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 journalArticle

24 Citations (Scopus)

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

Fingerprint

Myosin Type V
Fluorescence Resonance Energy Transfer
Energy Transfer
Actins
Energy transfer
Adenosine Diphosphate
Myosins
Nucleotides
Phosphates
Fluorescein
Actin Cytoskeleton
Flow measurement
Coloring Agents
Adenosine Triphosphate
Labels
Assays
Kinetics

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Sun, Mingxuan ; Oakes, Judy L. ; Ananthanarayanan, Shobana K. ; Hawley, Katherine H. ; Tsien, Roger Y. ; Adams, Stephen R. ; Yengo, Christopher M. / Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 9. pp. 5711-5717.
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Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer. / Sun, Mingxuan; Oakes, Judy L.; Ananthanarayanan, Shobana K.; Hawley, Katherine H.; Tsien, Roger Y.; Adams, Stephen R.; Yengo, Christopher M.

In: Journal of Biological Chemistry, Vol. 281, No. 9, 03.03.2006, p. 5711-5717.

Research output: Contribution to journalArticle

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T1 - Dynamics of the upper 50-kDa domain of myosin V examined with fluorescence resonance energy transfer

AU - Sun, Mingxuan

AU - Oakes, Judy L.

AU - Ananthanarayanan, Shobana K.

AU - Hawley, Katherine H.

AU - Tsien, Roger Y.

AU - Adams, Stephen R.

AU - Yengo, Christopher M.

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N2 - 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.

AB - 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.

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