Exploring the Self-Assembly of Encapsulin Protein Nanocages from Different Structural Classes

TL;DR: In this article, the disassembly/reassembly properties of three encapsulin nanocages were characterized using spectroscopic techniques and electron microscopy, and it was found that all encapsulins exhibited varying sensitivities to the denaturant guanidine hydrochloride (GuHCl), extreme pH, and elevated temperature.

Abstract: 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 characterise 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 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.

Summary

INTRODUCTION

• Protein nanocages (e.g., virus-like particles (VLPs), ferritins, heat-shock proteins) selfassemble from multiple protein subunits into highly-organised macromolecular structures, that exhibit well-defined inner cavities, outer surfaces, and interfaces between subunits.
• 17 Unravelling the self-assembly mechanisms of protein nanocages is complicated, especially if they exhibit highly symmetric homooligomeric structures, like encapsulins.
• One study revealed that ferritin disassembles at extremely acidic pH 1.5, 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.
• 14 Specifically, Tm-Enc has been found to disassociate in strong acidic and alkaline conditions, or high concentrations of denaturing agents (e.g., guanidine hydrochloride, GuHCl).

MATERIALS AND METHODS

• All chemicals and reagents used in this study were purchased from Sigma-Aldrich, unless stated otherwise.
• Protein samples were mixed in a 1:4 ratio with 4X Native-PAGE sample buffer (ThermoFisher Scientific) and loaded into NativePAGE™ 3–12% Bis-Tris protein gels (ThermoFisher Scientific).
• Excitation and emission bandwidths of 5 nm, response of 0.2 s, medium sensitivity, data interval of 0.1 nm, scan speed of 100 nm/min, and 4 measurement accumulations were averaged, also known as The measurement parameters were.
• Samples with a final encapsulin concentration of 5 µM were prepared as described above.
• The tip-sample contact point between the AFM cantilever and the encapsulin was performed independently for each FD curve analysed using methods previously described 29.

RESULTS AND DISCUSSION

• Monitoring encapsulin assembly/disassembly using intrinsic tryptophan fluorescence Unloaded Tm-Enc, Mx-Enc, and Qt-Enc were produced in E. coli and purified by SEC and anion exchange chromatography prior to biophysical characterisation.
• This result suggests that Mx-Enc is both disassembled and largely unfolded at GuHCl concentrations ≥.
• 3 M. However, for Tm-Enc and Qt-Enc, absence of the intact encapsulin macrostructure occurs at lower GuHCl concentrations than the major redshift in Trp emission.
• 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.
• When cooled back to 20°C, DLS and TEM indicated Qt-Enc reassembled to its original size and ITF showed a blue-shift of emission .

Conclusion

• In summary, this study characterised the conditions facilitating the disassembly and reassembly of Tm-Enc, Mx-Enc, and Qt-Enc, with each encapsulin system varying in degree of sensitivity to GuHCl, pH, and temperature.
• The authors results suggest that disassembly by GuHCl and pH 13 affect different interfacial interactions.
• Due to being disassembled under high acidic conditions, a lot of ferritin is lost during this process and therefore it has a low loading efficiency.
• Ferritin variant subunit interfaces were engineered to display His6 motifs, which allowed nanocages to reassemble in the presence of either transition metal ions or pH 10, which increased loading efficiency by 1.6-3.6 times compared with previous acid disassembly.
• In summary, the findings of this study advance their understanding of encapsulins by providing critical insight into their unique disassembly/reassembly dynamics.

Figures

Figure 4. Reassembly timeframes of encapsulins. (a) ITF shift in emission wavelength (∆360/320) 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 emission wavelength of disassembled Enc (normalised to 1) and assembled Enc at 75 min (normalised to 0) was plotted. Error bars represent the mean ± standard deviation; n = 3 from three independent experiments. (b) Heatmap 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 normalised so that 1 = expected assembled size and 0 = disassembled encapsulin.

Figure 5. AFM imaging and elasticity comparing self-assembled/reassembled encapsulins. (a) Nanoscale AFM images obtained for individual encapsulins comparing 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 Hertz Fit of encapsulins used to determine Young’s elastic modulus (E) using Hertz equation to convert experimentally derived force versus distance curves into indentation data (coloured circles) and to fit the elastic (non-linear) region to calculated E values (black lines).

Figure 3. Comparison of Enc spectra of self-assembled, disassembled in GuHCl 6 M or 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).

