The conformation of microtubule-bound paclitaxel has been examined by fluorescence and solid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3'-N-debenzoyl-3'-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promoting activity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino group did not adversely affect the ligand-receptor association. The distance between the fluorophore N-AB-PT and the colchicine binding site on tubulin polymers was determined through time-resolved measurements of fluorescence resonance energy transfer to be 29 +/- 2 A. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the Stokes shift as a function of solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in protic solvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokes shift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from the effects of the solvent. These data are best explained by considering the solution-state conformational properties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the nature of the solvent, and these fluorescence data are consistent with the molecule adopting a "hydrophobic collapsed" conformation in protic solvents and an "extended" conformation in aprotic solvents. The Stokes shift of microtubule-bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was also investigated by solid-state NMR. Paclitaxel was labeled with (19)F at the para position of the C-2 benzoyl substituent and with (13)C and (15)N in the side chain. Distances between the fluorine and carbon nuclei were determined by REDOR. The distance between the fluorine and the 3'-amide carbonyl carbon was 9.8 +/- 0.5 A, and the distance between the fluorine atom and the 3'-methine carbon was 10. 3 +/- 0.5 A. These spectroscopic data were used in conjunction with molecular modeling to refine the microtubule-bound conformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxel binding site.

The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.

The 15N{19F} REDOR NMR spectra of two fluorolumazines complexed to the 1-MDa β60 capsid of lumazine synthase have been obtained at 20.3 and 50.7 MHz. Distances from CF3 groups of the ligands to six side- and main-chain nitrogens have been measured. These distances were used in combination with the X-ray crystal coordinates of wild-type lumazine synthase, complexed to a related substrate ligand, in a series of distance-restrained molecular dynamics simulations. The result is a model of the binding site of lumazine synthase that has sufficient detail to predict the absolute configuration at C-7 of complexed 7-hydroxy-8-d-ribityl-6,7-bis(trifluoromethyl)-7,8-dihydropteridine-2,4(1H,3H)-dione, a fluorinated analogue of an unstable, hypothetical intermediate in the reactions catalyzed by both lumazine synthase and riboflavin synthase.