Characterizing the Dynamic Disassembly/Reassembly Mechanisms of Encapsulin Protein Nanocages.

Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, represent a versatile set of tools for nanobiotechnology. However, a comprehensive understanding of the mechanisms underlying encapsulin self-assembly, disassembly, and reassembly is lacking. Here, we characterize the disassembly/reassembly properties of three encapsulin nanocages that possess different structural architectures: T = 1 (24 nm), T = 3 (32 nm), and T = 4 (42 nm). Using spectroscopic techniques and electron microscopy, encapsulin architectures were found to exhibit varying sensitivities to the denaturant guanidine hydrochloride (GuHCl), extreme pH, and elevated temperature. While all three encapsulins showed the capacity to reassemble following GuHCl-induced disassembly (within 75 min), only the smallest T = 1 nanocage reassembled after disassembly in basic pH (within 15 min). Furthermore, atomic force microscopy revealed that all encapsulins showed a significant loss of structural integrity after undergoing sequential disassembly/reassembly steps. These findings provide insights into encapsulins' disassembly/reassembly dynamics, thus informing their future design, modification, and application.

Introduction

Protein nanocages [e.g., virus-like particles (VLPs), ferritins, heat-shock proteins] self-assemble from multiple protein subunits into highly organized macromolecular structures, which exhibit well-defined inner cavities, outer surfaces, and interfaces between subunits. Their capacity to encapsulate cargo, coupled with the ability to genetically and/or chemically modify their structures, has enabled protein nanocages to be custom-engineered for a multitude of applications, including biocatalysis, materials synthesis, sensing, vaccines, and drug delivery.[1,2]

Encapsulins are an emerging class of protein nanocages found inside many archaea and bacteria. They self-assemble from identical protein subunits into hollow icosahedral nanocages that structurally resemble the major capsid protein gp5 of the HK97 virus.[3,4] Based on their triangulation number (T), all encapsulins exhibit one of the following three symmetrical icosahedral architectures: T = 1 (24 nm, 60-mer, 12 pentameric units), T = 3 (32 nm, 180-mer, 12 pentameric and 20 hexameric units), and T = 4 (42 nm, 240-mer, 12 pentameric and 30 hexameric units).[5−8] In nature, encapsulins house cargo enzymes that mediate oxidative stress resistance, iron storage, anaerobic ammonium oxidation, or sulfur metabolism.[9−12] Uniquely, encapsulins selectively self-assemble around cargo enzymes tagged with a small encapsulation signal peptide (ESig), packaging them.[5] This mechanism has been adapted to load foreign cargo into encapsulins, reprograming their functionality for different practical applications.[51]

Encapsulin subunits autonomously assemble, with extraordinary fidelity, into macromolecular nanocages. Such self-assembly is driven not only by folding of the individual polypeptide chains but also by dynamic noncovalent interactions between the different polypeptide chains both within subunits and at the interfaces between subunits in the assembled supramolecular structure.[16] Unraveling the self-assembly mechanisms of protein nanocages is complicated, especially if they exhibit highly symmetric homo-oligomeric structures, such as encapsulins.[17] Nevertheless, multiple analytical techniques now allow the molecular mechanisms underlying protein nanocage assembly (e.g., protein folding) to be characterized and subsequently exploited.

For instance, the disassembly/reassembly of protein nanocages belonging to the ferritin family have been studied via a combination of intrinsic tryptophan fluorescence (ITF), circular dichroism (CD), and UV/vis spectroscopy and synchrotron small-angle X-ray scattering measurements to assess the protein conformation;[18−21] transmission electron microscopy (TEM) and dynamic light scattering (DLS) to evaluate the structural integrity, shape, and size distribution; and laser light scattering to monitor the assembly kinetics.[22] One study revealed that ferritin disassembles at an extremely acidic pH 1.5 and then shows a rapid reassembly upon return to neutral pH 7.0, accompanied by folding, followed by a slow phase in which the final 24-mer nanocage is formed.[22] Importantly, this fundamental work led to the rational redesign of ferritin subunit interfaces, resulting in engineered nanocages capable of disassembly at a more amenable pH 4.0.[23] Such modification now permits labile compounds (e.g., small-molecule drugs) to be controllably loaded into ferritin nanocages in a facile and nondestructive manner, enabling downstream applications (e.g., drug delivery).[23]

In contrast, experimental data pertaining to encapsulins’ ability to disassemble/reassemble and the mechanisms that underpin this natural phenomenon are sparse. The most characterized system is the T = 1 encapsulin from Thermotoga maritima (Tm-Enc), whose disassembly/reassembly has been primarily inspected via CD, polyacrylamide gel electrophoresis (PAGE), and TEM.[13] Specifically, Tm-Enc has been found to disassociate under strong acidic and alkaline conditions or at high concentrations of denaturing agents (e.g., guanidine hydrochloride, GuHCl). Furthermore, Tm-Enc was shown to spontaneously reassemble upon returning to the initial conditions (i.e., neutral pH or the absence of denaturants).[13,15] Interestingly, the reassembly of encapsulins in the presence of ESig-tagged cargo (e.g., proteins, nanomaterials) can enable selective encapsulation in vitro.[13−24] Based on these findings and the growing number of novel encapsulin structures, a better understanding of the biophysical mechanisms and physicochemical factors that underlie their disassembly/reassembly is needed. This includes characterizing the differences between structurally different encapsulins, specifically disassembly/reassembly conditions, reassembly timescales, and the impact these processes have on structural stability.

Motivated by this absence of information, we selected encapsulins with structures representing each of the three known architectures and then interrogated their disassembly/reassembly. These nanocages included Tm-Enc (T = 1) and the larger and more structurally complex encapsulins from Myxococcus xanthus (Mx-Enc, T = 3) and Quasibacillus thermotolerans (Qt-Enc, T = 4) which are less understood. We combined ITF spectroscopy, DLS, PAGE, and TEM to accurately monitor the assembly states of all three encapsulins under varying physicochemical conditions, including exposure to extreme pH, strong denaturants (GuHCl), and elevated temperatures. Furthermore, the effect disassembly/reassembly had on the nanocages’ structural integrity was evaluated by atomic force microscopy (AFM). Together, this work presents critical insights into the dynamic mechanisms that govern the disassembly/reassembly of different encapsulin structures, which will help to expedite and broaden their future design, modification, and practical applications.

