A sufficiently large population of αTm molecules is required for reliable measurement of L_p.

<p>L_p was calculated for each of 200 subsets of N αTm (N = 10, 20, 40…) randomly selected from a total of 1852 molecules. The mean L_p is plotted against N. A consistent overestimation of L_p up to 8 nm was observed for small N (<80–160). This overestimation can be attributed to the biased distribution of contour shapes, where highly bent configurations of the molecule are relatively rare. In case of small N, therefore, the highly bent configurations are under- or un-sampled, which leads to overestimation of L_p based on a biased population of relatively straight molecules.</p

Tangent angle correlation analysis shows that L_p of WT human cardiac αTm equals 40.6−45.8 nm.

<p>ln() data obtained from three separate samples independently prepared under identical conditions are plotted as a function of segment length along the molecular contour. The slope of this plot is –1/2L_p. L_p for WT Tm from this analysis were 45.8±0.8 nm (N = 741, R² = 0.99), 43.5±0.8 nm (N = 628, R² = 0.98) and 40.6±0.8 nm (N = 798, R² = 0.98). The variation in the L_p values represents the uncertainty inherent to our experimental setup and the tangent angle correlation analysis.</p

Summary of L_c, L_e-e, and L_p from tangent angle correlation, second moment and end-to-end length analyses.

*<p>0.01% p-Lys deposition on mica by 30 s incubation.</p>**<p>0.01% p-Lys deposition on mica by 300 s incubation.</p

Image processing procedure to extract the molecular contour of αTm from a typical AFM scan.

<p>An αTm molecule was selected from a typical 512 nm ×512 nm scan (A) and cropped into a smaller image (B). The image was filtered by a Gaussian box-car filter (C), thresholded (D), and skeletonized into a 1-pixel wide connected contour (E, F). A refined skeleton with coordinates defined at sub-pixel precision was generated by fitting the perpendicular height profiles to a Gaussian function (G), which was then fitted with a 5^th order polynomial. The continuous contour defined by the polynomial conformed very well with the shape of the original molecule (H). Contour length (L_c) and end-to-end length (L_e-e) of the molecule shown were 41.7 nm and 38.4 nm, respectively.</p

AFM images of α-tropomyosin (αTm) molecules.

<p>Wildtype human cardiac αTm was imaged dry on poly-lysine coated mica (A). Collage of 20 αTm molecules (B) show that molecular contours were smooth and continuous. One of the αTm molecules in the collage was processed as described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone-0039676-g002" target="_blank">Figure 2</a> C) and overlayed with the x-ray structure of αTm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone.0039676-Phillips2" target="_blank">[38]</a> on the same scale (B, expanded on right), which is evidence that the AFM images were good representations of single αTm molecules.</p

Deposition time study suggests L_p measurements were stable with incubation time of 300 s or longer.

<p>L_p values obtained by tangent angle correlation analysis (blue asterisks, left axis) and the corresponding number of molecules considered (black dots, right axis) were plotted against incubation times. An overestimation of L_p was observed at incubation times below 300 s, which may be due to the shorter incubation time and/or smaller number of molecules available for the analysis. L_p measurements were stable at incubation times above 300 s, where variation was comparable to the inherent uncertainty in our methodology. This implies an incubation time of 300 s was sufficient for surface equilibration of αTm on p-Lys coated mica substrate.</p

Selective Assembly and Guiding of Actomyosin Using Carbon Nanotube Network Monolayer Patterns

We report a new method for the selective assembly and guiding of actomyosin using carbon nanotube patterns. In this method, monolayer patterns of the single-walled carbon nanotube (swCNT) network were prepared via the self-limiting mechanism during the directed assembly process, and they were used to block the adsorption of both myosin and actin filaments on specific substrate regions. The swCNT network patterns were also used as an efficient barrier for the guiding experiments of actomyosin. This is the first result showing that inorganic nanostructures such as carbon nanotubes can be used to control the adsorption and activity of actomyosin. This strategy is advantageous over previous methods because it does not require complicated biomolecular linking processes and nonbiological nanostructures are usually more stable than biomolecular linkers

Deposition rate of αTm on p-Lys substrate shows the process is diffusion driven and irreversible.

<p>The number ratio between αTm molecules adhered to p-Lys coated mica and in 1 cm³ of the bulk solution, <i>N_s/N_B(t = 0)</i>, increased with incubation time up to 300 s, as shown in both linear (main graph) and logarithmic (inset) scales. 5% of total number of αTm molecules in the bulk solution were deposited on the substrate by the 300 s incubation; the absence of discernible change at longer incubation times of 450 s and 600 s suggests the top layer of the bulk solution was depleted of αTm molecules <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone.0039676-Rivetti1" target="_blank">[26]</a>. Fitting data from incubation time 10 s to 300 s to Eq. 1 (solid lines) returned estimates of two parameters: exponent parameter <i>p</i> equals 0.49, in close accordance to an irreversible and diffusion driven deposition process <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone.0039676-Rivetti1" target="_blank">[26]</a>; and diffusion constant parameter <i>D</i> of αTm equals 2.2×10⁻⁷ cm²/s, consistent with previous estimates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#pone.0039676-Cantor1" target="_blank">[43]</a>.</p

End-to-end length analysis shows L_p of WT human cardiac αTm consistent with tangent angle correlation analysis.

<p>Normalized end-to-end length (l_e-e) distributions from one of the WT αTm samples incubated on p-Lys coated mica substrate for 600 s (N = 798) fits to the WLC model (Eq. 3). <i>l_p</i> value from the fit was 1.0425±0.0505 (R² = 0.88). Errors were estimated by the jackknife method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039676#s2" target="_blank">Materials and Methods</a>).</p

Selective Assembly and Guiding of Actomyosin Using Carbon Nanotube Network Monolayer Patterns

We report a new method for the selective assembly and guiding of actomyosin using carbon nanotube patterns. In this method, monolayer patterns of the single-walled carbon nanotube (swCNT) network were prepared via the self-limiting mechanism during the directed assembly process, and they were used to block the adsorption of both myosin and actin filaments on specific substrate regions. The swCNT network patterns were also used as an efficient barrier for the guiding experiments of actomyosin. This is the first result showing that inorganic nanostructures such as carbon nanotubes can be used to control the adsorption and activity of actomyosin. This strategy is advantageous over previous methods because it does not require complicated biomolecular linking processes and nonbiological nanostructures are usually more stable than biomolecular linkers