The association of actin and myosin in the presence of gamma-amido-ATP proceeds mainly via a complex with myosin in the closed conformation.
The interaction of gamma-amido-ATP (ATPN) and its 2'(3')-O-methylanthraniloyl derivative (mantATPN) with skeletal myosin subfragment 1 (S1) and actomyosin (actoS1) was studied in stopped-flow experiments. Tryptophan fluorescence and fluorescence of the mant label or light scattering were measured simultaneously. Information about the binding of mant nucleotides was obtained from the quenching of tryptophan fluorescence by the mant label. The parameters of various kinetic models were fitted to the experimental traces. The high-fluorescence state of S1 forms with ATPN at a rate of 95 s-1 ("open-closed" transition); the transition is only slowly reversible, in contrast to the very fast equilibrium seen with its better known isomer AMPPNP [Urbanke, C., and Wray, J. (2001) Biochem. J. 358, 165-173]. The stabilization of the closed state of myosin by ATPN may be due to the formation of a complex with a pentacoordinated amido-gamma-phosphate, from which ATPN can dissociate at a rate of 0.005 s-1 or be hydrolyzed by cleavage of the beta-gamma bond at a rate of 2.5 x 10(-4) s-1. A corresponding actoS1-ATPN complex with myosin in the "closed" conformation is the first detectable intermediate in the association of actin and S1-ATPN, giving an experimental access to a state analogous to a key intermediate in the cross-bridge cycle.
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Jahnstrasse 29, D 69120 Heidelberg, Germany. Werner.Jahn@mpimf-heidelberg.mpg.de
Werner
W
Jahn
D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000266:Adenylyl Imidodiphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D004023:Dictyostelium / Q000378:metabolism; D005453:Fluorescence; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis; D062367:ortho-Aminobenzoates / Q000737:chemistry
C523356:2'(3')-O-methylanthraniloyl-gamma-amido-adenosine triphosphate; D000199:Actins; C523355:gamma-amido-adenosine triphosphate; D062367:ortho-Aminobenzoates; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; D014364:Tryptophan; D009218:Myosins