CRYSTAL-STRUCTURE OF A FLUORESCENT DERIVATIVE OF RNASE-A

The crystal structure of RNase A chemically modified with the fluoresc ent probe, odoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonic (1,5-IAE NS), has been solved and refined to high resolution. It yields informa tion on the mode of binding, the mobility of a probe commonly used in spectroscopic studies, and anion binding sites in RNase A. Trigonal cr ystals of the fluorescent derivative grown in sodium or cesium chlorid e and ammonium sulfate, pH 5.1, were nearly isomorphous with those of a semisynthetic RNase [DeMel, et al. (1992) J. Biol. Chem. 267, 247-25 6]. Refinement starting from semisynthetic RNase led to a model with R = 20% against 1.7-angstrom diffraction data from crystals in ammonium sulfate and another model with R = 17% against 1.9-angstrom data take n in the presence of 3 M NaCl. The second model contains three chlorid e ions: one is at the active site, and the other two are at molecular interfaces. Otherwise, the two models are very similar. The fluorophor e has very little effect on the protein conformation. It is found to b e covalently attached to the active site His-12 with the naphthyl grou p stacked on the imidazole ring of His-119. It remains largely accessi ble to solvent and in a polar environment on the protein surface, even though the fluorescence emission spectrum is blue shifted as it is in nonpolar solvents.