Results and Discussion

Monitoring Encapsulin Assembly/Disassembly Using ITF Spectroscopy

Unloaded Tm-Enc, Mx-Enc, and Qt-Enc were produced in Escherichia coli and purified by size exclusion chromatography (SEC) and anion-exchange chromatography prior to biophysical characterization. Purification and correct self-assembly were confirmed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blue native-PAGE, DLS, and TEM (Figures g and S1). TEM images of self-assembled Tm-Enc, Mx-Enc, and Qt-Enc displayed structures with a consistent shape and size (Figure g), and DLS analysis indicated a diameter of 23.7 ± 4.9 nm for Tm-Enc, consistent with its crystal structure data,[5] and a diameter of 32.0 ± 6.0 nm for Mx-Enc and 38.1 ± 7.3 nm for Qt-Enc, consistent with their cryogenic electron microscopy (cryo-EM) structures.[6,8]Mx-Enc was produced primarily in its T = 3 structure; however, populations of smaller, T = 1 like, structures were evident in Native-PAGE and TEM results (∼18 nm) (Figures S1 and 2g). This variation in the size of Mx-Enc has been previously observed, where recombinantly produced Mx-Enc without the presence of ESig-tagged cargo will form heterogeneous T = 1 and T = 3 populations.[8]

Figure 2

GuHCl, pH, and thermally induced disassembly of encapsulins. (a) ITF spectroscopy showed an observed shift in the emission wavelength (360/320) and indicated that Trp solvation begins from 1 M GuHCl for Mx-Enc and Qt-Enc and from 3 M for Tm-Enc. (b) DLS measurements indicated that Mx-Enc disassembled into its subunits (<0.5 nm) from 3 M GuHCl, and Tm-Enc and Qt-Enc disassembled from 4 M GuHCl. (c) ITF spectroscopy showed that Trp solvation increased significantly only under alkaline conditions from pH 12 for Tm-Enc, Mx-Enc, and Qt-Enc. (d) DLS measurements indicated that Mx-Enc began to disassemble at pH 12 (∼13 nm) and all Enc disassembled into their subunits at pH 13 (<0.7 nm). (e) ITF emission wavelengths (360/320) of Enc between 20 and 90 °C and cooled back to 20 °C. Tm-Enc remained stable with slight Trp solvation from 80 °C that reversed when cooled back to 20 °C. Mx-Enc began to display Trp solvation from 60 °C and Qt-Enc from 40 °C. (f) DLS measurements show that Tm-Enc remained assembled to 90 °C, and Mx-Enc and Qt-Enc began to disassemble into their subunits (<0.5 nm) at 80 °C. *A smaller population of intermediate Qt-Enc structures that remained at 80–90 °C. Upon cooling to 90 °C, Qt-Enc appeared to reassemble, whereas Mx-Enc became aggregated (indicated by the black square). (g–j) TEM images show “self-assembled” encapsulins compared to those “disassembled” (i.e., complete absence of visible nanocage structures) under varying conditions and then “reassembled” back into spherical nanocages; Mx-Enc (black triangle T = 3, white triangle T = 1) (scale bars = 50 nm). For ITF data, the difference in the emission wavelength of complete Trp solvation (normalized to 1) and assembled Enc (normalized to 0) was plotted. Error bars represent the mean ± standard deviation; n = 3 from three independent experiments. DLS results were normalized so that 1 = expected assembled size and 0 = disassembled encapsulin.

ITF spectroscopy is extremely sensitive to the local environment. As such, protein unfolding, disassembly, or conformational transitions often result in a change in the emission spectra of the Trp(s) within a protein, a lower maximum wavelength (blue-shifted) when the Trp(s) are buried, and a higher maximum wavelength (red-shifted) when solvent-exposed.[25]

Encapsulin subunits adopt a HK97-fold and have three conserved structural regions, a peripheral (P)-domain, an axial (A)-domain, and an elongated (E)-loop region (Figure S2).[3,4] Conveniently, each subunit of Tm-Enc, Mx-Enc, and Qt-Enc contains five, three, and two Trp residues, respectively. For each encapsulin, at least one of these Trp’s is located within the interface between subunits, and one is located within the hydrophobic core of a single subunit (Figure a–c). Therefore, these intrinsic Trp residues are likely to be suitable reporters for both assembly/disassembly of the encapsulin macrostructure (i.e., tertiary/quaternary structure) and folding of the individual subunit polypeptide chains. Indeed, ITF spectroscopy has previously been used to monitor the refolding of T = 1 encapsulin from Rhodococcus erythropolis N771 when desorbed from a zeolite substrate.[26] ITF spectroscopy is also an appealing technique to monitor the process of encapsulin assembly/disassembly owing to its relative simplicity and its ability to report on a dynamic ensemble of structures in solution due to its nondestructive nature, which enables measurements to be performed in real time and allows the same sample to be subject to additional techniques. ITF spectroscopy also provides a method to monitor encapsulins in more complex physiological solutions, such as blood, which would be beneficial in investigating their biomedical potential. Additionally, unlike fluorescence resonance energy transfer-based techniques, ITF spectroscopy does not require modification of the protein with any extrinsic labels that may alter the assembly/disassembly dynamics.

Figure 1

Monitoring encapsulin nanocage assembly states via ITF spectroscopy. Schematic diagram showing the assembled architectures of (a) Tm-Enc (PDB: 3DKT), (b) Mx-Enc (PDB: 4PT2), and (c) Qt-Enc (PDB: 6NJ8). For each structure, the pentameric and hexameric units are shown in light and saturated colors, respectively. Within the expanded pentameric or hexameric units, the tryptophan (W) residues belonging to individual subunits are highlighted in red: Tm: W19, W48, W70, W90, and W180; Mx: W17, W96, and W155; and Qt: W95 and W154. Molecular graphics were created using UCSF ChimeraX.[30] Using ITF spectroscopy, a shift in emission discriminated between the maximum emission wavelength of buried Trp in the “assembled” form: ∼334 nm for (a) Tm-Enc and (c) Qt-Enc, or ∼338 for (b) Mx-Enc, and solvent-exposed Trp in the “disassembled” form (∼354 nm) upon addition of 7 M GuHCl. Normalized fluorescence intensity (NFI) was achieved by making the maximum Trp emission wavelength (nm) = 1.

Figure shows the ITF spectroscopy of assembled Tm-Enc, Mx-Enc, and Qt-Enc overlayed with the spectra obtained after 1 h of incubation with 7 M GuHCl. For all three assembled encapsulins, blue-shifted Trp fluorescence spectra are observed (a ∼334 nm maximum emission wavelength for Tm-Enc and Qt-Enc and ∼338 nm for Mx-Enc), while in the presence of 7 M GuHCl, a dramatic red shift of the fluorescence spectra is seen (∼354 nm maximum emission wavelength). A maximum emission wavelength over 350 nm is observed, where the Trp’s within a protein are completely solvent-exposed, as in an unfolded polypeptide chain.[27] Therefore, it appears that 7 M GuHCl fully disassembles Tm-Enc, as has previously been demonstrated,[13] as well as both Mx-Enc and Qt-Enc, by completely unfolding the protein.

