ARTICLES SCIENCE CHINA Materials link.springer.com link.springer.com Published online 17 January 2022 | https://doi.org/10.1007/s40843-021-1914-0 Sci China Mater 2022, 65(6): 1662–1670 The spectrum of building block conformers sustains the biophysical properties of clinically-oriented self-assembling protein nanoparticles Eric Voltà-Durán1,2,3, Julieta M Sánchez1,2,3,4, Hèctor-López-Laguna1,2,3, Eloi Parladé1,2,3, Laura Sánchez-García1,2,3#, Alejandro Sánchez-Chardi5,6, Ario de Marco7, Ugutz Unzueta2,3,8, Esther Vázquez1,2,3 and Antonio Villaverde1,2,3* ABSTRACT Histidine-rich peptides confer self-assembling INTRODUCTION properties to recombinant proteins through the supramole- Histidine-rich peptides, when genetically fused to recombinant cular coordination with divalent cations. This fact allows the proteins, confer them the capability to self-assemble as different cost-effective, large-scale generation of microscopic and kinds of functional protein-only materials including fibers, macroscopic protein materials with intriguing biomedical nanoparticles (NPs) and microparticles, through their cross- properties. Among such materials, resulting from the simple molecular coordination with divalent cations [1]. This happens bioproduction of protein building blocks, homomeric nano- because the interactivity exhibited by the hexahistidine tag (H6) particles are of special value as multivalent interactors and and other His-rich tails with Ni2+ during the immobilized metal drug carriers. Interestingly, we have here identified that the affinity chromatographic (IMAC) purification of recombinant assembly of a given His-tagged protein might render distin- proteins [2,3] can be also exploited for a controlled cross- guishable categories of self-assembling protein nanoparticles. molecular assembly, when adding defined amounts of Zn2+, This fact has been scrutinized through the nanobody-con- Ca2+, Mn2+ or other divalent cations to solutions of pure His- taining fusion proteins EM1-GFP-H6 and A3C8-GFP-H6, tagged protein [4–6]. In fact, this principle sustains the forma- whose biosynthesis results in two distinguishable populations tion of the secretory granules in the mammalian endocrine of building blocks. In one of them, the assembling and dis- system [7,8] and of different types of amyloidal and non-amy- assembling is controllable by cations. However, a second po- loidal protein materials existing in nature [4,7,9–16]. The sim- pulation immediately self-assembles upon purification plicity of His-rich peptides used as architectonic agents at the through a non-regulatable pathway, rendering larger nano- nanoscale offers an interesting alternative to more refined particles with specific biological properties. The structural approaches to control protein self-assembling, that might analyses of both model proteins and nanoparticles revealed require more sophisticated protein engineering [17–21]. important conformational variability in the building blocks. We have previously generated a family of recombinant This fact renders different structural and functional categories modular proteins, based on an N-terminal cationic peptide and of the final soft materials resulting from the participation of a C-terminal polyhistidine (mainly H6), which, when flanking a energetically unstable intermediates in the oligomerization protein of interest, self-assemble as homomeric NPs of around process. These data illustrate the complexity of the His- 15–20 nm [22]. Since such an assembling platform is fully mediated protein assembling in recombinant proteins but transversal and highly robust, and the resulting NPs are stable they also offer clues for a better design and refinement of upon in vivo administration [23], these materials have been protein-based nanomedicines, which, resulting from biologi- developed as effective nanocarriers for conventional antitumoral cal fabrication, show an architectonic flexibility unusual drugs in colorectal cancer [24], lymphoma [25,26] and acute among biomaterials. myeloid leukemia [27]. In addition, the incorporation of pro- apoptotic peptides [28], toxins [29] or venoms [30] to the Keywords: recombinant proteins, self-assembling, protein ma- modular protein constructs allows generating