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A mutant heterodimeric myosin with one inactive head generates maximal displacement.

Kad NM, Rovner AS, Fagnant PM, Joel PB, Kennedy GG, Patlak JB, Warshaw DM, Trybus KM - J. Cell Biol. (2003)

Bottom Line: Proc.Natl.Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Health Science Research Facility, Burlington, VT 05405-0068, USA.

ABSTRACT
Each of the heads of the motor protein myosin II is capable of supporting motion. A previous report showed that double-headed myosin generates twice the displacement of single-headed myosin (Tyska, M.J., D.E. Dupuis, W.H. Guilford, J.B. Patlak, G.S. Waller, K.M. Trybus, D.M. Warshaw, and S. Lowey. 1999. Proc. Natl. Acad. Sci. USA. 96:4402-4407). To determine the role of the second head, we expressed a smooth muscle heterodimeric heavy meromyosin (HMM) with one wild-type head, and the other locked in a weak actin-binding state by introducing a point mutation in switch II (E470A). Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP. Optical trap measurements revealed that the heterodimer generated unitary displacements of 10.4 nm, strikingly similar to wild-type HMM (10.2 nm) and approximately twice that of single-headed subfragment-1 (4.4 nm). These data show that a double-headed molecule can achieve a working stroke of approximately 10 nm with only one active head and an inactive weak-binding partner. We propose that the second head optimizes the orientation and/or stabilizes the structure of the motion-generating head, thereby resulting in maximum displacement.

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Hydrolysis of ATP by E470A-HMM. (A) Time course of pyrene actin fluorescence quenching. 1.6 μM nucleotide-free E470A-HMM was added to 1 μM Pyr-actin at the time noted by the first arrow. Once a new steady-state level of fluorescence was reached, 0.6 μM MgATP was added (second arrow), causing a rapid increase in signal, followed by a slow decay, which was fit to a single exponential (gray line). (B) FPLC chromatograms from samples of E470A-HMM taken immediately (solid line) and 2 h after (dashed line) removal of all free ATP. The absorbance at 260 nm was normalized to that of the largest peak. The identities of the ATP and ADP peaks were established by comparison with trials conducted with pure standards.
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fig1: Hydrolysis of ATP by E470A-HMM. (A) Time course of pyrene actin fluorescence quenching. 1.6 μM nucleotide-free E470A-HMM was added to 1 μM Pyr-actin at the time noted by the first arrow. Once a new steady-state level of fluorescence was reached, 0.6 μM MgATP was added (second arrow), causing a rapid increase in signal, followed by a slow decay, which was fit to a single exponential (gray line). (B) FPLC chromatograms from samples of E470A-HMM taken immediately (solid line) and 2 h after (dashed line) removal of all free ATP. The absorbance at 260 nm was normalized to that of the largest peak. The identities of the ATP and ADP peaks were established by comparison with trials conducted with pure standards.

Mentions: To determine the role of each head in actin movement, we engineered a mutant heterodimeric HMM with one wild-type cycling head and one inactive weak-binding head, which was achieved by introducing a point mutation in switch II (E470A). The homodimeric E470A-HMM was first characterized to confirm the predominant biochemical state of the mutant head (Onishi et al., 1998a,b; Suzuki et al., 1998). When nucleotide-free phosphorylated E470A-HMM (see Materials and methods) was added to pyrene-labeled actin (Pyr-actin), it caused a large decrease in fluorescence (Fig. 1 A), indicating that the mutant can isomerize to the strong binding state (Criddle et al., 1985). Addition of ATP to ∼0.4 mol/mol of HMM caused a rapid increase in fluorescence, followed by a slow decay that was fit to a single exponential with a rate constant of ∼0.0004 s−1 (Fig. 1 A). Therefore, E470A-HMM binds strongly to actin, dissociates from actin on addition of MgATP, and then only very slowly returns to the strong actin-binding state.


