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. Author manuscript; available in PMC: 2024 Apr 22.
Published in final edited form as: Methods Mol Biol. 2023;2671:111–120. doi: 10.1007/978-1-0716-3222-2_6

Fabrication of Protein Macromolecular Frameworks (PMFs) and Their Application in Catalytic Materials

Masaki Uchida 1, Ekaterina Selivanovitch 2, Kimberly McCoy 3, Trevor Douglas 2
PMCID: PMC11034859  NIHMSID: NIHMS1977715  PMID: 37308641

Abstract

The construction of three-dimensional (3D) array materials from nanoscale building blocks has drawn significant interest because of their potential to exhibit collective properties and functions arising from the interactions between individual building blocks. Protein cages such as virus-like particles (VLPs) have distinct advantages as building blocks for higher-order assemblies because they are extremely homogeneous in size and can be engineered with new functionalities by chemical and/or genetic modification. In this chapter, we describe a protocol for constructing a new class of protein-based superlattices, called protein macromolecular frameworks (PMFs). We also describe an exemplary method to evaluate the catalytic activity of enzyme-enclosed PMFs, which exhibit enhanced catalytic activity due to the preferential partitioning of charged substrates into the PMF.

Keywords: Virus-like particle (VLP), Protein array, Protein macromolecular framework (PMF), Superlattice, Self-assembly, Enzyme encapsulation, Collective behavior

1. Introduction

Protein cages, including virus-like particles (VLPs), provide unique platforms for encapsulation of functional cargos and have been utilized in various applications including catalysis and vaccine development [1, 2]. For example, we have demonstrated encapsulation of various enzymes inside the VLPs derived from P22 bacteriophage [3–6]. The enzyme cargos maintain their catalytic activities, while the capsid shell serves as a protective layer for the enzymes [4, 7], leading to the development of more robust catalytic materials. A key feature of protein cages, their easy functionalization via chemical or genetic modification, also makes them ideal building blocks to construct higher-order assemblies with designed functionalities [2, 8]. A range of interactions, including electrostatic [9–15], hydrophobic [16], complementary DNA [17–19], metal-ligand coordination [20], and specific protein-protein [21, 22] interactions have been exploited to mediate higher-order assembly of protein cages into three-dimensional (3D) arrays.

We have recently developed a new class of 3D protein arrays, protein macromolecule framework (PMF), by using P22 VLP and a capsid decoration protein (Dec) as building blocks (Fig. 1). The P22 VLP has three well-characterized morphologies, procapsid (PC) [23], expanded (EX) [24], and wiffleball (WB) [25, 26]. Dec is a homotrimeric protein and binds to the quasi and true threefold symmetry sites (80 binding sites per VLP) on the EX and WB forms of P22 capsid [27, 28]. Point mutation of Dec, in which the C-terminal serine is replaced with a cysteine (DecS134C), results in the formation of a head-to-head dimer of trimers via disulfide bond formation upon oxidation, enabling DecS134C to act as a ditopic linker bridging between P22 VLPs (Fig. 1) [29]. We have demonstrated that DecS134C bridges neighboring VLPs in an ordered array of P22 VLPs and stabilizes the lattice, resulting in the formation of the PMF (Figs. 1 and 2) [21, 22], which is conceptually analogous to a metal-organic framework (MOF) [30] but is constructed entirely of protein building blocks.

Fig. 1.

Fig. 1

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Schematic cartoon depicting protein macromolecular framework (PMF) formation process. Negatively charged P22-E2 VLPs are mixed with either (a) DecS134C linker protein or (b) positively charged PAMAM generation 6 dendrimers (G6), resulting in formation of amorphous array or an ordered array, respectively. When ionic strength is increased, (c) the array disassembles. (d) DecS134C linker proteins are added to G6 templated array to lock the structure in place while concomitantly increasing the lattice parameter approximately 4 nm. (e) Ionic strength is then increased, removing the G6 from the array but preserving the structure, yielding a PMF. (Adapted from Ref. 21 with permission)

Fig. 2.