It is also interesting to note that the overall fluorescence emission spectral shape represents an average of all Trp environments present in the protein.[28] Where a protein contains multiple Trp’s, these may exist in different environments and therefore emit at different wavelengths. While we cannot draw conclusions about the foldedness/assembly of Tm-Enc, Mx-Enc, and Qt-Enc relative to one another (as they all contain a different number of Trp’s), changes in the spectral shape for the same encapsulin may discern encapsulin assembly (i.e., interactions between subunits) as opposed to the folding of individual subunit polypeptide chains. This is particularly true for Qt-Enc. As can be seen in Figure f, the peak shape for assembled Qt-Enc is flattened. This is consistent with its two Trp residues being located in different solvation environments such that in the spectra, two Trp’s overlap but are not resolved (i.e., Trp95 being more buried in a helix at the pentamer/hexamer interface than Trp154, which is located on a loop in the A-domain, nestled within the hydrophobic core of a single Qt-Enc subunit) (Figures c and S2).

Instead of reporting the maximum fluorescence wavelength, herein, we have presented much of our ITF data as a fluorescence intensity ratio of emission at 360 and 320 nm (360/320). This ratio is used as a proxy for the shift in overall Trp fluorescence emission peak and avoids any bias arising from the spectral peak shape.[29] This approach has allowed us to expand the repertoire of methods that can be combined to monitor encapsulin assembly/disassembly across a range of conditions.

GuHCl-Induced Encapsulin Disassembly

GuHCl is a denaturant that affects the protein structure by disrupting noncovalent interactions, including hydrogen bonding and hydrophobic effects.[31] Our initial ITF spectroscopy results indicated that 7 M GuHCl completely unfolds the Tm-Enc, Qt-Enc, and Mx-Enc protein polypeptide chains (Figure ) and thus disassembles the encapsulin macrostructure. To further examine the effect of GuHCl on encapsulin folding and assembly, all encapsulins were incubated in varying concentrations of GuHCl (0–7 M) for 1 h before performing ITF and DLS analyses.

The observed shift in the Trp emission peak for Tm-Enc, Mx-Enc, and Qt-Enc in the presence of GuHCl is shown in Figure a. As expected from our previous results, all three encapsulins show a blue to red shift from the assembled (0 M GuHCl, Δ360/320 = 0) to unfolded state (7 M GuHCl, Δ360/320 = 1). However, the concentration of GuHCl at which this transition occurs varies significantly between the different encapsulins. Tm-Enc Trp solvation began from approximately 3 M GuHCl, with complete Trp solvation from 6 M GuHCl, while Mx-Enc and Qt-Enc Trp solvation began at a lower concentration of 1 M GuHCl, with complete Trp solvation from 4 and 5 M GuHCl, respectively. This variation may reflect structural complexity, with the smaller and pentameric Tm-Enc being the most robust to GuHCl compared to the larger pentameric and hexameric Mx-Enc and Qt-Enc.

To complement these ITF spectroscopy results, DLS analysis was performed to characterize the size distribution of encapsulins in solution for the same samples (Figure b, Table S2). For Mx-Enc, no intact encapsulin macrostructure is detectable by DLS at concentrations ≥3 M GuHCl, which also correlates with the significant red-shifted Trp emission at 3 M GuHCl (Δ360/320 = 0.88). This result suggests that Mx-Enc is both disassembled and largely unfolded at GuHCl concentrations ≥3 M. However, for Tm-Enc and Qt-Enc, the absence of the intact encapsulin macrostructure occurs at lower GuHCl concentrations than the major red shift in Trp emission. For example, at 4 M GuHCl, the Tm-Enc macrostructure is not detectable by DLS, but the Trp emission remains significantly blue-shifted (Δ360/320 = 0.20), corresponding to a native-like fold. A similar result is seen for Qt-Enc at 3 M GuHCl (Δ360/320 = 0.27). TEM and native-PAGE images of each encapsulin after incubation in the lowest GuHCl concentration required for significant Trp solvation also show the absence of nanocage macrostructures, confirming their disassembly. Upon dialysis in the reassembly buffer (50 mM HEPES, 1 mM DTT) overnight, all three encapsulins subsequently reassembled into their original macrostructure (Figures h, S4, and S3).

Taken together, these results point to the existence of an intermediate state(s) in Tm-Enc and Qt-Enc GuHCl-induced disassembly, in which the nanocage breaks down into smaller (<0.5 nm) entities with a native-like fold. There is insufficient evidence from ITF or DLS analysis to define the stoichiometry or structure of this intermediate state(s). However, a previous native mass spectrometry study of the T = 1 encapsulin from Brevibacterium linens suggested that reassembly occurs via the formation of stable dimers prior to the final nanocage.[32] It is possible that disassembly of Tm-Enc and Qt-Enc proceeds via a similar mechanism. We also cannot rule out that Mx-Enc proceeds via an equivalent intermediate state upon GuHCl-induced disassembly. However, if this is the case, our data suggest that the chemical stability of the nanocage and the intermediate for Mx-Enc are more closely matched, and less chemically stable, than those of Tm-Enc or Qt-Enc such that both disassembly and complete unfolding occur at close to 3 M GuHCl. This potential model of encapsulin disassembly warrants further investigation as disassembly of the nanocage utilizing lower GuHCl concentrations, without completely unfolding the encapsulin protein, may provide an effective scheme for loading and/or releasing more chemically sensitive cargo.

Overall, our results for GuHCl-induced encapsulin disassembly suggest that folding plays a critical role for encapsulin nanocage assembly/disassembly. The smaller, less complex Tm-Enc structure requires more GuHCl for unfolding than the larger, more complex Qt-Enc and Mx-Enc. This agrees with the difference in the high stability of ferritin, which is a small protein nanocage comprising 24 subunits, compared to the high sensitivity of larger and more structurally complex VLPs in denaturants. Ferritin is stable up to 6 M GuHCl[33] and requires 8 M of the denaturant urea for disassembly.[34] A concentration of 2 M GuHCl is not strong enough to disassemble ferritin but is able to unfold the α-helices surrounding ferritin channels, thereby enlarging its pores to allow entry of cargo without the need for complete disassembly.[35] Contrastingly, the larger P22 VLP, which assembles from 420 subunits into a T = 7 nanoparticle-like structure that is 56 nm in diameter, dissociates at just 3 M GuHCl.[36] However, the chemical stability of the encapsulins tested is not directly related to their size as Qt-Enc requires more GuHCl for unfolding than the smaller Mx-Enc. Hence, other factors, such as the symmetry and stability of intermediate state(s), also need to be taken into consideration.

