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Different degrees of lever arm rotation control myosin step size.

Köhler D, Ruff C, Meyhöfer E, Bähler M - J. Cell Biol. (2003)

Bottom Line: We analyzed the step size of rat myosin 1d (Myo1d) and surprisingly found that this myosin takes unexpectedly large steps in comparison to other myosins.Engineering the length of the light chain binding domain of rat Myo1d resulted in a linear increase of step size in relation to the putative lever arm length, indicative of a lever arm rotation of the light chain binding domain.These results demonstrate that differences in myosin step sizes are not only controlled by lever arm length, but also by substantial differences in the degree of lever arm rotation.

View Article: PubMed Central - PubMed

Affiliation: Institute for General Zoology and Genetics, Westfälische Wilhelms-University, Schlossplatz 5, 48149 Münster, Germany.

ABSTRACT
Myosins are actin-based motors that are generally believed to move by amplifying small structural changes in the core motor domain via a lever arm rotation of the light chain binding domain. However, the lack of a quantitative agreement between observed step sizes and the length of the proposed lever arms from different myosins challenges this view. We analyzed the step size of rat myosin 1d (Myo1d) and surprisingly found that this myosin takes unexpectedly large steps in comparison to other myosins. Engineering the length of the light chain binding domain of rat Myo1d resulted in a linear increase of step size in relation to the putative lever arm length, indicative of a lever arm rotation of the light chain binding domain. The extrapolated pivoting point resided in the same region of the rat Myo1d head domain as in conventional myosins. Therefore, rat Myo1d achieves its larger working stroke by a large calculated approximately 90 degrees rotation of the light chain binding domain. These results demonstrate that differences in myosin step sizes are not only controlled by lever arm length, but also by substantial differences in the degree of lever arm rotation.

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Single molecule step sizes of different rat Myo1d constructs. (A) Displacement records showing the interaction of a single actin filament with recombinant rat Myo1d molecules encompassing variable numbers of light chain binding sites. For each construct, representative traces of raw needle displacements (top trace) and the corresponding running variance recordings (bottom trace) are shown. During the attachment of a motor molecule, the fluctuation of the bead–filament–needle complex is significantly reduced. The records contain many actomyosin interactions of variable length; in each panel we highlighted one interaction by an asterisk. Attachments occur over the entire range of the free needle positions. According to Molloy and coworkers (Molloy et al., 1995), the positions of several events show a Gaussian distribution shifted for a distance equivalent to the step size. (B) Distribution of events for the different constructs, summarizing the (oriented) results of several measurements. (C) Step sizes are plotted against the putative lever arm lengths of the various rat Myo1d proteins (circles). The putative lever arm length was calculated by assuming an α-helical conformation of the light chain binding domain. The corresponding solid line represents the best fit of a linear regression analysis. For comparison, previously reported data of D. discoideum myosin II (Dd II, squares; Ruff et al., 2001) and chicken smooth muscle myosin II (sm II, triangles; Warshaw et al., 2000) are also included. The Myo 1d-head construct possesses a COOH-terminal linker of five amino acids that contributes 0.75 nm to the putative lever arm helix. FLAG-tag of Myo1d and (His)8-tag of D. discoideum myosin II (Ruff et al., 2001) are not included in this calculation. (D) Analysis for possible substep displacements. Example showing synchronized and averaged records from the interaction of a single actin filament with Myo1d-1IQ (n = 69). We detect no substeps with this analysis, as the average beginning and ending positions differ by <1 nm. In agreement with the histogram analysis, this analysis predicts a step size of just below 10 nm.
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fig3: Single molecule step sizes of different rat Myo1d constructs. (A) Displacement records showing the interaction of a single actin filament with recombinant rat Myo1d molecules encompassing variable numbers of light chain binding sites. For each construct, representative traces of raw needle displacements (top trace) and the corresponding running variance recordings (bottom trace) are shown. During the attachment of a motor molecule, the fluctuation of the bead–filament–needle complex is significantly reduced. The records contain many actomyosin interactions of variable length; in each panel we highlighted one interaction by an asterisk. Attachments occur over the entire range of the free needle positions. According to Molloy and coworkers (Molloy et al., 1995), the positions of several events show a Gaussian distribution shifted for a distance equivalent to the step size. (B) Distribution of events for the different constructs, summarizing the (oriented) results of several measurements. (C) Step sizes are plotted against the putative lever arm lengths of the various rat Myo1d proteins (circles). The putative lever arm length was calculated by assuming an α-helical conformation of the light chain binding domain. The corresponding solid line represents the best fit of a linear regression analysis. For comparison, previously reported data of D. discoideum myosin II (Dd II, squares; Ruff et al., 2001) and chicken smooth muscle myosin II (sm II, triangles; Warshaw et al., 2000) are also included. The Myo 1d-head construct possesses a COOH-terminal linker of five amino acids that contributes 0.75 nm to the putative lever arm helix. FLAG-tag of Myo1d and (His)8-tag of D. discoideum myosin II (Ruff et al., 2001) are not included in this calculation. (D) Analysis for possible substep displacements. Example showing synchronized and averaged records from the interaction of a single actin filament with Myo1d-1IQ (n = 69). We detect no substeps with this analysis, as the average beginning and ending positions differ by <1 nm. In agreement with the histogram analysis, this analysis predicts a step size of just below 10 nm.

