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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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Related in: MedlinePlus

Schematic representation of the domain organization of wild-type rat Myo1d (wt) and the three recombinant constructs used in this work. Motor domains (gray), IQ motifs (white), and tail domain (black) are indicated. All recombinant constructs contain a COOH-terminal FLAG-epitope (circle). Amino acids of rat Myo1d, linker residue(s) (italics), and the FLAG-epitope sequence (underlined) included in the three different constructs are given.
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fig1: Schematic representation of the domain organization of wild-type rat Myo1d (wt) and the three recombinant constructs used in this work. Motor domains (gray), IQ motifs (white), and tail domain (black) are indicated. All recombinant constructs contain a COOH-terminal FLAG-epitope (circle). Amino acids of rat Myo1d, linker residue(s) (italics), and the FLAG-epitope sequence (underlined) included in the three different constructs are given.

Mentions: To investigate whether rat Myo1d is indeed a motor, and if so, whether its step size is determined by the same parameters as in conventional class II myosins, we expressed three recombinant rat Myo1d motors with different numbers of light chain binding sites in HeLa cells (Fig. 1) . For ease of purification, all our Myo1d constructs were FLAG-tagged at their COOH termini. Affinity purification yielded virtually pure Myo1d head domain and Myo1d head domains encompassing either one or two IQ motifs (Fig. 2 A). Endogenous calmodulin was copurified with the rat Myo1d head domains containing one or two IQ motifs (Fig. 2 B and Table I). The molar ratios of calmodulin to rat Myo1d were determined to be 1.03 ± 0.05 for Myo1d head-1IQ and 1.74 ± 0.15 for Myo1d head-2IQ, respectively. No calmodulin was copurified with the Myo1d head domain that contained no IQ motif (Fig. 2 B). All three constructs exhibited actin-activated ATPase activity. In the absence of actin, the ATPase activities were barely measurable (0.01–0.06 s−1). However, in the presence of actin, the ATPase activities reached Vmax values of 2–3 s−1 with KM values for actin of 35–40 μM (Table I and Fig. 2 C). Notably, the addition of one or two IQ motifs to the Myo1d head did not significantly change ATPase rates and actin affinities.


Different degrees of lever arm rotation control myosin step size.

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

Schematic representation of the domain organization of wild-type rat Myo1d (wt) and the three recombinant constructs used in this work. Motor domains (gray), IQ motifs (white), and tail domain (black) are indicated. All recombinant constructs contain a COOH-terminal FLAG-epitope (circle). Amino acids of rat Myo1d, linker residue(s) (italics), and the FLAG-epitope sequence (underlined) included in the three different constructs are given.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Schematic representation of the domain organization of wild-type rat Myo1d (wt) and the three recombinant constructs used in this work. Motor domains (gray), IQ motifs (white), and tail domain (black) are indicated. All recombinant constructs contain a COOH-terminal FLAG-epitope (circle). Amino acids of rat Myo1d, linker residue(s) (italics), and the FLAG-epitope sequence (underlined) included in the three different constructs are given.
Mentions: To investigate whether rat Myo1d is indeed a motor, and if so, whether its step size is determined by the same parameters as in conventional class II myosins, we expressed three recombinant rat Myo1d motors with different numbers of light chain binding sites in HeLa cells (Fig. 1) . For ease of purification, all our Myo1d constructs were FLAG-tagged at their COOH termini. Affinity purification yielded virtually pure Myo1d head domain and Myo1d head domains encompassing either one or two IQ motifs (Fig. 2 A). Endogenous calmodulin was copurified with the rat Myo1d head domains containing one or two IQ motifs (Fig. 2 B and Table I). The molar ratios of calmodulin to rat Myo1d were determined to be 1.03 ± 0.05 for Myo1d head-1IQ and 1.74 ± 0.15 for Myo1d head-2IQ, respectively. No calmodulin was copurified with the Myo1d head domain that contained no IQ motif (Fig. 2 B). All three constructs exhibited actin-activated ATPase activity. In the absence of actin, the ATPase activities were barely measurable (0.01–0.06 s−1). However, in the presence of actin, the ATPase activities reached Vmax values of 2–3 s−1 with KM values for actin of 35–40 μM (Table I and Fig. 2 C). Notably, the addition of one or two IQ motifs to the Myo1d head did not significantly change ATPase rates and actin affinities.

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