pH-Induced Encapsulin Disassembly

To investigate the role of electrostatic interactions in maintaining the encapsulin macrostructure, ITF and DLS analyses were also performed for the three encapsulins at varying pH (Figure c,d). The theoretical pI of Tm-Enc, Mx-Enc, and Qt-Enc are 4.90, 5.45, and 5.02, respectively.[37] If electrostatic interactions play a major role in the mechanism of encapsulin assembly, we would expect to see disassembly of the encapsulin nanocage at pH < pI (where the overall charge on the protein would be positive rather than negative, as at neutral pH). However, this is not the case. ITF spectroscopy indicates that all encapsulins remain relatively stable across a broad pH range, with a significant change in Trp exposure only observed under extreme alkaline conditions (pH 12–13) (Figure c). DLS demonstrates the presence of assembled encapsulins at all pHs, with the exception of pH 13, and smaller structures (∼13 nm) at pH 12 for Mx-Enc (Figure d, Table S3). Disassembly of the nanocage structures at pH 13 was also confirmed by TEM (Figure i). These results support a recent study that used Native-PAGE analysis to confirm that Qt-Enc is stable between pH 4.5 and 8.5.[24] Therefore, loss of electrostatic interactions between and/or within encapsulin subunits is unlikely to be a major driving force for disassembly. Disassembly more likely arises at extreme alkaline pH due to deprotonation of the protein side chains, resulting in a loss of the hydrogen bonding that holds the protein scaffold together.[38]

Thermal Encapsulin Disassembly

To assess encapsulin thermostability, assembled samples were heated to 90 °C with ITF and DLS measurements taken every 10 °C up to 90 °C as well as upon return to 20 °C. Additionally, encapsulins were heated to 90 °C with TEM samples prepared both immediately after heating and after being cooled at 20 °C for 1 h. ITF results showed that the Tm-Enc encapsulin remained stable throughout heating, with only a very slight red shift in the emission wavelength observed at temperatures above 80 °C, which was recovered when cooled back to 20 °C (Figure e). No significant change in the size of the Tm-Enc nanocages was observed by DLS, and TEM images showed assembled cages after heating to 90 °C, suggesting that the Tm-Enc nanocage is resistant to thermal disassembly (Figures f,j and S4; Table S4).

In contrast to Tm-Enc, both Mx-Enc and Qt-Enc disassemble at elevated temperatures. Mx-Enc Trp solvation began from 60 °C and disassembled from 80 °C, as shown by DLS (Figure e,f, Table S4) and appeared disassembled at 90 °C via TEM imaging (Figure j). Additionally, in contrast to Tm-Enc, disassembly of Mx-Enc by temperature is not reversible as when cooled back to 20 °C, only large aggregates were found (Figure f, Table S4). Similarly, ITF spectroscopy showed Qt-Enc began Trp solvation from 40 °C, and DLS indicated that the majority of the sample disassembled at 80 and 90 °C. However, even at 90 °C, a smaller population (approx. 16%) of Qt-Enc subunits remained in intermediate structures of ∼14 nm and another population (approx. 16%) at the assembled size of ∼44 nm (Figures f and S4; Table S4). When cooled back to 20 °C, DLS and TEM indicated that Qt-Enc reassembled primarily back into its original size (DLS data also showed a smaller population of approx. 32% at ∼15 nm) and ITF spectroscopy showed a blue shift of emission (Figure e,f,j and S4; Table S4).

The difference in the thermal stability and reassembly of Tm-Enc, Mx-Enc, and Qt-Enc points to kinetic complexity in the encapsulin assembly pathway. For Mx-Enc, aggregation of the unfolded/disassembled state is likely the main obstacle to achieving proper reassembly. Our GuHCl data also suggest that the Mx-Enc unfolded/disassembled state(s) is less stable than for Tm-Enc or Qt-Enc. While Qt-Enc began Trp solvation at 40 °C, DLS showed that it did not change in size until 80 °C, indicating that Qt-Enc undergoes structural change from 40 °C but remains assembled until 80 °C. The stabilities of Tm-Enc and Qt-Enc at high temperatures are consistent with them being derived from thermophilic bacteria.[5,6] However, Mx-Enc is derived from bacteria that live in soil,[39] which may explain its lower tolerance to temperature.

Alternate Disassembly Conditions

We have begun to use the ITF technique, developed herein, to investigate other chemical additives that may alter the folding and/or assembly of encapsulins, including redox conditions (10 mM DTT or H2O2) and ionic environments (1 M NaCl). However, we are yet to find another condition to induce disassembly. All encapsulins remained stable in NaCl, and only Mx-Enc displayed a small blue shift in emission in H2O2 and an observable increase in diameter after incubation in either DTT or H2O2 (Figure S5, Table S7). This may be due to reduction/oxidation of disulfide bonds causing a weaker association between subunits, resulting in swelling of the nanocage.[40]

Encapsulin Reassembly

Based on the above results, high concentrations (6 M) of GuHCl and pH 13 conditions were selected for further analysis of the Tm-Enc, Mx-Enc, and Qt-Enc reassembly mechanism. Notably, these conditions disassemble encapsulins to differing degrees. ITF spectroscopy revealed maximum peak emissions for disassembly with pH 13 to be ∼341 nm for Tm-Enc and ∼347 nm for Mx-Enc and Qt-Enc, whereas disassembly with 6 M GuHCl resulted in a maximum peak emission at ∼354 nm for all encapsulins (Figure a–c). As previously discussed, a Trp emission maximum ≥350 nm (as observed for all encapsulins in 6 M GuHCl) is expected for the unfolded protein. However, at pH 13, all encapsulins display a Trp max emission <350 nm, suggesting that at least some of their subunits’ secondary structure remains intact.

Figure 3

Comparison of Enc spectra of self-assembly, disassembly in 6 M GuHCl or at pH 13, and subsequent reassembly. (a) Tm-Enc ITF emission when self-assembled (blue), disassembled with 6 M GuHCl (dotted red), reassembled after 6 M GuHCl disassembly (solid red), disassembled with pH 13 (dotted green), and reassembled after pH 13 disassembly (solid green). (b) Mx-Enc ITF emission when self-assembled (purple), disassembled with 6 M GuHCl (dotted red), reassembled after 6 M GuHCl disassembly (solid red), disassembled with pH 13 (dotted green), and reassembled after pH 13 disassembly (solid green). (c) Qt-Enc ITF emission when self-assembled (orange), disassembled with 6 M GuHCl (dotted red), reassembled after 6 M GuHCl disassembly (solid red), disassembled with pH 13 (dotted green), and reassembled after pH 13 disassembly (solid green). NFI was achieved by making the maximum Trp emission wavelength (nm) = 1.

Reassembly of all encapsulins after disassembly in either 6 M GuHCl or pH 13 was initiated via overnight dialysis into the reassembly buffer. Following disassembly in 6 M GuHCl, Tm-Enc, Mx-Enc, and Qt-Enc all reassembled back to their original structures, as indicated by ITF maximum emission and DLS size (Figures a–c and S4). In contrast, after disassembly in pH 13, only Tm-Enc reassembled to its original structure. For Mx-Enc and Qt-Enc, a blue shift in ITF emission, resembling the Trp maximum emission of the original self-assembled material, was observed. However, native-PAGE results showed no bands (Figure S3), and TEM analysis found no structures for Mx-Enc, and only a single nanocage was imaged for Qt-Enc (Figure i). Indeed, DLS analysis of pH 13 reassembled Mx-Enc and Qt-Enc showed the presence of either large aggregates or smaller structures (∼18 nm for Mx-Enc and ∼9–25 nm for Qt-EncFigure S4), which suggests that the subunits may be reassembling into smaller cages and/or intermediate structures at a concentration too low to detect by native-PAGE and TEM.