Mentions: Having established that these three Myo1d constructs exhibit biochemical properties of a motor molecule, we tested them for mechanical activities by single molecule measurements using a combined microneedle laser trap system (Ruff et al., 2001). All three constructs were able to produce directed force along actin filaments as monitored by microneedle displacements that were correlated with a reduction in running variance of the microneedle to below 5 nm2 (Fig. 3 A), proving that rat Myo1d is a bona fide motor molecule. Interestingly, the Myo1d motor constructs showed step sizes about three times as large as those reported previously for conventional class II myosins (Molloy et al., 1995; Mehta et al., 1997; Ruff et al., 2001). The step size of the Myo1d head was 4.9 nm, the step size of the Myo1d head-1IQ was 9.3 nm, and finally, the step size of the Myo1d head-2IQ was 13.9 nm (Fig. 3 B and Table I). Only one step per power stroke was resolved when records from all three constructs were analyzed for step amplitudes at the start and end of the interactions with a time resolution of better than 5 ms (Fig. 3 D). For all three constructs, we observed differences at the beginning and end of average records of <1 nm, suggesting that Myo1d either moves in a single step, or the substeps follow in rapid succession (<5 ms separation) that we could not detect them. This distinguishes the movement of rat Myo1d from the movements observed for chicken Myo1a and rat Myo1b, which moved in two discrete steps per power stroke (Veigel et al., 1999).


Different degrees of lever arm rotation control myosin step size.

Köhler D, Ruff C, Meyhöfer E, Bähler M - J. Cell Biol. (2003)

Single molecule step sizes of different rat Myo1d constructs. (A) Displacement records showing the interaction of a single actin filament with recombinant rat Myo1d molecules encompassing variable numbers of light chain binding sites. For each construct, representative traces of raw needle displacements (top trace) and the corresponding running variance recordings (bottom trace) are shown. During the attachment of a motor molecule, the fluctuation of the bead–filament–needle complex is significantly reduced. The records contain many actomyosin interactions of variable length; in each panel we highlighted one interaction by an asterisk. Attachments occur over the entire range of the free needle positions. According to Molloy and coworkers (Molloy et al., 1995), the positions of several events show a Gaussian distribution shifted for a distance equivalent to the step size. (B) Distribution of events for the different constructs, summarizing the (oriented) results of several measurements. (C) Step sizes are plotted against the putative lever arm lengths of the various rat Myo1d proteins (circles). The putative lever arm length was calculated by assuming an α-helical conformation of the light chain binding domain. The corresponding solid line represents the best fit of a linear regression analysis. For comparison, previously reported data of D. discoideum myosin II (Dd II, squares; Ruff et al., 2001) and chicken smooth muscle myosin II (sm II, triangles; Warshaw et al., 2000) are also included. The Myo 1d-head construct possesses a COOH-terminal linker of five amino acids that contributes 0.75 nm to the putative lever arm helix. FLAG-tag of Myo1d and (His)8-tag of D. discoideum myosin II (Ruff et al., 2001) are not included in this calculation. (D) Analysis for possible substep displacements. Example showing synchronized and averaged records from the interaction of a single actin filament with Myo1d-1IQ (n = 69). We detect no substeps with this analysis, as the average beginning and ending positions differ by <1 nm. In agreement with the histogram analysis, this analysis predicts a step size of just below 10 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172898&req=5