Fig. 2

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Small-angle X-ray scattering of the templated assembly of PMFs. P22-E2 assembled with G6 results in an ordered array with a face-centered cubic (FCC) structure (black). When this ordered array is transferred into a high ionic strength buffer (I = 329 mM), structure factor contributions are significantly reduced, indicating disassembly of the array into individual VLPs (red). Addition of DecS134C linker protein to a G6 templated array (blue) shows preservation of long-range order and a shift in peak positions (vertical dashed lines). This peak shift corresponds to an increase in the lattice parameter from 92.6 nm to 96.8 nm. When PMFs is transferred into a high ionic strength buffer (purple), long-range order and peak position are preserved. (Adapted from Ref. 21 with permission)

We have shown that PMFs can also be fabricated from enzymeen-capsulated P22 VLPs [21, 22]. The robustness and versatility of the PMFs allow us to impart desired functionality by encapsulating any enzyme inside individual P22 VLPs, while the interstitial space and porosity of the PMF provides room for the diffusion of substrates with size- and charge-based selectivity. Because P22 VLP carries a negative exterior surface charge, the interstitial space of the PMF is highly negatively charged. Thus, positively charged substrates can be preferentially taken up into the PMF. This selectivity leads to a unique collective behavior where the catalytic activity of the PMFs is significantly higher than the enzyme-enclosed free VLPs for charged substrates [22]. Here we provide a protocol for the fabrication of PMFs from P22 VLPs and the Dec linkers. We also provide an exemplary protocol for a catalytic activity assay by using a PMF constructed from alcohol dehydrogenase D (AdhD) encapsulated P22 VLPs.

2. Materials

All solutions were prepared using ultrapure water (resistivity of 18.2 MΩcm at 25 °C) unless otherwise noted. Filter all buffer solutions through 0.2 μm filters.

2.1. Preparation of VLP Building Blocks

  1. P22 PMF is typically constructed with P22-E2, a mutant of P22 VLP with two repeats of the hepta-peptide sequence (VAALEKE) fused to the C-terminus of the coat protein and displayed on the exterior surface of the VLP [13]. The procapsid form of P22-E2 VLP and an enzyme encapsulated P22-E2 VLP were heterologously expressed in, and purified from, E. coli (BL21 (DE3)). The cloning, transformation, expression, and purification procedure was described in previous papers [3, 21, 22, 31].

  2. A mutant of the decoration protein (DecS134C) was heterologously expressed in, and purified from E. coli (BL21 (DE3)). The cloning, transformation, expression, and purification procedure was described in previous papers [21, 22, 29].

  3. Phosphate buffers: Phosphate buffers with two different ionic strengths (I) are prepared. (a) 50 mM sodium phosphate, 100 mM sodium chloride (I = 206 mM), pH 7.0. Weigh 2.92 g NaH2PO4•H2O, 7.73 g Na2HPO4•7H2O and 5.84 g NaCl. Dissolve them in 900 mL water. (b) 80 mM sodium phosphate, 160 mM sodium chloride (I = 329 mM), pH 7.0. Weigh 4.67 g NaH2PO4•H2O, 12.37 g Na2HPO4•H2O and 9.35 g NaCl. Dissolve them in 900 mL water. Adjust pH of the both solutions to 7.0 with 1 M NaOH. Make up to 1.0 L with water. Filter through 0.22 μm membrane filter. Store at room temperature.

  4. 0.2% (w/v) sodium dodecyl sulfate (SDS) in I = 206 mM phosphate buffer.

  5. Water bath with capability to heat up to 75 °C.

  6. Dithiothreitol (DTT).

  7. Copper (II) sulfate (CuSO4).

  8. 12–14 kDa molecular weight cutoff dialysis tubing and closures.

2.2. Construction of PMFs

  1. Polyamidoamine (PAMAM) generation 6 (G6) dendrimer.

  2. 12–14 kDa molecular weight cutoff dialysis tubing and closures.

  3. Phosphate buffers: Phosphate buffers with 50 mM sodium phosphate, 100 mM sodium chloride, pH 7.0 (I = 206 mM) and 80 mM sodium phosphate, 160 mM sodium chloride, pH 7.0 (I = 329 mM) (see subheading 2.1).

2.3. Activity Assay of AdhD-Enclosed PMF with Charged Substrates

  1. PMFs constituted with the EX form of AdhD-encapsulated P22-E2.

  2. Phosphate buffers: Phosphate buffers with three different ionic strengths (I), i.e., low salt (LS, I = 41 mM), intermediate salt (IS, I = 206 mM), and high salt (HS, I = 508 mM), are used for the activity assay. LS has 10 mM sodium phosphate, 20 mM sodium chloride, pH 7.0. IS has 50 mM sodium phosphate, 100 mM sodium chloride, pH 7.0. HS has 50 mM sodium phosphate, 400 mM sodium chloride, pH 7.0.