As previously discussed, at pH 13, hydrolysis of the peptide bonds can occur, leading to protein misfolding and/or aggregation,[38] which may account for some of this inefficiency in encapsulin reassembly. Interestingly, although the position of the Trp emission peak for the original self-assembled, GuHCl reassembled, and pH 13 reassembled samples of Qt-Enc is not significantly different, a difference in spectral shape is noted for the pH 13 reassembled sample. The blue-shifted shoulder of the Qt-Enc Trp emission peak, which is thought to correspond to Trp95 buried in the pentamer/hexamer interface of the intact nanocage, is lower in intensity for the pH 13 reassembled sample. This result is consistent with reassembly of a different structure with a native-like fold, instead of the original intact T = 4 Qt-Enc nanocage, upon reassembly from pH 13. The reassembly of Mx-Enc and Qt-Enc after pH 13 disassembly into smaller nanocages and/or alternative structures in inefficient quantities suggests that this pathway, in its native form, may not be suitable for some applications.

Timescale of Encapsulin Reassembly

To advance encapsulins as a cargo-carrying platform, the disassembled encapsulin needs to be able to be reassembled on a viable timescale. As the assembly rate of encapsulins inside host cells (native or recombinant) cannot be accurately monitored, in vitro reassembly can provide insights into their self-assembly in vivo. The encapsulin nanocages were disassembled for 1 h via 6 M GuHCl or pH 13 before reassembly was initiated using dialysis, with samples measured by ITF and DLS analyses every 15 min over a 75 min time frame. The rate of reassembly appeared to vary between each Enc. Tm-Enc reassembled faster than Mx-Enc and Qt-Enc, with DLS indicating that Tm-Enc reached its assembled diameter within 15 min after being disassembled via pH 13 and between 15 and 30 min when disassembled via 6 M GuHCl (Figure a,b, Tables S5 and S6). The faster reassembly after pH 13 disassembly compared to GuHCl may be explained by the subunits still maintaining some structure after incubation under pH 13 conditions. However, Tm-Enc reassembly after pH 13 disassembly displayed a more gradual Trp burial and a slight decrease in size at 45 min (Figure a,b, Table S6). This suggests that reassembly after pH 13 may follow a more dynamic pathway, with rapid nanocage formation, followed by restructuring. In contrast, after 6 M GuHCl disassembly, Qt-Enc reassembled into its original diameter between 30 and 45 min, and Mx-Enc appeared to partially assemble between 45 and 60 min but only completely assembled between 60 and 75 min (Figure a,b, Table S5). The difference in rates between encapsulins may reflect the overall stability of the complex; that is, complexes that are more stable may reassemble quicker. In a recent report, the in vivo loading of cargo proteins into Tm-Enc during its self-assembly was found to be ∼8 times less efficient than with Mx-Enc.[41] The authors suggested that this striking difference was partly due to the smaller Tm-Enc self-assembling at a faster rate than Mx-Enc, thus limiting the available contact time between ESig-tagged cargo and the nanocage’s interior surface. This hypothesis is now supported by our observation that Tm-Enc reassembles up to 4 times quicker than the larger Mx-Enc (Figure ). This therefore highlights the critical role assembly time frames play in cargo-loading efficiency and that modifying such properties can enhance cargo packing, density, and/or stoichiometry.

Figure 4

Reassembly time frames of encapsulins. (a) ITF spectral shift in the emission wavelength (Δ360/320) of Tm-Enc, Mx-Enc, and Qt-Enc disassembled via 6 M GuHCl and Tm-Enc disassembled via pH 13 and reassembly measured every 15 min. The difference in the emission wavelength of disassembled encapsulin (normalized to 1) and assembled encapsulin at 75 min (normalized to 0) was plotted. Error bars represent the mean ± standard deviation; n = 3 from three independent experiments. (b) Heat map of DLS measurements of Tm-Enc, Mx-Enc, and Qt-Enc disassembled via 6 M GuHCl and Tm-Enc disassembled via pH 13 and reassembly measured every 15 min. DLS results were normalized so that 1 = expected assembled size and 0 = disassembled encapsulin.

The rapid burial of Trp residues for all encapsulins within 15 min, as shown by ITF spectroscopy, prior to the formation of the cages, suggests a reassembly pathway where subunits first fold into an intermediate structure before gradually forming the cage. A previous study on encapsulins from B. linens (T = 1) suggested that reassembly occurred with subunits first forming stable dimers prior to the final nanocage formation, with a preference for even-numbered stoichiometries as demonstrated via mass spectrometry analysis.[32] Additionally, ferritin has been found to have a biphasic reassembly, where an initial fast step occurs with folding of subunits and unknown stable intermediates, followed by the slower restructuring of intermediates into the nanocage.[22]

Data of the pH 13 reassembly rate for Mx-Enc and Qt-Enc were not included as the presence of aggregation prevented extraction of accurate DLS size values. This suggests that reassembly from the pH 13 disassembled state for the larger and more complex Mx-Enc and Qt-Enc may be prone to protein misfolding pathways and/or partial assembly and thus, without additional re-engineering, may not be ideal for use in future applications.

These results also highlight the value of adding ITF spectroscopy to complement the existing repertoire of methods to monitor disassembly/reassembly of encapsulins. Although CD may provide more specific secondary structural information, ITF spectroscopy can monitor global structural changes (i.e., tertiary/quaternary) due to information about the degree of local solvation.[42] This may be a more genuine representation of encapsulin macrostructure assembly. In addition, unlike ITF spectroscopy, CD spectroscopy is not compatible with the high concentration of GuHCl required to completely unfold encapsulins.[43]

Nanomechanical Stability of Self-Assembled versus Reassembled Encapsulins

To understand any changes in the structural integrity of Tm-Enc, Mx-Enc, and Qt-Enc after reassembly under the conditions tested above, AFM was utilized to compare the morphology, rupture point, and elasticity between self-assembled and reassembled encapsulins. As the future application of encapsulins will likely require native solution conditions, it was pertinent to examine their structural integrity in solution and is why AFM was utilized over alternative techniques that examine proteins in the gas phase, such as native mass spectrometry. AFM has been employed to assess the mechanical stability of comparable bacterial microcompartments (i.e., carboxysomes)[44] and VLPs.[52] Indeed, previous AFM analysis has been done on the rigidity of Tm-Enc and encapsulins from B. linens (T = 1) and suggested that the presence of cargo within encapsulins from B. linens may lead to some destabilization, as indicated by a lower rupture force in loaded versus unloaded encapsulins.[32] However, the effect of reassembly on the nanomechanical stability on Tm-Enc as well as the larger and more structurally complex Mx-Enc and Qt-Enc is yet to be elucidated.

