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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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Myosin step size. Each circle represents the mean step size obtained for a single myosin molecule in a separate experiment based on mean-variance analysis (Fig. 3). The mean and SDs are indicated by the squares and associated error bars. A detailed summary of these values can be found in Table II.
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fig4: Myosin step size. Each circle represents the mean step size obtained for a single myosin molecule in a separate experiment based on mean-variance analysis (Fig. 3). The mean and SDs are indicated by the squares and associated error bars. A detailed summary of these values can be found in Table II.

Mentions: The unitary displacement of the heterodimeric E470A/wt-HMM was characterized at the single-molecule level in the optical trap, and compared with wt-HMM and S1-neo (Finer et al., 1994; see also Materials and methods). Representative raw data traces for these three constructs are presented in Fig. 3 (A, C, and E). All three constructs show displacement events that can be visually detected by the reduction in position variance that occurs on myosin strong binding to actin. These data were analyzed using mean variance methods (Patlak, 1993; Guilford et al., 1997), yielding the histograms seen in Fig. 3 (B, D, and F). Each histogram has two clear populations corresponding to baseline (B) when myosin is detached from actin and the displacement events themselves (E), which are separated both by mean position and by variance. Subtracting the mean position of the baseline population from that of the event population yields the myosin step size. Fig. 4 shows the mean step size for each myosin construct from multiple experiments. A clear difference is evident between the step sizes of wt-HMM and S1-neo (10.2 vs. 4.4 nm; Table II). Remarkably, E470A/wt-HMM, with one compromised head, produced a mean step size of 10.4 nm, almost identical to wt-HMM, but clearly larger than S1-neo.


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)

Myosin step size. Each circle represents the mean step size obtained for a single myosin molecule in a separate experiment based on mean-variance analysis (Fig. 3). The mean and SDs are indicated by the squares and associated error bars. A detailed summary of these values can be found in Table II.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Myosin step size. Each circle represents the mean step size obtained for a single myosin molecule in a separate experiment based on mean-variance analysis (Fig. 3). The mean and SDs are indicated by the squares and associated error bars. A detailed summary of these values can be found in Table II.
Mentions: The unitary displacement of the heterodimeric E470A/wt-HMM was characterized at the single-molecule level in the optical trap, and compared with wt-HMM and S1-neo (Finer et al., 1994; see also Materials and methods). Representative raw data traces for these three constructs are presented in Fig. 3 (A, C, and E). All three constructs show displacement events that can be visually detected by the reduction in position variance that occurs on myosin strong binding to actin. These data were analyzed using mean variance methods (Patlak, 1993; Guilford et al., 1997), yielding the histograms seen in Fig. 3 (B, D, and F). Each histogram has two clear populations corresponding to baseline (B) when myosin is detached from actin and the displacement events themselves (E), which are separated both by mean position and by variance. Subtracting the mean position of the baseline population from that of the event population yields the myosin step size. Fig. 4 shows the mean step size for each myosin construct from multiple experiments. A clear difference is evident between the step sizes of wt-HMM and S1-neo (10.2 vs. 4.4 nm; Table II). Remarkably, E470A/wt-HMM, with one compromised head, produced a mean step size of 10.4 nm, almost identical to wt-HMM, but clearly larger than S1-neo.

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