cytotoxic building terials, nanoparticles, biomaterials blocks that, if targeted through solvent-exposed ligands of 1 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain 2 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain 3 CIBER de Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra 08193, Barcelona, Spain 4 Instituto de Investigaciones Biológicas y Tecnológicas (IIBYT) (CONICET-Universidad Nacional de Córdoba), ICTA & Cátedra de Química Biológica, Departamento de Química, FCEFyN, UNC. Av. Velez Sarsfield 1611, X 5016GCA Córdoba, Argentina 5 Servei de Microscòpia, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain 6 Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain 7 Laboratory for Environmental and Life Sciences, University of Nova Gorica, Nova Gorica 5000, Slovenia 8 Biomedical Research Institute Sant Pau (IIB Sant Pau), Sant Antoni Mª Claret 167, 08025 Barcelona, Spain # Present address: Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, 08195 Sant Cugat del Vallès, Spain * corresponding author (email: antoni.villaverde@uab.cat) 1662 © The Author(s) 2022. This article is published with open access at link.springer.com. June 2022 | Vol. 65 No. 6 SCIENCE CHINA Materials ARTICLES tumoral markers, result in NPs with selective, build-in anti- 20°C overnight, upon addition of isopropyl β-D-1-thiogalacto- tumoral activity for precision medicine [31]. The polyhistidine pyranoside (IPTG, at 0.1 mmol L−1 for A3C8-GFP-H6 and tail, apart from being an architectonic agent, allows the one-step 1 mmol L−1 for EM1-GFP-H6). Bacterial lysates were obtained purification of the recombinant building blocks from the bac- by French Press (Thermo FA-078A) disruption at 1200 psi (1 psi terial cell extracts, upon their mechanical disruption. = 6.89 × 103 Pa) in wash buffer (20 mmol L−1 Tris-HCl, During the production of these types of NPs, we have occa- 500 mmol L−1 NaCl, 10 mmol L−1 imidazole, pH 8.0) with the sionally observed some structural variability in the oligomers protease inhibitor cOmplete ethylene diamine tetraacetic acid resulting from self-assembling [32]. Although from a production (EDTA)-free (Roche, USA). A recombinant version of ricin, point of view, this fact can be simply overcome by selecting the namely T22-mRTA-H6, was produced and purified as pre- desired material population in the chromatography, the extent, viously described [34]. causes and biological significance of such variability are not An ÄKTA Pure FPLC system (GE Healthcare, USA) was used known. In a recent development of two versions of fluorescent for the IMAC in a His-Trap HP column (GE Healthcare, USA). and nanostructured nanobody fusions [33], such variability was Protein elution was achieved by lineally increasing the molar specially apparent. Therefore, we decided to take these two amount of imidazole in the column through an elution buffer proteins (A3C8-GFP-H6 and EM1-GFP-H6, Fig. 1a, b) as (20 mmol L−1 Tris-HCl, 500 mmol L−1 NaCl, 500 mmol L−1 models to investigate the categories of particle subpopulations imidazole, pH 8.0). Protein purity and integrity was assessed by generated through the H6-based platform, why they assemble in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- disparate material versions and how they functionally perform PAGE), western blot (anti-His, Santa Cruz Biotechnology, USA) in biological interfaces. and matrix assisted laser-desorption ionization time-of-flight (MALDI-TOF). Proteins were finally dialyzed against sodium EXPERIMENTAL SECTION bicarbonate with salt buffer (166 mmol L−1 NaHCO3, 333 mmol L−1 NaCl, pH 8.0). Protein concentration was deter- Protein design, production and purification mined by the Bradford assay and Nanodrop was used to measure Escherichia coli (E. coli) codon-optimized A3C8-GFP-H6 and the A280/A260 (absorbance at 280/260 nm) ratio, indicative of EM1-GFP-H6 genes were expressed as inserted in the plasmid DNA presence in the sample. pET22b. Both genes encode nanobody and green fluorescent protein (GFP) fusions in the form of His-tagged proteins [33]. Three-dimensional protein modeling and visualization A3C8 shows specificity towards the plant toxin ricin, while EM1 Structure prediction of each nanobody-based modular protein has