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Notes The authors declare no competing financial interest. Nanotechnology , 22 ,

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Charged residues such as aspartic acid, glutamic acid, and lysine are proposed to be involved in the interaction between the monomers of SbsB nanosheets, 23 suggesting that similar interaction occur between SbpA monomers. Because light scattering gives a dynamic readout of the nanosheet growth, we sought to predict nanosheet size over the range of solution conditions examined above and develop a mechanism for self-assembly.

These diagrams provide a guide for creating SbpA nanosheets of a distribution of sizes by varying the ionic strength of the solution and the reaction time. Self-assembly by SbpA corresponds to irreversible growth. The lengths were calculated using the equation for scattering by dielectric discs and using a sheet thickness of 10 nm. The color map is capped at nm due to the variability of the lengths calculated with high light scattering.

Fits to eq 4 are shown as dashed curves in the same color. We did not fit traces from concentrations below 4. These sheet lengths are consistent with the end point sheet sizes observed in the STEM images. The quality of the fits demonstrates that a substantial nucleation barrier is not necessary to explain the sigmoid shape of the light scattering traces; rather, the sigmoid shape is a natural consequence of the nonlinear dependence of light scattering on sheet size.

Our results highlight the usefulness of mapping out diagrams for understanding self-assembling systems and indicate several means to control the size and morphology of SbpA-based nanostructures. Previously, solution controls on protein self-assembly have been mapped out using phase diagrams constructed using end point turbidity measurements. Thus, we suggest that such diagrams coupled with modeling of the self-assembly process may provide a useful method to determine critical kinetic controls of proteins and polymers into nanostructures.

Additionally, our results identify conditions to initiate and halt self-assembly to produce SbpA nanosheets of an average size in high yield. This information provides a first example of using self-assembly to tune the size of 2D protein nanostructures with long-range order and will be useful for patterning inorganic nanomaterials at defined length scales, potentially opening the door to efforts to design self-assembling multicomponent, mesoscale S-layer lattices.

SbpA was purified as previously described. Because of the absorption of light by proteins at lower wavelengths, nanosheet self-assembly by SbpA was monitored via the scattering of light at nm. All buffers were made using Milli-Q water. Solutions were mixed and then added into wells on a Costar UV clear, well flat bottom plate and covered with an optically clear seal to prevent evaporation Corning Inc.

The calculated path length for each well is 2. Data were corrected by subtracting the absorbance at zero time, A nm, t 0. Data were exported to and analyzed in Origin 8. The solution of SbpA was incubated for 2 min at room temperature on the grid. The drop was removed by absorbing the liquid with filter paper. The grid was washed twice by pipetting Milli-Q water on the surface, then removing excess liquid with filter paper.

Finally, the protein sheets were stained for 2 min with one drop of NanoW solution Nanoprobes Inc. Images were captured using SmartSEM software.

The histogram of SbpA crystal lengths were plotted and fit to a Gaussian distribution in Origin 8. The refractive index, n , of the protein was set to 1. Measurements were repeated 3 times for each sample, and replicated 2—3 times on different days. The cell was then filled with the solution containing protein and sealed.

Measurements were taken within 1—2 min of preparation, prior to formation of crystals in solution. To calculate the light scattering signal as a function of nanosheet length, the square sheets were approximated as thin circular disks of equivalent cross section, thickness, and uniform dielectric constant. This approximation allows for the application of a known solution for the a scattering efficiency factor, Q , as detailed in the Supporting Information Methods.

The index of refraction for S-layer proteins is set to 1. A nm cutoff filter was mounted in front of the emission port in the instrument to prevent interference from harmonic doubling. The sample was illuminated at nm with 5 nm slit-width. Emission spectra were collected using 5 nm slit width. We used this concentration of SbpA to avoid self-assembly in solution and measure only the binding of ions to the proteins. The solution was excited at nm with a slit-width of 5 nm, and the emission was monitored at nm after being filtered through a nm cutoff filter.

Stock solutions of 2 and 10 mM TbCl 3 in water were used to titrate the ion in solution. The solution was allowed to equilibrate for 1 min after each titration, and then the shutters were open to take a reading for 30 s.

The fluorescence was recorded and corrected as described above. The data were fit to the general solution for competitive binding of two ligands to a macromolecule. Light scattering data were imported into and fit to the equations described below in Mathematica 7 Wolfram Research, Champaign, IL. The growth of sheets was modeled by assuming that a fraction f of proteins serves as nucleation sites, with subsequent growth driven by adsorption of proteins onto the perimeter of the sheets.

We assume that the growth of a sheet composed of N t monomers is given by. The last factor in eq 4 accounts for saturation when all monomers are incorporated into the growing sheet. In this case eq 4 can be written as. Combining the above results and integrating eq 5 yields the following solution for sheet size:.

These parameters were then used in fits of the light scattering calculation eq 1 to the light scattering data to determine the best-fit protein index of refraction. De Yoreo and R. Tscheliessnig for helpful discussions. Department of Energy under Contract No. This material is available free of charge via the Internet at http: National Center for Biotechnology Information , U. Published online Dec Author information Article notes Copyright and License information Disclaimer.

Received Jun 2; Accepted Dec This is an open access article published under an ACS AuthorChoice License , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

This article has been cited by other articles in PMC. Open in a separate window. Results and Discussion Light Scattering Experiments Measure Growth of Nanosheets of SbpA While the self-assembly of SbpA can be measured ex situ by scanning transmission electron microscopy STEM or atomic force microscopy AFM , the throughput of these techniques is too low to allow monitoring of self-assembly yield and dynamics over a wide range of conditions.

The Dynamics of Self-Assembly of SbpA Map out the Time-Evolution of Nanosheet Size and Are Consistent with Irreversible Growth from a Negligibly Small Nucleus Because light scattering gives a dynamic readout of the nanosheet growth, we sought to predict nanosheet size over the range of solution conditions examined above and develop a mechanism for self-assembly.

Light Scattering Assay Because of the absorption of light by proteins at lower wavelengths, nanosheet self-assembly by SbpA was monitored via the scattering of light at nm. Calculating Light Scattering from Nanosheets To calculate the light scattering signal as a function of nanosheet length, the square sheets were approximated as thin circular disks of equivalent cross section, thickness, and uniform dielectric constant. Acknowledgments We thank J.

Notes The authors declare no competing financial interest. Nanoscale , 2 , — Soft Matter , 8 , — Biogenesis and Functions of Bacterial S-Layers. Structural Research on Surface Layers: Nature , , — Nanotechnology , 22 , Biochemistry , 43 , — Lanthanide-Tagged Proteins—an Illuminating Partnership.

In Kinetic Analysis of Macromolecules: A Practical Approach ; Johnson K. ACS Nano , 5 , — You can uncheck the check box to stop trusting the certificate. If there is an empty check box next to the certificate you want to trust, you can click the check box to trust it. If there is a check mark but no check box, then you trust the certificate because your administrator has decided the trust for you. If no certificates appear, then the certificates cannot be found. Notes certificate authorities always use this naming style when issuing certificates.

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