University of Groningen Self-Assembly of Electrostatic Cocrystals from Supercharged Fusion Peptides and Protein Cages

Self-assembly is a convenient process to arrange complex biomolecules into large hierarchically ordered structures. Electrostatic attraction between the building blocks is a particularly interesting driving force for the assembly process, as it is easily tunable and reversible. Large biomolecules with high surface charge density, such as proteins and protein cages, are very promising building blocks due to their uniform size and shape. Assemblies of functional molecules with well-defined nanostructures have wide-ranging applications but are difficult to produce precisely by synthetic methods. Furthermore, obtaining highly ordered structures is an important prerequisite for X-ray structure analysis. Here we show how negatively charged ferritin and viral protein cages can adopt specific cocrystal structures with supercharged cationic polypeptides (SUPs, K72) and their recombinant fusions with green fluorescent protein (GFP-K72). The cage structures and recombinant proteins self-assemble in aqueous solution to large ordered structures, where the structure morphology and size are controlled by the ratio of oppositely charged building blocks and the electrolyte concentration. Both ferritin and viral cages form cocrystals with face centered cubic structure and lattice constants of 14.0 and 28.5 nm, respectively. The crystals are porous and the cationic recombinant proteins occupy the voids between the cages. Such systems resemble naturally occurring occlusion bodies and may serve as protecting agents as well as aid the structure determination of biomolecules by X-ray scattering. M the highly evolved functionalities of native biomolecules has been in the focus of research efforts, especially over the past decade. Besides chemical composition, functionalities of natural systems are typically based on the three-dimensional position of the molecules. Additionally, biomolecules are often large but can still adopt specific hierarchical structures with great selectivity. Production of synthetic materials that could achieve the same level of structural sophistication has, however, been challenging. Another way to harvest the designs of nature is to extract the molecules from natural sources and incorporate them into nanostructured materials. The restrictions of top-down methods to produce fine-structures can simultaneously be overcome, as many biological molecules form organized systems via self-assembly processes. The procedure is the basis of many natural phenomena like protein folding and can be used to produce functional materials with well-defined nanostructures. Self-assembly is typically carried out in liquid media, which allows the building blocks to diffuse without restraints. Noncovalent self-assembly is preferred in many cases as it is typically reversible, easy to control, and applicable to a large pool of molecules, allowing production of assemblies with varying chemical composition and physical dimensions. The assemblies can additionally be tuned by chemical modification of the assembling particles or changing the environment of the assembly. Several bottom-up synthesis methods have been recently studied to produce such nanostructured materials. Practical applications include tissue engineering, drug delivery, catalysis, and nanopatterning. Protein cages have been utilized as part of self-assembling systems due to their ability to retain functionality while complexed. They often possess uniform size and shape, making them ideal building blocks for crystalline assemblies. Many protein cages additionally carry an overall electric charge, which enables them to assemble via electrostatic interactions. Such assemblies are reversible and responsive to changes in both pH and salinity of the solution, allowing additional control over the system. To form complexes, the charged particles require counterparts with opposing charge. Polyelectrolytes are a noteworthy option as they possess high charge density. They also have the ability to provide proteins and enzymes with additional stability and have therefore been used in delivery systems. Copolymers enable even more possibilities for optimizing such systems, as block Received: January 10, 2018 Accepted: February 13, 2018 Published: February 19, 2018 Letter pubs.acs.org/macroletters Cite This: ACS Macro Lett. 2018, 7, 318−323 © 2018 American Chemical Society 318 DOI: 10.1021/acsmacrolett.8b00023 ACS Macro Lett. 2018, 7, 318−323 This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. D ow nl oa de d vi a U N IV G R O N IN G E N o n M ar ch 2 7, 2 01 9 at 1 4: 31 :0 2 (U T C ). Se e ht tp s: //p ub s. ac s. or g/ sh ar in gg ui de lin es f or o pt io ns o n ho w to le gi tim at el y sh ar e pu bl is he d ar tic le s.

