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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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Western blots of E470A/wt heterodimer. Left side reacted with anti-Flag antibody M2 (Sigma-Aldrich) and right side reacted with anti-(His)6-tag mAb (Sigma-Aldrich). Lanes 1–3 and 1′-3′ are purified FLAG- and (His)6-labeled HMM standards in loads of 25, 35, and 45 ng, respectively. Lane 4 and lane 5 on both blots are identical samples of the heterodimer containing 35 and 45 ng protein. The amount of Flag- and (His)6-reactive material in the heterodimer was determined by normalization to the staining intensity of the standards.
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fig2: Western blots of E470A/wt heterodimer. Left side reacted with anti-Flag antibody M2 (Sigma-Aldrich) and right side reacted with anti-(His)6-tag mAb (Sigma-Aldrich). Lanes 1–3 and 1′-3′ are purified FLAG- and (His)6-labeled HMM standards in loads of 25, 35, and 45 ng, respectively. Lane 4 and lane 5 on both blots are identical samples of the heterodimer containing 35 and 45 ng protein. The amount of Flag- and (His)6-reactive material in the heterodimer was determined by normalization to the staining intensity of the standards.

Mentions: Western blots of the purified E470A/wt-HMM (Fig. 2) provided additional evidence for homogeneity. Identical samples of the heterodimer were probed with FLAG- and (His)6-specific antibodies, and the intensities of the heterodimer bands were normalized by comparison to known quantities of purified FLAG- and (His)6-tagged wt HMMs. This analysis showed that the amounts of FLAG- and (His)6-reactive heavy chain in the heterodimer were equal to within 15%, the limits of measurement errors. This evidence further substantiates our assertion that the heterodimeric preparation is homogeneous.


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)

Western blots of E470A/wt heterodimer. Left side reacted with anti-Flag antibody M2 (Sigma-Aldrich) and right side reacted with anti-(His)6-tag mAb (Sigma-Aldrich). Lanes 1–3 and 1′-3′ are purified FLAG- and (His)6-labeled HMM standards in loads of 25, 35, and 45 ng, respectively. Lane 4 and lane 5 on both blots are identical samples of the heterodimer containing 35 and 45 ng protein. The amount of Flag- and (His)6-reactive material in the heterodimer was determined by normalization to the staining intensity of the standards.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Western blots of E470A/wt heterodimer. Left side reacted with anti-Flag antibody M2 (Sigma-Aldrich) and right side reacted with anti-(His)6-tag mAb (Sigma-Aldrich). Lanes 1–3 and 1′-3′ are purified FLAG- and (His)6-labeled HMM standards in loads of 25, 35, and 45 ng, respectively. Lane 4 and lane 5 on both blots are identical samples of the heterodimer containing 35 and 45 ng protein. The amount of Flag- and (His)6-reactive material in the heterodimer was determined by normalization to the staining intensity of the standards.
Mentions: Western blots of the purified E470A/wt-HMM (Fig. 2) provided additional evidence for homogeneity. Identical samples of the heterodimer were probed with FLAG- and (His)6-specific antibodies, and the intensities of the heterodimer bands were normalized by comparison to known quantities of purified FLAG- and (His)6-tagged wt HMMs. This analysis showed that the amounts of FLAG- and (His)6-reactive heavy chain in the heterodimer were equal to within 15%, the limits of measurement errors. This evidence further substantiates our assertion that the heterodimeric preparation is homogeneous.

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