X-RAY CRYSTAL-STRUCTURE AND SOLUTION FLUORESCENCE CHARACTERIZATION OFMG 2'(3')-O-(N-METHYLANTHRANILOYL) NUCLEOTIDES BOUND TO THE DICTYOSTELIUM-DISCOIDEUM MYOSIN MOTOR DOMAIN

Mant (2'(3')-O-(N-methylanthraniloyl)) labeled nucleotides have proven to be useful tools in the study of the kinetic mechanism of the myosi n ATPase by fluorescence spectroscopy. The sensitivity of the mant flu orophore to its local environment also makes it suitable to investigat e the exposure of bound nucleotides to solvent from collisional quench ing measurements. Here we present the crystal structure of mant-ADP an d beryllium fluoride complexed with Dictyostelium discoideum myosin mo tor domain (S1dC) at 1.9 Angstrom resolution. We complement the struct ural approach with an in investigation of the accessibility of the man t moiety to solvent using acrylamide quenching of fluorescence emissio n. Ln contrast to rabbit skeletal myosin subfragment 1, where the mant group is protected from acrylamide (K-sv= 0.2 M-1), the fluorophore i s relatively exposed when bound to Dictyostelium myosin motor domain ( K-sv = 1.4 M-1). Differences between the Dictyostelium structure and t hat of vertebrate skeletal subfragment 1, in the region of the nucleot ide binding pocket, are proposed as an explanation for the differences observed in the solvent accessibility of complexed mant-nucleotides. We conclude that protection of the mant group from acrylamide quenchin g does not report on overall closure of the nucleotide binding pocket but reflects more local structural changes. (C) 1997 Academic Press Li mited.