N-terminal domain swapping and metal ion binding in nitric oxide synthase dimerization.

Nitric oxide synthase oxygenase domains (NOS(ox)) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOS(ox) reveal that conformational changes in a switch region (residues 103-111) preceding a pterin-binding segment exchange N-terminal beta-hairpin hooks between subunits of the dimer. N-terminal hooks interact primarily with their own subunits in the 'unswapped' structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N-terminal hooks rearrange from intra- to intersubunit interactions in the 'swapped structure', and Cys109 forms a self-symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N-terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOS(ox) molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three-dimensional domain swapping of the N-terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.

Deciphering the fluorescence signature of daunomycin and doxorubicin.

The fluorescence characteristics of daunomycin (DNM), doxorubicin (DXR), and other anthracycline drugs are often used to monitor localization of the drug within lipid bilayers and liposomal delivery systems and to assess interaction of the drug with DNA and other macromolecules. However, the binding of DNM and DXR to proteins and membrane systems has been observed to exhibit variable effects on the anthracycline's fluorescence. We have delineated the spectroscopic response of DXR and DNM to their surroundings in several systems, including solvents of differing dielectric constant, aqueous solutions of varying pH or fluorophore concentration, and the reverse micellar system of AOT/heptane/water with a range of doxorubicin concentrations. We have observed that the ratio of fluorescence intestinal at the two principal lambda max values shows a parabolic dependence on solvent dielectric constant, i.e. inverted solvatochromism. This behavior has been overlooked by previous investigators and, together with the appearance of a long-wavelength band near 630 nm in solvents of low dielectric strength (also previously not reported), is key to understanding the partitioning of anthracyclines in membrane systems as well as resolving the conflicting interpretation of data in the literature.