Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer.
The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding dye (FlAsH) (MV 1IQ FlAsH). The enzymatic properties of MV 1IQ FlAsH were similar to those of unlabeled MV 1IQ except for a 3-fold reduced ADP-release rate. MV 1IQ FlAsH was also capable of moving actin filaments in the in vitro motility assay. To examine rotation of the upper 50-kDa region, we determined the difference in the degree of energy transfer from N-methylanthraniloyl (mant)-labeled nucleotides to FlAsH in both steady-state and transient kinetic experiments. The energy transfer efficiency was higher with mant-ATP (0.65 +/- 0.02) compared with mant-ADP (0.55 +/- 0.02) in the absence of actin. Stopped-flow measurements suggested that the energy transfer efficiency decreased with phosphate release (0.04 s(-1)) in the absence of actin. In contrast, upon mixing MV 1IQ FlAsH in the ADP.P(i) state with actin, a decrease in the energy transfer signal was observed at a rate of 13 s(-1), similar to the ADP release rate. Our results demonstrate there was no change in the energy transfer signal upon actin-activated phosphate release and suggest that actin binding alters the dynamics of the upper 50-kDa region, which may be critical for the ability of myosin to bind tightly to both ADP and actin.
Sun
Mingxuan
M
Department of Biology, University of North Carolina, Charlotte, NC 28223, USA.
Oakes
Judy L
JL
Ananthanarayanan
Shobana K
SK
Hawley
Katherine H
KH
Tsien
Roger Y
RY
Adams
Stephen R
SR
Yengo
Christopher M
CM
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001152:Arsenicals / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D024701:Myosin Type V / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D000199:Actins; D001152:Arsenicals; D005456:Fluorescent Dyes; D024701:Myosin Type V