Dynamics of the Upper 50-kDa Domain of Myosin V Examined with Fluorescence Resonance Energy Transfer

• Published in: The Journal of biological chemistry 2006-03, Vol.281 (9), p.5711-5717
• Main Authors: Sun, Mingxuan, Oakes, Judy L., Ananthanarayanan, Shobana K., Hawley, Katherine H., Tsien, Roger Y., Adams, Stephen R., Yengo, Christopher M.
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Amino Acid Sequence; Arsenicals - metabolism; Fluorescence Resonance Energy Transfer; Fluorescent Dyes - metabolism; Myosin Type V - chemistry; Myosin Type V - genetics; Myosin Type V - metabolism; Protein Binding; Protein Structure, Tertiary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M508103200

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·Pi 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.