Figure 2. GuHCl, pH, and thermally induced disassembly of Encs. (a) ITF showed an observed shift in emission wavelength (360/320) and indicated Trp solvation begins from 1 M GuHCl for MxEnc and Qt-Enc, and 3 M for Tm-Enc. (b) DLS measurements indicated 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 showed Trp solvation only significantly increased in alkaline conditions from pH 12 for Tm-Enc, MxEnc and Qt-Enc. (d) DLS measurements indicated 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 20–90°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 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 remained at 80-90°C. Upon cooling to 90°C, Qt-Enc appeared to reassemble, whereas Mx-Enc aggregated (indicated by black square). (g-j) TEM images show self-assembled encapsulins compared to disassembled encapsulins under varying conditions and subsequent reassembly back into spherical nanocages; Mx-Enc (black triangle T=3, white triangle T=1) (scale bars = 50 nm). For ITF data, the difference in emission wavelength of complete Trp solvation (normalised to 1) and assembled Enc (normalised to 0) was plotted. Error bars represent the mean ± standard deviation; n = 3 from three independent experiments. DLS results were normalised so that 1 = expected assembled size and 0 = disassembled encapsulin.

Figure 1. Monitoring encapsulin nanocage assembly states via ITF. 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 colours, 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; Qt: W95 and W154. Molecular graphics were created using UCSF ChimeraX 36. Using ITF, a shift in emission discriminated between the maximum emission wavelength of buried Trp in ‘assembled’ form: ~334 nm for (a) Tm-Enc and (c) Qt-Enc, or ~338 for (b) Mx-Enc; and solvent exposed Trp in ‘disassembled’ form (~354 nm) upon addition of 7 M GuHCl. Emission wavelengths are represented as Normalised Fluorescence Intensity (NFI).

Full text

Exploring the Self-Assembly of Encapsulin Protein
Nanocages from Different Structural Classes
India Boyton
⸸
†
, Sophia C. Goodchild
⸶
, Dennis Diaz
⸶
, Aaron Elbourne
‡
,
Lyndsey Collins-Praino
‡†
& Andrew Care
⸸
†§
*
⸸
School of Life Sciences, University of Technology Sydney, NSW 2007, Australia
⸶
Department of Molecular Sciences, Macquarie University, NSW 2109, Australia
‡
School of Science, College of Science, Engineering and Health, RMIT University,
Melbourne, VIC 3000, Australia
‡
Adelaide Medical School, The University of Adelaide, Adelaide, 5005, SA, Australia.
†
ARC Centre of Excellence for Nanoscale BioPhotonics, Macquarie University, NSW 2109,
Australia
§
ARC Centre of Excellence in Synthetic Biology, Macquarie University, NSW 2109,
Australia
*andrew.care@uts.edu.au
ABSTRACT
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 characterise 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 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.
KEYWORDS: encapsulin, protein nanocage, capsid assembly, self-assembly, intrinsic
tryptophan fluorescence, atomic force microscopy
.CC-BY-NC 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.06.447285doi: bioRxiv preprint

INTRODUCTION
Protein nanocages (e.g., virus-like particles (VLPs), ferritins, heat-shock proteins) self-
assemble from multiple protein subunits into highly-organised macromolecular structures, that
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.
9-11
Uniquely,
encapsulins selectively self-assemble around cargo enzymes tagged with a small encapsulation
signal peptide (ESig), packaging them.
12
This mechanism has been adapted to load foreign
cargo into encapsulins, reprogramming their functionality for different practical applications.
13-16
Encapsulin subunits autonomously assemble, with extraordinary fidelity, into
macromolecular nanocages. Such self-assembly is not only driven 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.
17
Unravelling the self-assembly mechanisms of protein nanocages is
complicated, especially if they exhibit highly symmetric homooligomeric structures, like
encapsulins.
18
Nevertheless, multiple analytical techniques now allow the molecular
mechanisms underlying protein nanocage assembly (e.g., protein folding) to be characterised
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), UV/VIS spectroscopy and synchrotron small-angle X-ray scattering (SAXS)
measurements to assess protein conformation
19-22
, transmission electron microscopy (TEM)
.CC-BY-NC 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.06.447285doi: bioRxiv preprint