. The amino acid sequences of K72 (a) and GFP-K72 (b) used in the study.
E.coli BLR (DE3) competent cells (Novagen) were transformed with the expression vectors containing protein of interests. Terrific Broth medium (for 1 L, 12 g tryptone and 24 g yeast extract) enriched with phosphate buffer (for 1 L, 2.31 g potassium phosphate monobasic and 12.54 g potassium phosphate dibasic) and glycerol (4 mL per liter TB) and supplemented with 100 µg/mL ampicillin, was inoculated with an overnight starter culture to an initial optical density at 600 nm (OD600) of 0.1 and incubated at 37 °C with orbital agitation at 250 rpm until OD600 reached 0.7. Protein expression was induced by shifting temperature to 30 °C. Cultures were then continued for additional 16 h post-induction. Cells were subsequently harvested by centrifugation (7,000 x g, 30 min, 4 ºC), resuspended in lysis buffer (50 mM sodium phosphate buffer, pH 8.0, 300 mM NaCl, 20 mM imidazole) to an OD600 of 100 and disrupted with a constant cell disrupter (Constant Systems Ltd., UK). Cell debris was removed by centrifugation (40,000 x g, 90 min, 4 ºC). Proteins were purified from the supernatant under native conditions by Nisepharose chromatography. Product-containing fractions were pooled and dialyzed against ultrapure water and then purified by anion exchange chromatography using a Q HP column. Protein-containing fractions were dialyzed extensively against ultrapure water. Purified proteins were frozen in liquid nitrogen, lyophilized and stored at -80 ºC until further use.

Protein Characterization with SDS-PAGE and Mass Spectrometry
The concentrations of the purified polypeptides were determined by measuring absorbance at 280 nm using a spectrophotometer (Spectra Max M2, Molecular Devices, Sunnyvale, USA). Product purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 15 % polyacrylamide gel. Afterwards, gels were stained with Coomassie staining solution (40 % methanol, 10 % glacial acetic acid, 1 g/L Brilliant Blue R250). Photographs of the gels after staining were taken with a LAS-3000 Image Reader (Fuji Photo Film GmbH, Dusseldorf, Germany). The results are shown as Figure  S2, where the supercharged polypeptides exhibit different electrophoretic mobility. Mass spectrometric analysis was performed using a 4800 MALDI-TOF Analyzer in the linear positive mode. The protein samples were mixed 1:1 v/v with α-Cyano-4-hydroxycinnamic acid (SIGMA) (100 mg/mL in 70 % ACN and 0.1 % TFA). Mass spectra were analyzed with the Data Explorer software (version 4.9). Values determined by mass spectrometry are in good agreement with the masses that are calculated (shown in Figure S3 and Table S1) based on the amino acid sequence.

Dynamic Light Scattering (DLS) and Zeta Potential
Scattering intensity and hydrodynamic radius of the studied complexes were measured using a Nano ZS ZEN3600 tabletop DLS device provided by Malvern Instruments. The same instrument was used for the zeta potential measurements. All measurements were carried out at room temperature using water as solvent.
The initial samples in DLS measurements were prepared by adding 10 µL of CCMV stock solution (1 g/L) into 490 µL of water or 5 µL of aFT stock solution (10 g/L in water) into 495 µL of water, making the concentration of CCMV solution 0.02 g/L and that of aFT solution 0.1 g/L. K72 or GFP-K72 were gradually titrated into the initial solutions until the count rate stabilized. As the concentrations of CCMV and aFT differed from each other, the concentration of K72 and GFP-K72 were announced as concentration ratio to CCMV or aFT to make comparison easier. The solution was mixed with a micropipette after each addition and before measuring. Brand semi-micro UV-Cuvettes (PMMA) were used for all measurements. Count rates and volume distributions were obtained using Zetaziser software by Malvern Instruments. The volume distribution curves were presented for the initial solution, the end of steep increase in count rate and the final stabilized state (denoted as 1, 2 and 3 in Figure 2, respectively). Afterwards, the solutions were titrated with 5 M NaCl solution in water to investigate the disassembly of the structures. The titration was carried out similarly as K72 and GFP-K72 titrations, gradually and with mixing after each titration step.
In zeta potential measurements the CCMV and aFT water solutions were prepared in a similar manner to the DLS samples, making the concentration of the CCMV solution 0.02 g/L and that of aFT solutions 0.1 a) b) g/L in water. 6 µL of K72 or GFP-K72 stock solution (1 g/L) were added to the CCMV solutions and 30 µL of the same K72 or GFP-K72 stock solutions to the aFT solutions. After the zeta potentials of these final solutions were measured, the K72 or GFP-K72 content was doubled by adding another 6 µL of the stock solutions to the CCMV samples and 30 µL to the aFT solutions in order to see if excessive cation concentration would have an impact on the measurements. The zeta potential curves are presented in Figure  S4. Figure S4. Zeta potential curves with ratio of K72 or GFP-K72 to CCMV or aFT a) 3:5 and b) 6:5.