A mutant heterodimeric myosin with one inactive head generates maximal displacement.

Kad NM, Rovner AS, Fagnant PM, Joel PB, Kennedy GG, Patlak JB, Warshaw DM, Trybus KM - J. Cell Biol. (2003)

Hydrolysis of ATP by E470A-HMM. (A) Time course of pyrene actin fluorescence quenching. 1.6 μM nucleotide-free E470A-HMM was added to 1 μM Pyr-actin at the time noted by the first arrow. Once a new steady-state level of fluorescence was reached, 0.6 μM MgATP was added (second arrow), causing a rapid increase in signal, followed by a slow decay, which was fit to a single exponential (gray line). (B) FPLC chromatograms from samples of E470A-HMM taken immediately (solid line) and 2 h after (dashed line) removal of all free ATP. The absorbance at 260 nm was normalized to that of the largest peak. The identities of the ATP and ADP peaks were established by comparison with trials conducted with pure standards.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172693&req=5

fig1: Hydrolysis of ATP by E470A-HMM. (A) Time course of pyrene actin fluorescence quenching. 1.6 μM nucleotide-free E470A-HMM was added to 1 μM Pyr-actin at the time noted by the first arrow. Once a new steady-state level of fluorescence was reached, 0.6 μM MgATP was added (second arrow), causing a rapid increase in signal, followed by a slow decay, which was fit to a single exponential (gray line). (B) FPLC chromatograms from samples of E470A-HMM taken immediately (solid line) and 2 h after (dashed line) removal of all free ATP. The absorbance at 260 nm was normalized to that of the largest peak. The identities of the ATP and ADP peaks were established by comparison with trials conducted with pure standards.
Mentions: To determine the role of each head in actin movement, we engineered a mutant heterodimeric HMM with one wild-type cycling head and one inactive weak-binding head, which was achieved by introducing a point mutation in switch II (E470A). The homodimeric E470A-HMM was first characterized to confirm the predominant biochemical state of the mutant head (Onishi et al., 1998a,b; Suzuki et al., 1998). When nucleotide-free phosphorylated E470A-HMM (see Materials and methods) was added to pyrene-labeled actin (Pyr-actin), it caused a large decrease in fluorescence (Fig. 1 A), indicating that the mutant can isomerize to the strong binding state (Criddle et al., 1985). Addition of ATP to ∼0.4 mol/mol of HMM caused a rapid increase in fluorescence, followed by a slow decay that was fit to a single exponential with a rate constant of ∼0.0004 s−1 (Fig. 1 A). Therefore, E470A-HMM binds strongly to actin, dissociates from actin on addition of MgATP, and then only very slowly returns to the strong actin-binding state.

Bottom Line: Proc.Natl.Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Health Science Research Facility, Burlington, VT 05405-0068, USA.

ABSTRACT
Each of the heads of the motor protein myosin II is capable of supporting motion. A previous report showed that double-headed myosin generates twice the displacement of single-headed myosin (Tyska, M.J., D.E. Dupuis, W.H. Guilford, J.B. Patlak, G.S. Waller, K.M. Trybus, D.M. Warshaw, and S. Lowey. 1999. Proc. Natl. Acad. Sci. USA. 96:4402-4407). To determine the role of the second head, we expressed a smooth muscle heterodimeric heavy meromyosin (HMM) with one wild-type head, and the other locked in a weak actin-binding state by introducing a point mutation in switch II (E470A). Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP. Optical trap measurements revealed that the heterodimer generated unitary displacements of 10.4 nm, strikingly similar to wild-type HMM (10.2 nm) and approximately twice that of single-headed subfragment-1 (4.4 nm). These data show that a double-headed molecule can achieve a working stroke of approximately 10 nm with only one active head and an inactive weak-binding partner. We propose that the second head optimizes the orientation and/or stabilizes the structure of the motion-generating head, thereby resulting in maximum displacement.

Show MeSH
Related in: MedlinePlus