fig3: Single molecule step sizes of different rat Myo1d constructs. (A) Displacement records showing the interaction of a single actin filament with recombinant rat Myo1d molecules encompassing variable numbers of light chain binding sites. For each construct, representative traces of raw needle displacements (top trace) and the corresponding running variance recordings (bottom trace) are shown. During the attachment of a motor molecule, the fluctuation of the bead–filament–needle complex is significantly reduced. The records contain many actomyosin interactions of variable length; in each panel we highlighted one interaction by an asterisk. Attachments occur over the entire range of the free needle positions. According to Molloy and coworkers (Molloy et al., 1995), the positions of several events show a Gaussian distribution shifted for a distance equivalent to the step size. (B) Distribution of events for the different constructs, summarizing the (oriented) results of several measurements. (C) Step sizes are plotted against the putative lever arm lengths of the various rat Myo1d proteins (circles). The putative lever arm length was calculated by assuming an α-helical conformation of the light chain binding domain. The corresponding solid line represents the best fit of a linear regression analysis. For comparison, previously reported data of D. discoideum myosin II (Dd II, squares; Ruff et al., 2001) and chicken smooth muscle myosin II (sm II, triangles; Warshaw et al., 2000) are also included. The Myo 1d-head construct possesses a COOH-terminal linker of five amino acids that contributes 0.75 nm to the putative lever arm helix. FLAG-tag of Myo1d and (His)8-tag of D. discoideum myosin II (Ruff et al., 2001) are not included in this calculation. (D) Analysis for possible substep displacements. Example showing synchronized and averaged records from the interaction of a single actin filament with Myo1d-1IQ (n = 69). We detect no substeps with this analysis, as the average beginning and ending positions differ by <1 nm. In agreement with the histogram analysis, this analysis predicts a step size of just below 10 nm.
Mentions: Having established that these three Myo1d constructs exhibit biochemical properties of a motor molecule, we tested them for mechanical activities by single molecule measurements using a combined microneedle laser trap system (Ruff et al., 2001). All three constructs were able to produce directed force along actin filaments as monitored by microneedle displacements that were correlated with a reduction in running variance of the microneedle to below 5 nm2 (Fig. 3 A), proving that rat Myo1d is a bona fide motor molecule. Interestingly, the Myo1d motor constructs showed step sizes about three times as large as those reported previously for conventional class II myosins (Molloy et al., 1995; Mehta et al., 1997; Ruff et al., 2001). The step size of the Myo1d head was 4.9 nm, the step size of the Myo1d head-1IQ was 9.3 nm, and finally, the step size of the Myo1d head-2IQ was 13.9 nm (Fig. 3 B and Table I). Only one step per power stroke was resolved when records from all three constructs were analyzed for step amplitudes at the start and end of the interactions with a time resolution of better than 5 ms (Fig. 3 D). For all three constructs, we observed differences at the beginning and end of average records of <1 nm, suggesting that Myo1d either moves in a single step, or the substeps follow in rapid succession (<5 ms separation) that we could not detect them. This distinguishes the movement of rat Myo1d from the movements observed for chicken Myo1a and rat Myo1b, which moved in two discrete steps per power stroke (Veigel et al., 1999).

Bottom Line: We analyzed the step size of rat myosin 1d (Myo1d) and surprisingly found that this myosin takes unexpectedly large steps in comparison to other myosins.Engineering the length of the light chain binding domain of rat Myo1d resulted in a linear increase of step size in relation to the putative lever arm length, indicative of a lever arm rotation of the light chain binding domain.These results demonstrate that differences in myosin step sizes are not only controlled by lever arm length, but also by substantial differences in the degree of lever arm rotation.

View Article: PubMed Central - PubMed

Affiliation: Institute for General Zoology and Genetics, Westfälische Wilhelms-University, Schlossplatz 5, 48149 Münster, Germany.

ABSTRACT
Myosins are actin-based motors that are generally believed to move by amplifying small structural changes in the core motor domain via a lever arm rotation of the light chain binding domain. However, the lack of a quantitative agreement between observed step sizes and the length of the proposed lever arms from different myosins challenges this view. We analyzed the step size of rat myosin 1d (Myo1d) and surprisingly found that this myosin takes unexpectedly large steps in comparison to other myosins. Engineering the length of the light chain binding domain of rat Myo1d resulted in a linear increase of step size in relation to the putative lever arm length, indicative of a lever arm rotation of the light chain binding domain. The extrapolated pivoting point resided in the same region of the rat Myo1d head domain as in conventional myosins. Therefore, rat Myo1d achieves its larger working stroke by a large calculated approximately 90 degrees rotation of the light chain binding domain. These results demonstrate that differences in myosin step sizes are not only controlled by lever arm length, but also by substantial differences in the degree of lever arm rotation.

Show MeSH
Related in: MedlinePlus