The effect of F-actin on the relay helix position of myosin II, as revealed by tryptophan fluorescence, and its implications for mechanochemical coupling.
The fluorescence properties of Dictyostelium discoideum (Dd) myosin II constructs containing a single tryptophan residue have revealed detailed information regarding nucleotide binding and hydrolysis steps. Here we extend these studies to investigate the influence of actin on nucleotide-induced fluorescence transients. The fluorescence from native actin tryptophan residues is not significantly perturbed on binding to myosin, although an apparent signal is detected as a consequence of a light scatter artifact. Actin has a minor effect on the response of W129, located at the entrance to the nucleotide-binding pocket, and reduces the forward rate constants for the isomerization(s) associated with binding of ATP, ATPgammaS, and ADP by 3-fold or less. The isomerization detected by W129 clearly precedes the dissociation of actin in the case of ADP and ATPgammaS binding. The fluorescence from the conserved W501 residue, located at the distal end of the relay helix, is very sensitive to the switch 2 and/or lever arm disposition. Consequently, the observed fluorescence emission intensity can be used to estimate the equilibrium constant between the pre- and post-power stroke conformations. Actin modulates this equilibrium by no more than 2-fold in the presence of nucleoside triphosphate. These data have implications for the mechanism of product release and suggest that actin activates another process in the mechanism, such as switch 1 movement and Pi release, rather than influencing the switch 2 equilibrium and lever arm position directly.
Conibear
Paul B
PB
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom. crb5@le.ac.uk
Málnási-Csizmadia
András
A
Bagshaw
Clive R
CR
D000199:Actins / Q000737:chemistry*; D000205:Actomyosin / Q000235:genetics / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D004023:Dictyostelium / Q000737:chemistry / Q000235:genetics; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024462:Myosin Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D000199:Actins; D020409:Molecular Motor Proteins; D015800:Protozoan Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000205:Actomyosin; D024462:Myosin Type II