Resonance energy transfer was used to determine separation distances between fluorescent derivatives of substrates for Klenow fragment and a unique sulfhydryl, cysteine 907, on the enzyme. Fluorescent derivatives of duplex DNA, deoxynucleotide triphosphates (dNTP), and deoxynucleotide monophosphates (dNMP), modified with aminonaphthalenesulfonates (ANS), served as energy-transfer donors to the fluorophore used to modify cysteine 907, 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The labeling of cysteine 907 with NBD caused no decrease in the enzyme's polymerase activity, suggesting that the probe did not significantly alter the conformation of the enzyme. The efficiency of singlet–singlet resonance energy transfer was determined from the quantum yield of the donor in the presence and absence of acceptor. By Förster's theory, the measured distances between cysteine 907 and binding sites for duplex DNA, dNTP, and dNMP were 25–39, 19–28, and 17–26 Å, respectively. As the fluorophores, attached to the substrates via a tether arm, are separated from the substrates by approximately 12 Å, the distances measured between binding sites are subject to this uncertainty. To measure the separation between binding sites for duplex DNA and dNMP, and to reduce the uncertainty introduced by the tether arm, two experiments were carried out. In the first, duplex DNA was labeled with the acceptor fluorophore NBD and used with the donor ANS-modified dNMP to yield a measured distance separating these two sites of 19–28 Å. In the second, polymerization of the NBD-labeled duplex by six base pairs increased the separation distance between these two sites to 26–41 Å, supporting the placement of the DNA duplex in the cleft of the large domain of the enzyme and the separation of polymerase and exonuclease sites. Collectively these distances permitted the construction of the model for the ternary complex whose overall dimensions are in satisfactory agreement with those obtained from the X-ray crystal structure from the binary KF·dTMP complex (Ollis et al., 1985).
All Science Journal Classification (ASJC) codes