Individual encapsulins were imaged with a scan size of ∼100 nm × 100 nm to visualize their topographical detail. Self-assembled encapsulins were found to be at their expected size (Tm-Enc, 24 nm; Qt-Enc, 42 nm), including both the T = 3 (32 nm) and T = 1 (17 nm) conformations of Mx-Enc (Figure a). However, clear morphological variation can be seen after reassembly, with all encapsulins demonstrating flattening of their surfaces. Additionally, Qt-Enc appeared more aggregated after reassembly.

Figure 5

AFM imaging and elasticity comparing self-assembled/reassembled encapsulins. (a) Nanoscale AFM images obtained for individual encapsulins comparing the morphologies of self-assembled Tm-Enc, Mx-Enc, and Qt-Enc with encapsulins disassembled via pH 13 or GuHCl (6 M) and subsequently reassembled. White scale bars represent 30 nm. (b) Representative indentation curves and the Hertz fit of encapsulins used to determine Young’s elastic modulus (E) using the Hertz equation to convert experimentally derived force versus distance curves into indentation data (colored circles) and to fit the elastic (nonlinear) region to calculated E values (black lines).

Being composed of similar constituents, approximately the same elasticity would be expected between each self-assembled encapsulin, which was found to be the case of Tm-Enc and Mx-Enc, with an average elasticity of 2.77 ± 0.96 and 2.12 ± 0.81 MPa, respectively (Figures b and S6). However, Qt-Enc displayed a higher average elasticity of 25.60 ± 14.92 MPa, indicating that it has greater structural integrity and is therefore more resilient to deformation (Figures b and S6). Qt-Enc may therefore be more strongly self-assembled than Tm-Enc and Mx-Enc, which could be attributed to its higher structural complexity, as the different symmetries and subunit–subunit contacts may influence the strength of interfacial interactions.[45] Upon reassembly following 6 M GuHCl disassembly, the elasticity of all encapsulins decreased significantly, with a Tm-Enc average elasticity of 1.16 ± 0.93 MPa (58.12% decrease), Mx-Enc 1.39 ± 0.78 MPa (34.43% decrease), and Qt-Enc 0.85 ± 0.62 MPa (96.68% decrease), indicating a significant reduction in structural integrity (Figure b and S6). Elasticity data of reassembled Tm-Enc after disassembly via pH 13 were not included as during testing, the sample became unstructured and did not retain any elasticity or show any elastic response. This suggests that even though Tm-Enc reassembled after disassembly via pH 13, these conditions may have irreversibly altered the protein by hydrolysis, thereby affecting stability.[38]

The rupture force represents the maximum force a protein cage withstands prior to puncture by the AFM tip.[46] The rupture force of all self-assembled encapsulins was between ∼0.1 and 0.2 nN, indicating that they are fairly fragile (Figure b). This is in contrast to AFM results from a previous study on self-assembled empty Tm-Enc and encapsulins from B. linens, which had rupture forces of 0.63 and 0.64 nN, respectively,[32] indicating that the encapsulins used in the current study are slightly more fragile. After reassembly, the rupture force decreased further by a factor of ∼5 for each encapsulin system tested (Figure b).

Together, these results show that after undergoing disassembly in either 6 M GuHCl or pH 13 and subsequent reassembly, all three encapsulin architectures significantly lose structural integrity. In terms of encapsulins loaded with cargo under in vitro conditions, the observed increased fragility could be beneficial if wanting a system that can be easily broken down to release cargo, such as intracellular drug delivery or immunotherapy. If in need of a more robust system (e.g., enzyme-loaded nanocages for industrial biocatalysis), however, these results demonstrate the importance of understanding and modifying encapsulins, so they can disassemble under more specific and benign conditions. Accordingly, future encapsulin engineering efforts could focus on the site-directed modification of specific amino acids and/or key structural domains within subunits or at the interface between subunits (e.g., the E-loop domain) to trigger disassembly/reassembly. For example, recently, a GALA peptide was inserted in the E-loop region of Qt-Enc which underwent a coil-to-helix conformational change at pH 5.5, allowing triggered reversible disassembly under more facile conditions.[24] This work represents a promising step forward to controlling encapsulin assembly; however, further insights into how modifications affect cargo loading efficiency, reassembly speed, and functional cargo proteins are required for a fully developed stimuli-responsive loading approach. Therefore, our results may provide information that could assist in improving such strategies.

Furthermore, to enhance understanding of the reassembly pathway of encapsulins, future studies could assess real-time assembly of the subunits. This has been achieved by using AFM with large bacterial microcompartments, where it was revealed that the shell facets assemble from preformed oligomers,[47] and the pH-dependent disassembly/reassembly of ferritin has also been monitored in real space.[48] Similarly, highly specialized efforts by MS researchers to combine structural MS techniques (i.e., Native MS) to determine the real-time stepwise assembly of oligomeric protein cages are also ongoing.[150] Such strategies might be complemented with cryo-EM and SAX methods to further probe the dynamic transitions of intermediate structures within encapsulin disassembly/reassembly pathways. The data presented in this report provide a clear framework to relate structural transitions observed by such non-solution-based techniques to the process of Enc assembly/disassembly in solution.

Conclusions

In summary, this study determined a set of physicochemical conditions that induce the disassembly/reassembly of Tm-Enc, Mx-Enc, and Qt-Enc, with each encapsulin system showing varying degrees of sensitivity to denaturants, pH, and temperature. As shown in Figure , the data generated also revealed several structural states and changes (i.e., protein structure, size, and foldedness) that occur during the dynamic process of encapsulin disassembly/reassembly, with clear differences between the disassembly/reassembly pathways of all three encapsulin architectures studied. Using AFM, we revealed a previously unknown effect of in vitro disassembly/reassembly on encapsulin stability, finding that the encapsulin loses a substantial amount of structural integrity when disassembled and subsequently reassembled. These findings have implications if requiring in vitro-loaded encapsulins for a particular application but may be beneficial when nanocage dissociation and cargo release are favored.

Figure 6

Proposed disassembly/reassembly pathways for each encapsulin architecture. Schematic diagram that compares and contrasts the proposed disassembly/reassembly dynamics, pathways, timescales, and potential intermediates of the three encapsulin architectures, as elucidated from our results. Encapsulin size was determined by DLS, and foldedness was based on ITF results.

