Femtosecond dynamics of a drug–protein complex: Daunomycin with Apo riboflavin-binding protein

Abstract

In this contribution, we report studies of the primary dynamics of the drug-protein complexes of daunomycin with apo riboflavinbinding protein. With femtosecond resolution, we observed the ultrafast charge separation between daunomycin and aromatic amino acid residues of the protein, tryptophan(s). Electron transfer occurs from tryptophan(s) to daunomycin with two reaction times, 1 ps and 6 ps, depending on the local complex structure. The formation of anionic daunomycin radical is crucial for triggering a series of chemical reactions in redox cycling. One of the subsequent reactions is the reduction of dioxygen to form active superoxide by the reduced daunomycin. This catalytic process was found to occur within 10 ps. In the absence of dioxygen, charge recombination takes a much longer time, more than 100 ps. These results, along with similar findings in DNA and nucleotides, elucidate that the ultrafast generation of reduced daunomycin radicals by photoactivation is a primary step for the observed photoenhancement of drug cytotoxicity by several orders of magnitude. We also studied the dependence of the dynamics on protein conformations at different ionic strengths and denaturant concentrations. We observe a sharp transition from the tertiary structure to the unfolding state at 2 M of denaturant concentration. D aunomycin (DM, Fig. 1), an anthracycline antibiotic, is one of the effective drugs used for cancer chemotherapy. It has been shown that the cytotoxicity of DM is enhanced by several orders of magnitude with irradiation of light, but the mechanism by photoactivation is still unknown (1-3). In recent studies from this group (X. Qu, C.W., H.-C. Becker, D.Z., and A.H.Z., unpublished work), we reported the striking observation of the primary dynamics of DM when it intercalates into GC base pairs of DNA or binds to nucleotides. Ultrafast electron-transfer (ET) reactions between DM and the base G were found to occur on the femtosecond to picosecond time scales (4). The reduced DM radicals can catalyze superoxide formation from dioxygen (O 2 ), which then triggers redox cycling. Thus, molecular oxygen becomes an integral part of the drug function.Here, we report our studies of the dynamics of DM with a protein. Many proteins have hydrophobic domains and can interact with DM molecules. Aromatic or sulfur-containing amino acid residues in proteins, such as Trp, Tyr, or cysteine, can often function as reductants and donate electrons. One of the flavoproteins, apo riboflavin-binding protein (apoRfBP) and a riboflavin carrier in plasma, has several Trp residues in its hydrophobic cleft (20 Å wide and 15 Å deep). This protein, a single polypeptide chain of 219 amino acids, was chosen as a prototype (Fig. 1) and the complex of DM-apoRfBP as a model system.As mentioned in the preceding paper (5), the x-ray crystal structure reveals a compact stack of riboflavin (vitamin B 2 ) sandwiched between Trp-156 and Tyr-75 at an equal interplanar distance of 3.7 Å in the hydrophobic cleft of apoRfBP (6). Ultrafast ET between rib...

Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

“…This interaction is reminiscent of the substrate pocket of riboflavin-binding protein in which the ligand riboflavin forms a compact stack between Trp-156 and Tyr-75 with equal interplanar distances of 3.7 Å (40, 41). DNR and riboflavin have similar planar three-ring anthraquinone-type structures, and it has been suggested that DNR inserts in the riboflavin-binding protein-binding site (42). The distribution of the spin label quenching sites, particularly along helix 3, reflects a diffuse location of bound DNR.…”

Dynamical perspective of protein-DNA interaction

“…The fluorescence anisotropy, r(t), which can decay in time because of the rotational motion of the molecules and consequently leads to depolarization of the fluorescence is fitted to biexponential decay function. The faster time constant indicates the local/inertial motion of the probe and the slower one reveals the overall tumbling motion of the entire protein (47). The estimated rotational time constant (τ) for the repressor associated with the overall λ-repressor tumbling is estimated to be 27 ns using the Stokes-Einstein-Debye (SED) equation (48).…”

Drug/protein interactions studied by time-resolved fluorescence spectroscopy

“…[2][3][4][5][6]. These have shown the influence of the biological nanocavities not only on the diffusional/orientational motions of the drug, but also on its solvation dynamics and excited state processes such as energy, electron and proton transfer.…”