10 research outputs found
Comparison between actin decorated with wild-type smooth muscle myosin S1 and actin decorated with a R403Q smooth muscle myosin mutant.
<p>A: Wild-type smooth muscle actomyosin in the presence of ADP. The top row shows a 4-nm thick central slice perpendicular to the filament axis. The second row shows a 4-nm thick central slice parallel to the filament axis (cutting plane indicated by the left line in top row). The pointed end is to the top of the figure in the two lower rows. The bottom row shows an adjacent 4-nm thick slice (cutting plane indicated by right line in top row). This slice goes primarily through the S1 molecules. B: R403Q mutant smooth muscle actomyosin in the presence of ADP. Organization of rows as in (A). C: Wild-type smooth muscle actomyosin in the absence of nucleotide (apo). The approximate location of the motor domain (MD) and the light chain (LC) regions are labeled in each view. Organization of rows as in A. The approximate outline of the actin portion of the filament is indicated. D: Apo R403Q mutant smooth muscle actomyosin. Organization of rows as in (A). Bar:10 nm.</p
Difference and structural variability maps.
<p>4-nm wide slices perpendicular to the helix axis of several maps are shown on the left. Only peaks significant at a confidence level of 99.99% are shown. A: Wild-type smooth muscle actomyosin in the absence of nucleotide. The motor domain (MD) and light-chain (LC) regions are labeled. A faint ghost image of this map is also overlaid on C and E to aid visualization. B: A difference map generated by subtracting the R403Q mutant smooth muscle actomyosin apo state reconstruction from the R403Q mutant smooth muscle actomyosin ADP state reconstruction. A clear difference peak can be identified in the light-chain region. A faint ghost image of the wild-type ADP state reconstruction is overlaid to aid visualization. This image is also overlaid on D and F. C: Difference map between two independently generated R403Q mutant apo state reconstructions. Only occasional, randomly distributed, isolated pixels can be seen, no coherent difference peaks exist. D: Difference map between two independently generated R403Q mutant ADP state reconstructions. E: Structural variability (AVID map) of R403Q mutant apo state reconstruction. Only randomly distributed peaks can be seen, there is no consistent structural variability in any confined region. F: Structural variability (AVID map) of R403Q mutant ADP state reconstruction. Bar:10 nm. G: Surface representation of the difference map shown in B. The cyan density represents additional density in the R403Q mutant ADP state reconstruction if compared to the R403Q mutant apo state reconstruction. The apo state (pink) and ADP state (blue wireframe) wild-type reconstructions are also shown. The difference between the mutant reconstructions is located in the light-chain region and correlates with the changes observed in wild-type smooth muscle actomyosin.</p
Surface representations of smooth muscle actomyosin constructs.
<p>The pointed end of the filaments is towards the top of the figure. A: Wild-type smooth muscle actomyosin in the presence of ADP. B: Wild-type smooth muscle actomyosin in the apo state. The contour level for A and B is chosen to represent the correct molecular mass. Note the well defined density and angle of the light-chain region (LC) C: R403Q mutant smooth muscle actomyosin in the presence of ADP. D: R403Q mutant smooth muscle actomyosin in the apo state. The contour level for C and D is chosen to show as much of the light chain domains as possible without completely obscuring the shape of the motor domain. E: Overlay of the maps in A–D. Color code and contouring as in A–D. F, G: Watershed segmentation of the maps in C (F) and D (G). These results reconfirm the orientation of the light-chain domains that correspond to those of the wild-type reconstructions (sketches) and the better definition of boundaries in the center of the filaments: the actin subunits are well segmented while there is no sub-segmentation of myosin domains as can be obtained for wild-type reconstructions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001123#pone.0001123-Volkmann4" target="_blank">[33]</a>. The sketches show central lines extracted from the density of the wild-type (grey) and the segmentation of the R403Q (black). Only the line segments extracted for the corresponding light-chain regions (LC) are shown, line segments corresponding to the motor domain region (MD) overlap almost completely for all maps.</p
Proximity of residue Arg<sup>406</sup> (Arg<sup>403</sup> in cardiac myosin) to the actin interface.