  3. Nicotinamide adenine dinucleotide (NADH) conjugated with PAMAM G0.5, G1.5, or their variants. They are referred to as NADH-x (y) in this chapter, where x and y represent the generation and charge of a dendrimer conjugated to NADH, respectively. The synthesis of the NADH variants has been described elsewhere [22, 32].

  4. Acetoin.

  5. UV-Vis spectrometer or Plate reader.

3. Methods

3.1. Transformation of the P22 VLP Morphology from PC to EX Form

  1. Adjust the concentration of P22-E2 PC to 1 mg/mL in I = 206 mM phosphate buffer.

  2. Add a freshly prepared 0.2% (w/v) SDS solution in I = 206 mM phosphate buffer to an equal volume of 1 mg/mL P22-E2 PC solution.

  3. Incubate the mixed solution for 5 min at room temperature.

  4. Centrifuge the sample at 17,000 g for 5 min at room temperature to remove aggregated protein.

  5. Pellet P22-E2 by ultracentrifugation at approximately 209,000 g (45,000 rpm) (F50L-839 rotor, Piramoon Technologies) for 50 min at 4 °C. Discard the supernatant to remove SDS. Resuspend the P22-E2 pellet into I = 329 mM phosphate buffer. Repeat this ultracentrifugation and resuspension process twice to ensure the removal of SDS [22, 33].

  6. Adjust the concentration of the EX form of P22-E2 to 1 mg/mL (see Note 1).

3.2. Transformation of the P22 VLP Morphology from PC to WB Form

  1. Adjust the concentration of P22-E2 PC to 1 mg/mL in I = 206 mM phosphate buffer.

  2. Heat the P22 sample in a 75 °C water bath for 25 min, then cool on ice for at least 15 min [21, 26].

  3. Centrifuge the sample at 17,000 g for 5 min at room temperature to remove aggregated protein.

  4. Pellet P22-E2 by ultracentrifugation at approximately 209,000 g (45,000 rpm) (F50L-8×9 rotor) for 50 min at 4 °C to remove scaffolding protein and coat protein released from the VLPs [25], followed by resuspension into I = 329 mM phosphate buffer.

  5. Adjust the concentration of the WB sample to 1 mg/mL with the same buffer (see Note 1).

3.3. Preparation of Ditopic Protein Linker from DecS134C

  1. Adjust the concentration of DecS134C to 1 mg/mL in I = 206 mM phosphate buffer.

  2. Add DTT to the DecS134C solution to obtain a final concentration of 5 mM DTT.

  3. Incubate the DecS134C solution for 3 h at room temperature (see Note 2).

  4. Transfer the DecS134C solution into a 12–14 kDa molecular weight cutoff dialysis tubing and dialyzed against I = 206 mM phosphate buffer at 4 °C overnight. The buffer solution should be replaced once with fresh buffer to ensure the removal of DTT from the DecS134C solution.

  5. Add CuSO4 to the reduced DecS134C solution to obtain a final concentration of 20 μM CuSO4, which will oxidize DecS134C.

  6. Incubate the DecS134C solution at 4 °C overnight, then heat at 60 °C for 20 min to form the head-to-head Dec dimer via disulfide bonds (see Note 3).

  7. Centrifuge the solution at 17,000 g for 5 min at room temperature to remove aggregated protein.

  8. Transfer the supernatant into a 12–14 kDa molecular weight cutoff dialysis tubing and dialyze against I = 206 mM phosphate buffer at 4 °C overnight to remove CuSO4.

3.4. Construction of PMFs from the EX or WB Form of P22 VLP

  1. Prepare a fresh solution of PAMAM G6 dendrimer in a 1:4 dilution of I = 329 mM phosphate buffer.

  2. Slowly add the dendrimer solution to the solution containing P22-E2 VLPs (either EX or WB form) at room temperature in a 1000-fold excess of dendrimers to VLP (see Note 4).

  3. Transfer the mixed solution into a 12–14 kDa molecular weight cutoff dialysis tubing and dialyze against I = 206 mM phosphate buffer at room temperature for at least 3 h (see Note 5).

  4. Adjust the concentration of DecS134C to 2 mg/mL with I = 206 mM phosphate buffer (see Note 6).

  5. Add the DecS134C solution to the P22-G6 solution at room temperature in a 160: 1 molar ratio of trimeric DecS134C: P22 VLP (see Note 7).

  6. Incubate the sample solution at room temperature for at least 30 min to ensure binding of DecS134C linkers to P22 VLPs, which will lead to the formation of the PMFs constituted of DecS134C linked P22 VLPs.