and dynamic light scattering (DLS) to evaluate structural integrity, shape and size distribution,
and laser light scattering to monitor assembly kinetics.
23
One study revealed that ferritin
disassembles at extremely acidic pH 1.5, 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.
23
Importantly, this fundamental work led to the rational re-design of ferritin subunit
interfaces, resulting in engineered nanocages capable of disassembly at a more amenable pH
4.0.
24, 25
Such modification now permits labile compounds (e.g. small-molecule drugs) to be
controllably loaded into ferritin nanocages in a facile and non-destructive manner, enabling
downstream applications (e.g., drug delivery).
24, 25
In contrast, experimental data pertaining to encapsulins’ ability to disassemble/reassemble
and the mechanisms that underpin this natural phenomenon are sparse. The most characterised
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.
14
Specifically, Tm-Enc has been found to disassociate in
strong acidic and alkaline conditions, or 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 absence of denaturant).
27
Interestingly, Tm-Enc can be reassembled in the presence of ESig-tagged cargo (e.g., proteins,
nanomaterials), resulting in their selective encapsulation in vitro, and thus further expanding
encapsulins’ utility.
8, 27, 28 29
Despite these promising findings, key questions concerning the
biophysical mechanisms and physicochemical factors that underlie encapsulin
disassembly/reassembly, and how they might be controlled, remain unanswered, especially for
the T = 3 and T = 4 nanocages.
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). We combined intrinsic tryptophan fluorescence (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 provides critical insights into the dynamic
.CC-BY-NC 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.06.447285doi: bioRxiv preprint

mechanisms that govern the disassembly/reassembly of differing encapsulin structures, which
will help to expedite and broaden their future design, modification, and practical 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 optimised for expression in Escherichia coli and custom synthesised
as gBlock Gene Fragments (Integrated DNA Technologies). Encapsulins from Thermotoga
maritima (Tm) (UniProt: TM_0785), Myxococcus xanthus (Mx) (UniProt: MXAN_3556) and
Quasibacillus thermotolerans (Qt) (UniProt: QY95_01592) were each synthesised 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), summarised in Supplementary 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 Supplementary Table S1).
Recombinant Protein Production
Protein expression experiments were performed in 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/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/200–250 rpm for 4
.CC-BY-NC 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.06.447285doi: bioRxiv preprint

h and then cells were harvested via centrifugation (8,000 x g, 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 8,000 x
g, 4°C for 15 min. Supernatant containing soluble protein was heat treated in a water bath at
65°C for 15 min before centrifugation (10,000 x g, 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 10,000 x g 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 size exclusion
chromatography (SEC) using a HiPrep 26/60 Sephacryl S-500 HR column (GE Healthcare)
equilibrated with 50 mM HEPES pH 7.4. 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 linear gradient of 0–0.3 M NaCl and 0.3–
1M NaCl (Supplementary Figure S1). Fractions containing encapsulin, identified via sodium
dodecyl sulphate polyacrylamide gel electrophoresis (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 cut-off. Lastly, 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 visualised using SDS-PAGE, with molecular
weights compared with a commercial protein ladder (Precision Plus Protein, BioRad). The 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 at 300 rpm. Electrophoresis was performed at 200
.CC-BY-NC 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.06.447285doi: bioRxiv preprint

Frequently asked questions

Q1. What have the authors contributed in "Exploring the self-assembly of encapsulin protein nanocages from different structural classes" ?

While all 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. 

Q2. What is the effect of reassembly on the encapsulins?

clear morphological variation can be seen after reassembly, with all encapsulins demonstrating flattening of their surfaces. 

Q3. Why is ferritin able to disassemble under acid conditions?

For instance, due to being disassembled under high acidic conditions, a lot of ferritin is lost during this process and therefore it has a low loading efficiency. 

Q4. How did the encapsulins reassemble after disassembly?

TmEnc, Mx-Enc, and Qt-Enc were all able to reassemble within 75 min after disassembly with GuHCl; however, only Tm-Enc was able to reassemble after disassembly at pH 13 (within 15 min). 

Q5. How long does it take to reassemble encapsulins?

To advance encapsulins as a cargo-carrying platform, disassembled encapsulin needs to be able to be reassembled on a viable timescale. 

Q6. How was the reassembly of encapsulins initiated?

Reassembly of all encapsulins after disassembly in either 6 M GuHCl or pH 13 was initiated via overnight dialysis into reassembly buffer. 

Q7. What was the reaction used to purify the encapsulins?

The three encapsulins used in this study were purified via size exclusion chromatography (SEC) using a HiPrep 26/60 Sephacryl S-500 HR column (GE Healthcare) equilibrated with 50 mM HEPES pH 7.4. 

Q8. What is the rate of reassembly of Tm-Enc?

In a recent report, the in vivo loading of cargo proteins into Tm-Enc during its selfassembly was found to be ~8 times less efficient than with Mx-Enc.47 

Q9. What is the chemical stability of the encapsulins tested?

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. 

Q10. What conditions are used to alter the folding and/or assembly of encapsulins?

The authors 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). 

Q11. What is the robust encapsulin to GuHCl?

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.