Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer

Mingxuan Sun, Judy L. Oakes, Shobana K. Ananthanarayanan, Katherine H. Hawley, Roger Y. Tsien, Stephen R. Adams, Christopher M. Yengo

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)5711-5717
Number of pages7
JournalJournal of Biological Chemistry
Volume281
Issue number9
DOIs
StatePublished - Mar 3 2006

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer'. Together they form a unique fingerprint.

Cite this