Encapsulation of Transketolase into In Vitro-Assembled Protein Nanocompartments Improves Thermal Stability

Abstract

Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritima T = 1 and Myxococcus xanthus T = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthus T = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritima T = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritima T = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization.

Keywords: cargo loading; encapsulin; in vitro assembly; nanocompartment; protein; stability; transketolase.

Figure 1

In vitro reassembly of T. maritima and M. xanthus encapsulins. (A) Capsid structures and sizes of T. maritimaT = 1 encapsulin and M. xanthusT = 3 and T = 1 encapsulins. (B, C) BN-PAGE of Tm_encap and Mx_encap reassembly reaction conditions (not SEC purified). A = assembled, D = denatured, R = reassembled in reassembly buffer, RA = reassembly buffer +250 mM l-arginine, RAG = reassembly buffer + 250 mM l-arginine + 20% v/v glycerol, RS = reassembly buffer + 0.5 M sorbitol. (D, E) SEC A₂₈₀ profiles of Tm_encap and Mx_encap, assembled, disassembled, and reassembled with denaturation conditions indicated. (F–H) TEM micrographs of Tm_encap, in vivo assembled, disassembled (0.15 M NaOH), and reassembled, images from pooled SEC fractions 8.5–14 mL. Scale bar = 100 nm. (I–K) TEM micrographs of Mx_encap, in vivo assembled, disassembled (8 M urea), and reassembled, images from pooled SEC fractions 8.5–12 mL. Scale bar = 100 nm.

Figure 2

In vitro sfGFP cargo loading specificity and scaffolding effect. (A) Workflow of in vitro sfGFP cargo loading method. Reassembly of encapsulin proteins was initiated by 10 times dilution of denaturant condition with reassembly buffer (0.3 M Tris-Cl pH 7.5, 0.15 M NaCl) to a final monomer concentration of 10 μM. (B, C) sfGFP loading into the Tm_encap (B) and sfGFP loading into the Mx_encap (C). Molar ratio of sfGFP to encapsulin monomer 0.2, 1, 5, and 10 to 1, respectively. M = molecular weight marker. Top black and white image shows fluorescence signal of sfGFP. Bottom image shows Coomassie-stained BN-PAGE gel. Full BN-PAGE is shown in Figure S2. (D) sfGFP cargo loading into Mx_encap (disassembled in 8 M urea) at increasing concentrations of sfGFP showing a decrease of assembly with high sfGFP concentration. Top black and white image shows fluorescence signal of sfGFP, bottom image shows Coomassie-stained BN-PAGE gel. M = molecular weight marker, vivo = in vivo-loaded encapsulins, A = assembled (before denaturation), and numbers in lanes indicate molar ratio of sfGFP to encapsulin monomer.

Figure 3

In vitro sfGFP cargo loading and scaffolding effect on M. xanthus encapsulin. All data shown are from SEC purified samples. (A, B) SDS-PAGE densitometry of in vitro- versus in vivo-loaded encapsulin. Schematic below A and B shows summary of loading capacity of in vitro versus in vivo sfGFP-loaded encapsulins. Data were derived from a single experiment. (C–G) TEM micrographs of the following: in vitro-loaded Tm_encap with sfGFP-TmCLP, molar ratios indicated (C, D); in vivo-loaded Tm_encap with sfGFP-TmCLP (E); in vitro-loaded Mx_encap with sfGFP-MxCLP (F); and in vivo-loaded Mx_encap with sfGFP-MxCLP (G). TEM scale bar = 100 nm. (H) Violin plot of TEM diameter measurement of capsids before and after in vitro loading of sfGFP versus in vivo loading. Measurements of 100 particles per sample. Associated T = 3 and T = 1 size range indicated. (I) sfGFP:capsid monomer concentration affects in vitro capsid assembly efficiency and capsid properties. Increasing sfGFP concentrations lead to misformed Tm_encap and higher proportion of T = 3 over T = 1 Mx_encap species (scaffolding effect), followed by a decrease in Mx_encap assembly when ratio is above 5:1.

Figure 4

In vitro loading and enzyme activity of transketolase (TK) cargo. (A) Denaturing SDS-PAGE showing TK loading into T. maritima encapsulin. (B) TEM micrograph of TK-loaded T. maritima encapsulins at 1:1 molar ratio, from SEC fractions 8.5–14 mL (Figure S5) (scale bar 100 nm). Some incomplete capsids can be seen. (C) Erythrulose reaction with TK (yellow) loaded into Tm_encap, TK cofactor thiamine pyrophosphate (TPP) and Mg²⁺. TK activity was measured as the formation of erythrulose from the 3-carbon ketol donor β-hydroxypyruvate (HPA) and 2-carbon acceptor glycolaldehyde (GA). (D) Transketolase activity comparing free TK (100%), free TK-TmCLP and encapsulated TK-TmCLP in Tm encapsulin (TK-TmCLP). Column plots show the mean values with error bars from standard deviations based on three replicates.

Figure 5

FRET assay of in vitro-loaded transketolase. (A) TK homodimer with K603pAzF-labeled position shown in green and magenta, illustrating the FRET donor AF488 and acceptor AF594, respectively. The α carbon distance indicated at 19 Å. (B) SDS-PAGE of Tm-encapsulin only and encapsulated FRET dye-labeled TK-TmCLP K603pAzF. White arrows highlighting two bands associated with labeled and unlabeled TK-TmCLP K603pAzF. (C) Normalized 350/330 nm ITF ratio of transketolase (TK), TK fused with TmCLP (TK-TmCLP), and TK-TmCLP with NcAA incorporation (TK-TmCLP K603pAzF). Thermal ramp rate at 4 °C per minute. (D, E) Normalized FRET donor data at 520 nm emission of free (D) and encapsulated (E) labeled TK-TmCLP K603pAzF. Normalized plots fitted Van‘t Hoff two-state transition. Different colored data points indicate individual repeats. (F,G) Normalized FRET acceptor data at 620 nm emission of free (F) and encapsulated (G) labeled TK-TmCLP K603pAzF. 620 nm emission showed a multistate transition and could not be fitted. Different colored data points indicate individual repeats. (H) FRET emission ratio 520/620 of free and encapsulated labeled TK-TmCLP K603pAzF with the same thermal ramp rate. (I) Luminal view on the T. maritima encapsulin at the three-symmetry axis. Bound FMN is shown in yellow, and residues involved in cargo binding are highlighted in magenta.