Self-Sorting of Foreign Proteins in a Bacterial Nanocompartment

Nature uses bottom-up approaches for the controlled assembly of highly ordered hierarchical structures with defined functionality, such as organelles, molecular motors, and transmembrane pumps. The field of bionanotechnology draws inspiration from nature by utilizing biomolecular building blocks such as DNA, proteins, and lipids, for the (self-) assembly of new structures for applications in biomedicine, optics, or electronics. Among the toolbox of available building blocks, proteins that form cage-like structures, such as viruses and virus-like particles, have been of particular interest since they form highly symmetrical assemblies and can be readily modified genetically or chemically both on the outer or inner surface. Bacterial encapsulins are a class of <i>nonviral</i> protein cages that self-assemble <i>in vivo</i> into stable icosahedral structures. Using teal fluorescent proteins (TFP) engineered with a specific native C-terminal docking sequence, we report the molecular self-sorting and selective packaging of TFP cargo into bacterial encapsulins during <i>in vivo</i> assembly. Using native mass spectrometry techniques, we show that loading of either monomeric or dimeric TFP cargo occurs with unprecedented high fidelity and exceptional loading accuracy. Such self-assembling systems equipped with self-sorting capabilities would open up exciting opportunities in nanotechnology, for example, as artificial (molecular storage or detoxification) organelles or as artificial cell factories for <i>in situ</i> biocatalysis

Dynamic evolution of mechanical degrees of freedom and survival probability for CCMV shell.

<p>Panel (a) exemplifies the dynamics of Hertzian deformation <i>x</i>_<i>H</i> and beam-bending deformation <i>x</i>_<i>b</i> vs. <i>X</i> in the Hertzian regime I and in the transition regime II. Model calculations are performed using parameter values obtained from the fit of theoretical <i>FX</i>-curves to the simulated average <i>FX</i>-spectra for CCMV nanoindentation along the 2-fold symmetry axis (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#pcbi.1004729.t001" target="_blank">Table 1</a>). The solid curves correspond to the exact method of parameter estimation; the dashed and dashed-dotted curves are for the (piece-wise) approximate method of estimation. Snapshots exemplify the local flattening of CCMV structure under the tip for <i>X</i> = 1 nm and 5 nm deformation. Panel (b) displays the results of overlap function <i>χ</i>-based estimation of the survival probability <i>s</i>(<i>X</i>) from simulations of CCMV nanoindentation (<i>ν</i>_<i>f</i> = 1.0 <i>μ</i>m/s, <i>R</i>_<i>tip</i> = 20 nm, and <i>κ</i> = 0.05 N/m; <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#pcbi.1004729.s007" target="_blank">S3a Fig</a>) along the 2-fold (red), quasi-3-fold (blue), and quasi-2-fold symmetry axes (green). The theoretical profiles of <i>s</i>(<i>X</i>) (solid curves; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#pcbi.1004729.e055" target="_blank">Eq (16)</a>) are compared with the simulated profiles of <i>χ</i>(<i>X</i>) (data points; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#pcbi.1004729.e053" target="_blank">Eq (15)</a>). The model parameters are summarized in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#pcbi.1004729.t001" target="_blank">Table 1</a>. The values of are obtained using Lagrange multipliers and the approximate method of parameter estimation (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#sec010" target="_blank">Discussion</a>).</p

Stress distribution on CCMV shell surface.

<p>Map of the Cauchy stress tensor projections along the direction of out-of-plane bending deformation (left) and tangential in-plane stretching (right) for different deformation <i>X</i> of the CCMV shell and corresponding indentation force <i>F</i> (indentation along the 2-fold symmetry axis with <i>R</i>_<i>tip</i> = 20 nm and <i>ν</i>_<i>f</i> = 1.0 <i>μ</i>m/s). For each amino acid residue (<i>C</i>_<i>α</i>-particle), the stress components are averaged over amino acids within a sphere of radius <i>R</i>_<i>C</i> = 15 Å (color denotation is presented in the graph). Also shown are formation and subsequent evolution of microscopic cracks in the side portion (particle barrel) of CCMV structure (shown in red circle/ellipse).</p

Deformation and collapse of biological particles—CCMV, TrV, and AdV.

<p>Accumulated are the Young’s moduli for Hertzian <i>E</i>_<i>H</i> and bending <i>E</i>_<i>b</i> deformations, the beam strength and the cooperativity parameter <i>m</i>. The first (second) entries correspond to the exact (approximate) methods of parameter estimation. The model predictions for <i>F</i>^<i>col</i> are compared with the peak forces (in parenthesis) from the spectra (Figs <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#pcbi.1004729.g004" target="_blank">4</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#pcbi.1004729.g005" target="_blank">5</a>). For TrV and AdV particles, the shell thickness was estimated as described in the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004729#pcbi.1004729.s003" target="_blank">S3 Text</a>.</p

Types of mechanical excitations exemplified using the CCMV shell.

<p>(a)-(c) Hertzian deformation <i>x</i>_<i>H</i> with normal displacements <i>u</i>_<i>tip</i> and <i>u</i>_<i>par</i> (scheme on (a)) under the influence of force (vertical arrow). Dashed contour lines show the tip and particle in their undeformed states. Structures in (b)—the native (left) and partially deformed (right) states show an amplitude of <i>x</i>_<i>H</i> ≈ 3 nm. (c) CCMV shell profile showing parts of the structure with high potential energy (>3 kcal/mol per residue; red) and low potential energy (blue). (d)-(f) Bending deformation. The side portion of the structure (barrel) is partitioned into curved beams (top-side view on (d)). Structures in (e)—the partially deformed (left) and pre-collapse (middle and right) states reveal the amplitude of <i>x</i>_<i>b</i> ≈ 4.3 nm. (f) CCMV shell profile under Hertzian and bending deformations showing the potential energy distribution.</p