X-ray crystal structure and solution fluorescence characterization of Mg.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 myosin ATPase by fluorescence spectroscopy. The sensitivity of the mant fluorophore to its local environment also makes it suitable to investigate the exposure of bound nucleotides to solvent from collisional quenching measurements. Here we present the crystal structure of mant-ADP and beryllium fluoride complexed with Dictyostelium discoideum myosin motor domain (S1dC) at 1.9 A resolution. We complement the structural approach with an investigation of the accessibility of the mant moiety to solvent using acrylamide quenching of fluorescence emission. In contrast to rabbit skeletal myosin subfragment 1, where the mant group is protected from acrylamide (Ksv=0.2 M-1), the fluorophore is relatively exposed when bound to Dictyostelium myosin motor domain (Ksv= 1.4 M-1). Differences between the Dictyostelium structure and that of vertebrate skeletal subfragment 1, in the region of the nucleotide 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 quenching does not report on overall closure of the nucleotide binding pocket but reflects more local structural changes.
Bauer
C B
CB
Institute for Enzyme Research and Department of Biochemistry, University of Wisconsin, 1710 University Avenue, Madison, WI 53705, USA.
Kuhlman
P A
PA
Bagshaw
C R
CR
Rayment
I
I
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001608:Beryllium / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004023:Dictyostelium / Q000737:chemistry*; D005453:Fluorescence; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011817:Rabbits; D012996:Solutions; D062367:ortho-Aminobenzoates / Q000737:chemistry* / Q000378:metabolism
D000178:Acrylamides; D012996:Solutions; D062367:ortho-Aminobenzoates; D020106:Acrylamide; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D009218:Myosins; D008274:Magnesium; D001608:Beryllium; D005459:Fluorides