Agarose Gel Electrophoresis Mobility Shift Assay (EMSA)
Electrophoresis mobility of CCMV was followed as a function of K72 and GFP-K72 concentrations. The gel was prepared by dissolving 1 g of agarose into 100 mL of 1 x TAE buffer in a microwave oven. The solution was stained with 100 µL of 0.625 g/L ethidium bromide, poured into a mold and allowed to set while cooling down to room temperature. Samples were prepared by mixing CCMV and GFP-K72 solutions in water as presented in Table S2.

Small-angle X-ray Scattering (SAXS)
Scattering was measured using a system consisting of rotating anode Bruker Microstar microfocus X-ray source (Cu Kα radiation, λ = 1.54 Å), Montel multilayer focusing monochromator (Incoatec), four collimating slits (JJ X-Ray, resulting in beam size of less than 1 mm at the sample position) and Hi-Star 2D area detector (Bruker, sample to detector distance 1.59 m). The instrumentation except for the detector were under high vacuum to prevent scattering from the air. Silver behenate standard was used for the calibration of the scattering vector (magnitude of = 4 / , where 2 is the scattering angle) and the onedimensional SAXS data was obtained by azimuthally averaging the 2D scattering data. The samples were prepared by mixing together 1.0 µL of K72 or GFP-K72, 4 µL of water and 5 µL of CCMV stock solution (1 g/L) or 2.5 µL of K72 or GFP-K72, 2.5 µL water and 5.0 µL of aFT stock solution (1 g/L). Thus the weight ratios were 1:0.2 for CCMV samples and 1:0.5 for aFT samples. The sample solutions were sealed within a steel slug sealed from both sides with Kapton foil.

Cryo-Transmission Electron Microscopy (Cryo-TEM)
The transmission electron microscopy (TEM) images were collected using JEM 3200FSC field emission microscope (JEOL) operated at 300 kV in bright field mode with Omega-type Zero-loss energy filter. Samples were imaged on plasma cleaned 200-mesh copper grids with either holey carbon (CF-Quantifoil) or lacey carbon support film. 3.0 µL of the aqueous dispersion was placed on a grid and plunge freezed in 1/1 (v/v) liquid propane/ethane mixture using Vitrobot™ with 3s blotting time under 100 % humidity. The images were acquired with GATAN DIGITAL MICROGRAPH software while the specimen temperature was maintained at -187 °C.

Optical Microscopy
The optical microscopy imaging was done using Leica DM4500 P microscope combined with Canon EOS 60D camera in transmission mode. Samples were prepared in tris(hydroxymethyl)aminomethane (Tris) buffer with varying NaCl concentration to observe the effect of electrolyte. The samples were prepared by making a Tris buffer in water and adding NaCl, K72 or GFP-K72 and CCMV or aFT into it (in that order) and mixing gently with a pipette. All of the solutions had 5.0 mM of Tris and 100 mg/L of aFT or 50 mg/L of CCMV. CCMV solutions had 10 mg/L of K72 or GFP-K72 and aFT solutions 60 mg/L of K72 or GFP-K72. The sample compositions are presented in Table S3 and obtained images in Figure S5. The samples were left to crystallize at 6 °C for 10 days before imaging. Samples with NaCl concentration of 100 mM or higher had no assemblies large enough to be detected by an optical microscope.

Fluorescence Spectroscopy
The complex composition was studied by quenching the fluorescence of GFP-K72 via digestion of GFP using trypsin. Fluorescence of free GFP-K72 and that of CCMV and aFT complexes were measured for three hours in both absence and presence of trypsin. The measurements were conducted using an Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer equipped with Varian PCB 1500 Water Peltier System. All the spectra were recorded in Brand semi-micro UV-Cuvettes (PMMA) at 37 °C using excitation wavelength of 400 nm. Sample compositions are presented in Table S4 and the measured spectra in Figure  S6. The samples were prepared by combining all the components of each sample except for trypsin in a cuvette and mixing gently with a pipette. Trypsin was added last to the specified samples and solutions were stirred quickly with a pipette tip. All the samples were equilibrated in the sample holder for 5 minutes to reach the measurement temperature before starting the measurement.  Figure S6. Fluorescence spectroscopy curves of GFP-K72, GFP-K72 -CCMV and GFP-K72 -aFT complexes in both presence and absence of trypsin. The intensities are presented as percentage values of the initial intensity (t = 5 min after mixing) detected for each sample.