At high concentrations of GuHCl, all three encapsulins completely unfold and refold efficiently; however, these harsh conditions pose limitations for applications involving in vitro loading of cargo that may be sensitive to these conditions as it could be destroyed in the process. Furthermore, the use of harsh denaturants and extreme pH to disassemble encapsulins may limit their downstream utility, especially in biomedicine, where biocompatibility is paramount. With an increased understanding of the encapsulin structure, disassembly/reassembly conditions, stability, and stoichiometry of intermediate structures and having established clear differences of these between the encapsulins assessed herein, this study provides the groundwork to test the effect of altering the native Enc sequences. For example, by identifying critical interactions between nanocage subunits that may affect the assembly process, future genetic engineering efforts of subunits may lead to enhanced control over conditions that trigger disassembly/reassembly, size, structure, and cargo-loading efficiency of encapsulins.[49,50]

In summary, the findings of this study advance our understanding of encapsulins by providing critical insight into their unique disassembly/reassembly dynamics. This knowledge provides a roadmap toward an encapsulin “tool kit” comprising nanocages with varying structural architectures and biochemical/biophysical properties, which can be readily selected and further customized for a specific nanobiotechnological application.

Materials and Methods

Materials

All chemicals and reagents used in this study were purchased from Sigma-Aldrich unless stated otherwise.

Molecular Cloning of Constructs

All inserts were codon-optimized for expression in E. coli and custom-synthesized as gBlock Gene Fragments (Integrated DNA Technologies). Encapsulins from T. maritima (Tm) (UniProt: TM_0785), M. xanthus (Mx) (UniProt: MXAN_3556), and Q. thermotolerans (Qt) (UniProt: QY95_01592) were each synthesized with flanking restriction sites (NcoI/BamHI). For gene expression in E. coli, Tm-Enc was cloned into pETDuet-1 (Novagen, Merck), and Mx-Enc and Qt-Enc were cloned into pACYC-Duet-1 (Novagen, Merck), summarized in Table S1. E. coli α-Select (Bioline, UK) was used for general plasmid storage and propagation. Gene insertion was confirmed by PCR using primer pairs pETUpstream/DuetDOWN (Merk). E. coli BL21 (DE3) cells (New England Biolabs) were used for recombinant protein expression. Herein, cells were transformed with the appropriate plasmids, and the resulting transformants were selected on Luria–Bertani (LB) agar supplemented with either 100 mg/mL of carbenicillin or 50 mg/mL of chloramphenicol (see Table S1).

Recombinant Protein Production

Protein expression experiments were performed in the LB medium supplemented with carbenicillin (100 mg/mL) or chloramphenicol (50 mg/mL). Briefly, strains were streaked out on LB agar plates and grown overnight at 37 °C. A starter culture (1 colony in 5 mL LB) was grown for 16 h at 37 °C and used to inoculate 500 mL of LB media. Cultures were incubated at 37 °C with flasks shaking at 200–250 rpm until an optical density at 600 nm (OD600) of 0.5–0.6 was reached. Protein synthesis was then induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. Induced cultures were incubated at 37 °C with flasks shaking at 200–250 rpm for 4 h and then cells were harvested via centrifugation (8000g, 4 °C, 15 min). The resulting cell pellets were stored at −30 °C until further use.

Protein Purification

Cell pellets from 500 mL encapsulin-producing cultures were thawed and resuspended in 30 mL of lysis buffer [50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH 7.4 (Chem-Supply Pty) and Benzonase nuclease 10 U/mL]. Cells were lysed by three rounds of passage through a French pressure cell at 1000 psi and centrifuged at 8000g and 4 °C for 15 min. The supernatant containing the soluble protein was heat-treated in a water bath at 65 °C for 15 min before centrifugation (10,000g, 4 °C, 10 min). Protein precipitation was initiated by adding 10% (w/v) PEG8000 and 2% (w/v) NaCl to the supernatant, followed by incubation on ice for 30 min. Next, the sample was spun down at 10,000g for 10 min at 4 °C. The precipitated protein was resuspended in 2.5 mL of HEPES buffer (50 mM, pH 7.4) and filtered through a 0.22 mm syringe filter.

All purifications were carried out on an ÄKTA start chromatography system (GE Healthcare). The three encapsulins used in this study were purified via SEC using a HiPrep 26/60 Sephacryl S-500 HR column (GE Healthcare) equilibrated with 50 mM HEPES pH 7.4. This column is traditionally used to separate large macromolecules and not typically used for proteins, however, this is the standard column used for encapsulins due to their large size.[6,51] Fractions showing the corresponding encapsulin band on SDS-PAGE were pooled and subjected to further purification via anion-exchange chromatography using a HiPrep Q 16/10 column (GE Healthcare) equilibrated with 50 mM HEPES pH 7.4. Encapsulin proteins were eluted with a linear gradient of 0–0.3 M NaCl and 0.3–1 M NaCl (Figure S1). Fractions containing encapsulins, identified via SDS-PAGE, were pooled, concentrated, and buffer-exchanged into 50 mM HEPES buffer pH 7.4 using Amicon Ultra-15 centrifugal filter units with a 100 KDa cutoff. Last, purified encapsulin Enc proteins were filtered through a 0.22 mm syringe filter and stored at −30 °C until further use.

PAGE Analysis and Protein Quantification

Protein samples were denatured, separated, and visualized using SDS-PAGE, with molecular weights compared with a commercial protein ladder (Precision Plus Protein, BioRad). A Bio-Rad mini-protean system (Bio-Rad laboratories) was used for SDS-PAGE analysis. Protein samples were mixed in a 1:1 ratio with 2X Laemmli sample buffer with 50 mM 1,4-dithiothreitol and heated at 99 °C for 10 min with shaking at 300 rpm in a ThermoMixer (Eppendorf). Electrophoresis was performed at 200 V for 30 min on a 4–20% polyacrylamide gel (Mini-PROTEAN TGX, BioRad) in SDS running buffer (25 mM Tris, 192 mM glycine, 1% (w/v) SDS, pH 8.3). Gels were stained following the Coomassie G-250 safe stain protocol. Encapsulin assembly was visualized via nondenaturing Blue Native-PAGE (BN-PAGE). An XCell SureLock Mini-Cell Electrophoresis System (Thermo Fisher Scientific) was used for BN-PAGE analysis. Protein samples were mixed in a 1:4 ratio with 4× Native-PAGE sample buffer (Thermo Fisher Scientific) and loaded into NativePAGE 3–12% Bis-Tris protein gels (Thermo Fisher Scientific). BN-PAGE was performed using two different running buffers: 1× anode buffer (NativePAGE running buffer 20×, Thermo Fisher Scientific) in the outer buffer chamber and 1X dark-blue cathode buffer (1× anode buffer, 0.02% (w/v) Coomassie G-250) in the inner buffer chamber. Last, the samples were run on ice at 150 V for 90 min, followed by a second run at 250 V for 30 min. Protein concentration was determined by measuring the absorbance at 280 nm on a NanoDrop 2000 Spectrophotometer instrument (Thermo Fisher Scientific).