<p>Arg<sup>406</sup> (red spheres) is immediately adjacent to residues of the cardiomyopathy loop that were previously implicated in actin binding by docking studies (407–414; green). While the conformation of the Arg<sup>406</sup> in the smooth muscle myosin crystal structure points away from the interface (A), it can easily reach actin by simple, stereochemically permitted bond angle rotations (B). The resulting conformation does not generate serious clashes with other myosin residues. Myosin is shown in blue, the interacting actin filament subunits in grey. Residue Pro<sup>333</sup> of actin, the closest to myosin Arg<sup>406</sup>, is shown as spheres.</p
Data collection
a<p>This counts split data (i.e. far and near side) as different data sets (see text for details).</p>b<p>All statistics are calculated over the respective number of data sets.</p>c<p>The phase residual is a measure for the homogeneity and quality of the data. Values below 45° are considered excellent; values below 55° are acceptable.</p
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Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots
We
develop magnetic cytoskeleton affinity (MiCA) purification,
which allows for rapid isolation of molecular motors conjugated to
large multivalent quantum dots, in miniscule quantities, which is
especially useful for single-molecule applications. When purifying
labeled molecular motors, an excess of fluorophores or labels is usually
used. However, large labels tend to sediment during the centrifugation
step of microtubule affinity purification, a traditionally powerful
technique for motor purification. This is solved with MiCA, and purification
time is cut from 2 h to 20 min, a significant time-savings when it
needs to be done daily. For kinesin, MiCA works with as little as
0.6 μg protein, with yield of ∼27%, compared to 41% with
traditional purification. We show the utility of MiCA purification
in a force-gliding assay with kinesin, allowing, for the first time,
simultaneous determination of whether the force from each motor in
a multiple-motor system drives or hinders microtubule movement. Furthermore,
we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD
(DDB-QD), ordinarily a difficult protein-complex to purify
Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots
We
develop magnetic cytoskeleton affinity (MiCA) purification,
which allows for rapid isolation of molecular motors conjugated to
large multivalent quantum dots, in miniscule quantities, which is
especially useful for single-molecule applications. When purifying
labeled molecular motors, an excess of fluorophores or labels is usually
used. However, large labels tend to sediment during the centrifugation
step of microtubule affinity purification, a traditionally powerful
technique for motor purification. This is solved with MiCA, and purification
time is cut from 2 h to 20 min, a significant time-savings when it
needs to be done daily. For kinesin, MiCA works with as little as
0.6 μg protein, with yield of ∼27%, compared to 41% with
traditional purification. We show the utility of MiCA purification
in a force-gliding assay with kinesin, allowing, for the first time,
simultaneous determination of whether the force from each motor in
a multiple-motor system drives or hinders microtubule movement. Furthermore,
we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD
(DDB-QD), ordinarily a difficult protein-complex to purify
Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots
We
develop magnetic cytoskeleton affinity (MiCA) purification,
which allows for rapid isolation of molecular motors conjugated to
large multivalent quantum dots, in miniscule quantities, which is
especially useful for single-molecule applications. When purifying
labeled molecular motors, an excess of fluorophores or labels is usually
used. However, large labels tend to sediment during the centrifugation
step of microtubule affinity purification, a traditionally powerful
technique for motor purification. This is solved with MiCA, and purification
time is cut from 2 h to 20 min, a significant time-savings when it
needs to be done daily. For kinesin, MiCA works with as little as
0.6 μg protein, with yield of ∼27%, compared to 41% with
traditional purification. We show the utility of MiCA purification
in a force-gliding assay with kinesin, allowing, for the first time,
simultaneous determination of whether the force from each motor in
a multiple-motor system drives or hinders microtubule movement. Furthermore,
we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD
(DDB-QD), ordinarily a difficult protein-complex to purify
Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots
We
develop magnetic cytoskeleton affinity (MiCA) purification,
which allows for rapid isolation of molecular motors conjugated to
large multivalent quantum dots, in miniscule quantities, which is
especially useful for single-molecule applications. When purifying
labeled molecular motors, an excess of fluorophores or labels is usually
used. However, large labels tend to sediment during the centrifugation
step of microtubule affinity purification, a traditionally powerful
technique for motor purification. This is solved with MiCA, and purification
time is cut from 2 h to 20 min, a significant time-savings when it
needs to be done daily. For kinesin, MiCA works with as little as
0.6 μg protein, with yield of ∼27%, compared to 41% with
traditional purification. We show the utility of MiCA purification
in a force-gliding assay with kinesin, allowing, for the first time,
simultaneous determination of whether the force from each motor in
a multiple-motor system drives or hinders microtubule movement. Furthermore,
we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD
(DDB-QD), ordinarily a difficult protein-complex to purify
Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots
We
develop magnetic cytoskeleton affinity (MiCA) purification,
which allows for rapid isolation of molecular motors conjugated to
large multivalent quantum dots, in miniscule quantities, which is
especially useful for single-molecule applications. When purifying
labeled molecular motors, an excess of fluorophores or labels is usually
used. However, large labels tend to sediment during the centrifugation
step of microtubule affinity purification, a traditionally powerful
technique for motor purification. This is solved with MiCA, and purification
time is cut from 2 h to 20 min, a significant time-savings when it
needs to be done daily. For kinesin, MiCA works with as little as
0.6 μg protein, with yield of ∼27%, compared to 41% with
traditional purification. We show the utility of MiCA purification
in a force-gliding assay with kinesin, allowing, for the first time,
simultaneous determination of whether the force from each motor in
a multiple-motor system drives or hinders microtubule movement. Furthermore,
we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD
(DDB-QD), ordinarily a difficult protein-complex to purify