  7. Centrifuge the PMF sample solution at 5000 g for 3 min at room temperature and then gently remove the supernatant without disturbing the a small while pellet containing the PMFs.

  8. To remove the PAMAM G6 dendrimers from the PMF, resuspend the pellet in I = 329 mM phosphate buffer, followed by gentle centrifugation at 5000 g for 3 min at room temperature. Carefully remove the supernatant containing released PAMAM G6 dendrimers (see Note 8). Repeat this process twice to ensure that all dendrimers are removed from PMFs. Resuspend PMFs in I = 206 mM phosphate buffer and stored at 4 °C until further use.

3.5. Activity Assay of AdhD-Enclosed PMF with Charged Substrates

  1. Prepare acetoin in the three different buffers (LS, IS, and HS) with concentrations ranging from 0 to 120 mM (see Note 9).

  2. Prepare NADH variants, NADH0.5(−), NADH0.5(+), NADH1.5(−), and NADH1.5(+), in water at a high concentration, typically around 16 mM.

  3. Adjust the AdhD-enclosed PMF concentration to 1 mg/mL of P22 VLPs.

  4. Centrifuge the PMF at 5000 g for 3 min at room temperature to form a pellet, and then remove the supernatant without disturbing the pellet.

  5. Resuspend the pellet in the same volume of LS, IS, or HS buffer containing 0–120 mM acetoin and transfer to reaction vials.

  6. Add the NADH variants to the reaction vials in a 1:100 dilution just before the activity assay. The final concentration of NADH is typically around 160 μM.

  7. Constantly agitate the sample solutions containing PMFs, acetoin, and NADH variants to prevent the PMFs from settling during the enzyme activity assay at room temperature.

  8. Upon reaching each individual time-point, remove the respective aliquots from the reaction vials and centrifuge (17,000 × g, 1 min) (see Note 10). Recover the supernatant.

  9. Measure the absorbance at 340 nm (oxidation of NADH to NAD+ ) of the supernatant using a UV-Vis spectroscope or a plate reader (see Note 11). Repeat this step a total minimum of three times for each condition.

  10. Use the Michaelis-Menten kinetics model to extract the Michaelis-Menten constant (KM) and the turnover rate (kcat) (see Note 12).

Acknowledgments

This work was supported by a grant from the Human Frontier Science Program (HFSP) 4124801. M.U. was supported in part by the National Science Foundation grant CMMI-1922883. E.S. was partially supported by the Graduate Training Program in Quantitative and Chemical Biology under Award T32 GM109825 and Indiana University. T.D. was additionally supported by the National Science Foundation through grant 1720625.

Footnotes

1.

Morphological transformation of PC to EX and WB forms can be confirmed with native agarose gel electrophoresis as described in previous papers [21, 22, 25, 26, 34].

2.

The C-terminal cysteine of DecS134C tends to form disulfide bonds with other thiol containing molecules such as glutathione and cysteine during the expression process. DecS134C needs to be reduced to increase thiol availability for the desired formation of the head-to-head Dec dimer.

3.

It is common that some aggregates form during this step.

4.

The mixed solution should be clear, indicating no higher-order assembly in the I = 329 mM buffer.

5.

The mixed solution should become turbid in the I = 206 mM buffer, indicating formation of a higher-order assembly (Figs. 1 and 2).

6.

DecS134C can be concentrated by using a centrifugal filter device.

7.

There are 80 total Dec binding sites per P22 VLP [28]. Addition of 160-fold Dec per P22 VLP results in a twofold excess of Dec per binding site.

8.

The PMF of P22 VLP should stay as an assembled array material in the I = 329 mM buffer as the DecS134C linker cements the assembly (Figs. 1 and 2). The assembly without addition of DecS134C, on the other hand, disassembles in the same buffer [21].

9.

Concentration of acetoin for the activity assay is typically 0, 1, 3, 10, 25, 50, 80, and 120 mM.

10.

Typically, the solutions are aliquoted 3, 6, 9, 15, 21, 30, 60, and 120 min after addition of the NADH variants.

11.

Wavelength at 600 nm is also monitored to ensure that the PMFs are removed from the solution. The optical density at 600 nm of the supernatant should be nearly zero. The presence of the PMFs results in a higher value of the optical density due to light scattering.

12.

The concentration of AdhD was determined by measuring the absorbance at 280 nm and the molecular weight values of AdhD-encapsulated P22 VLP determined by SEC-MALS, as previously described [3, 35].

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