In Vitro Disassembly/Reassembly of Encapsulins

To characterize the in vitro disassembly of encapsulins, the presence of a denaturing agent (GuHCl), changes in pH, and thermally induced disassembly methods were used. GuHCl was added to the encapsulin sample to final concentrations between 1 and 7 M. For compatibility and practicality with small volumes, in pH-induced disassembly experiments, 50 mM HEPES buffer solutions with varying pH values (pH 3–13) were prepared via dropwise adjustment with HCl/NaOH, with pH confirmed using pH indicator strips. Once under their respective conditions, encapsulin samples were incubated for 1 h at room temperature and the same sample was subject to all subsequent analysis methods. For thermally induced disassembly, the encapsulin sample was subjected to a temperature ramp from 20–90 °C at a rate of 2 °C/min. In all disassembly methods, the final encapsulin concentration was 5 μM and dithiothreitol (DTT) was added to the encapsulin sample to a final concentration of 1 mM. In GuHCl- and thermally induced disassembly experiments, the desired final volumes were reached by the addition of 50 mM HEPES pH 7.4. For stability experiments, 5 μM of encapsulins was incubated for 1 h at room temperature under reducing (10 mM DTT), oxidizing (10 mM H2O2), and high-ionic strength (1 M NaCl) conditions.

The subsequent reassembly of encapsulins was initiated by returning the same sample to original conditions. Briefly, the samples were dialyzed against 50 mM HEPES buffer pH 7.4 and 1 mM DTT at room temperature overnight using a 3.5K MWCO SnakeSkin dialysis tubing (Thermo Fisher Scientific). To measure the reassembly rate, samples were removed from dialysis every 15 min for 75 min and centrifuged for 5 min at 10,000g to remove any aggregated proteins.

ITF Spectroscopy

ITF spectroscopy measurements of encapsulins in their varying states of assembly were performed with an FP-8500 spectrofluorometer (JASCO) using a 3 mm pathlength microvolume quartz cuvette. Samples (60 μL) were prepared in triplicate with a final encapsulin concentration of 5 μM. Samples were excited at 290 nm, and emission spectra were collected from 300 to 450 nm. The measurement parameters were as follows: excitation and emission bandwidths of 5 nm, a response of 0.2 s, medium sensitivity, a data interval of 0.1 nm, a scan speed of 100 nm/min, and 4 measurement accumulations were averaged. To investigate the effect of temperature on the different encapsulins, spectra were collected from 20 to 90 °C with a temperature ramp of 2 °C/min. The obtained spectra were further processed by buffer spectra subtraction using Spectra Manager software (JASCO), and the ratio between the fluorescence intensity at 360 and 330 nm (360/330) was calculated and plotted in Microsoft Excel. NFI was achieved by making the maximum emission wavelength (nm) = 1.

Transmission Electron Microscopy

To visualize the morphology, size, and state of encapsulin assembly, TEM was performed using a Philips CM10 microscope (100 kV accelerating voltage). Briefly, encapsulin samples (0.2 mg/mL) were adsorbed onto Pioloform-coated 200 mesh copper grids (ProSciTech) for 2 min. Prior to imaging, samples were negatively stained for 1 h using uranyl acetate replacement (UAR-EMS, Electron Microscopy Sciences), washed with ultrapure water, and allowed to dry for at least 15 min.

Dynamic Light Scattering

To measure the diameter of encapsulins, DLS was performed with a Malvern Zetasizer ZSP instrument equipped with a 633 nm laser. Samples with a final encapsulin concentration of 5 μM were prepared as described above. Three measurements were performed at 25 °C using a plastic microcuvette (ZEN0040, Malvern), with 173° backscatter and automatic attenuator selection. Data analysis was performed in Zetasizer Nano software. All DLS sizes reported, herein, are size by number values calculated using distribution analysis. A 1 cm quartz cuvette was used for temperature ramp experiments. Heat maps were made using GraphPad Prism 9.

Atomic Force Microscopy

Images and force curve measurements were obtained using a Cypher ES Atomic Force Microscope (Oxford Instrument, Asylum Research, Santa Barbara, CA, USA) at room temperature (25 °C). Protocols were adapted from Collett et al.[52,53] For imaging, the instrument was operated in amplitude-modulated AFM (AM-AFM), while force measurements were obtained in the contact mode. Biolever BL-AC40TS cantilevers (Oxford Instruments, Asylum Research, Santa Barbara, CA, USA, nominal spring constant kc = 0.09 N/m) were used for all measurements. All experiments were completed within a droplet of 50 mM HEPES buffer 7.4 (∼100 μL) with a concentration of ∼100 ng of encapsulins deposited onto the freshly cleaved muscovite surface (the supporting substrate). Prior to experimentation, each cantilever was calibrated via the thermal spectrum method and the lever sensitivity was determined using force spectroscopy. Processing of AFM data involved using a combination of the Asylum Research software, custom MATLAB codes, and the Gwyddion software package.[54]

Determining Elasticity of Intact Encapsulins

Force versus distance curves (FD curves) were obtained from the central region of encapsulins. Following image location, FD curves were first obtained on an area of bare mica next to the nanocage to ensure that linear (nonelastic) FD curves were observed, which provided a reference for determining the elasticity of the particles. The spring constant (kc) of the cantilever was determined for each cantilever used as described above (values in the range of 0.05–0.1 N/m were obtained for all cantilevers). The tip was then directed to the central region of the particle to obtain accurate indentation data. Upward of 50 FD curves were recorded across several particles for each sample.

The tip–sample contact point between the AFM cantilever and the encapsulin was performed independently for each FD curve analyzed using methods previously described.[55] Specifically, the contact point between the AFM cantilever tip and the encapsulin is defined as the point at which the cantilever first makes physical contact with the surface adsorbed particle. Following this point, the cantilever bends in response to interactions with the encapsulin, while the nanocage itself is indented. In a raw FD curve [z-displacement (Z) versus cantilever deflection (d)], this point of first contact is mathematically defined as Z0 and d0, respectively. This is observed in the AFM force curve as an increase in force (y-axis), above the zeroed baseline, as a function of distance (x-axis prior to zeroing the indentation).

The indentation (δ) is then calculated viaand transformed into indentation versus force curves using Hooke’s law

The elastic response of the samples is then fit to the Hertz/Sneddon eq to obtain Young’s elastic modulus (E) for each of the fitted curves. Using custom MATLAB scripts, curves were fitted to the equationsandwhere F is the loading force, δ is the indentation depth, α is the cone opening angle, E* is the apparent elastic modulus, and v is the Poisson ratio. A cone tip angle of 34.4° and a Poisson ratio of 0.5 were used for processing all force curves. All elasticity values were obtained from curves fitted with an R2 value of 0.9 or above. Data falling below this quality were rejected for further analysis.