that occurs when the next correct nucleotide binds to the enzyme-DNA complex and anneals to the nascent DNA strand. The intensity of the fluorescence signal resulting from the binding of the DNA template usually washes out the signal accompanying nucleotide binding. We hypothesized that by attaching MDCC to a residue on the surface of HIV-1 RT farther from the active site that underwent a drastic conformational change during catalysis, we would be able to generate a signal for nucleotide binding that was comparable in fluorescent intensity to the signal associated with DNA template strand binding. Our results for one labelling position were consistent with this hypothesis. Furthermore, our data confirms work conducted in past studies that showed how conformational changes in HIV-1 RT were related to catalytic activity at the active site. A brief literature review discussing past research describing this relationship, along with its implications, is included.