specificity towards the Her2 receptor, overexpressed in was performed to estimate the size of the building block, using breast cancer cells. Proteins were produced in E. coli Origami B the RosettaCM [35] high-resolution approach via the Robetta (BL21, OmpT−, Lon−, TrxB−, Gor−) (Novagen, Germany) at webserver [36]. Specifically, EM1, A3C8 and GFP-H6 domains Figure 1 Protein materials. (a) Modular disposition of A3C8-GFP-H6 and EM1-GFP-H6 chimeric proteins. Amino acid sequences are shown. An N- terminal nanobody (A3C8, in dark red; EM1, in dark purple) is fused to a GFP (in green) with a C-terminal hexa-histidine tag (H6, in yellowish orange) through a short peptidic linker (L, in grey). Color legend is maintained hereafter. (b) Three-dimensional models of the monomeric form of each protein. Black lines indicate the estimated main and cross axis distances (in nm) extracted from the models. (c) IMAC purification chromatograms of both proteins. The first peak corresponds to untagged bacterial proteins that are weakly attached to the column through nonspecific binding. Two pure populations were obtained in each case, eluting at different concentrations of imidazole. Left y-axis and right y-axis indicate the A280 nm (AU) and the imidazole concentration (mmol L−1), respectively, when elution of each mL (x-axis) occurs. (d) Volume size distribution (nm) of each purified population measured by DLS. Monomeric populations of each protein are represented in gray, whereas NPs are colored as previously stated. Values depicted indicate the mode size of each peak. (e) Table summarizing the properties of both proteins, in their respective monomeric or NP forms. Data are indicated as mean ± standard error of the mean. June 2022 | Vol. 65 No. 6 © The Author(s) 2022. This article is published with open access at link.springer.com. 1663 ARTICLES SCIENCE CHINA Materials were modelled individually and manually assembled in UCSF dried, and immediately observed without coating in a field Chimera (v1.14) to serve as quality templates for the prediction emission scanning electron microscope (FESEM) Merlin (Zeiss, of the whole modular construct. Parameters were set to 100 Oberkochen, Germany) operating at 1 kV and equipped with a sampling models, 1 register shift, and a probability of 0.1 of high-resolution in-lens secondary electron detector. Repre- sampling fragments within template regions. Best candidates sentative images of general fields and nanostructure details were were chosen by the highest confidence score and lowest error collected at three magnifications (180,000×, 240,000× and estimates. UCSF ChimeraX (v1.1) was used to visualize the 500,000×). For negative staining, drops of 10 μL of the same three-dimensional structures [37]. samples were deposited 1 min in 200 mesh copper grids coated with carbon, contrasted with uranyl acetate 2% (Polysciences Assembly and disassembly of NPs Inc.) during 1 min, air dried and observed with a transmission The monomeric populations of A3C8-GFP-H6 and EM1-GFP- electron microscope (TEM) JEM-1400 (Jeol Ltd.) operating at H6 were assembled into protein Zn NPs by the addition of 120 kV and equipped with a Gatan Orius SC200 CCD camera 0.22 μm-filtered ZnCl2 at 1:3 molar ratio with the histidine (Gatan Inc.). Representative images of general fields and residues in the H6 tag. Proteins were set to 0.2 mg mL−1 and nanostructure details were collected at three magnifications ZnCl2 was added at a final concentration of 8.5 mmol L−1 for (5000×, 10,000× and 50,000×). A3C8-GFP-H6 and 8.3 mmol L−1 for EM1-GFP-H6, depending on their molecular weights. Controlled disassembly of protein Thermal stability Zn NPs was subsequently obtained by adding EDTA at the same DLS (using the settings and equipment afore-mentioned) was molar concentration. The spontaneous protein NPs based on used to evaluate the thermal stability of A3C8-GFP-H6 and A3C8-GFP-H6 and EM1-GFP-H6 were disassembled by the EM1-GFP-H6 at their different oligomerization states. Tem- addition of 0.5% v/v Triton X-100. Addition of 2% anionic SDS perature was initially set at 25°C and progressively increased up detergent also resulted in NP disassembly. to 90°C. To evaluate the thermal behavior of A3C8-GFP-H6 and EM1-GFP-H6 at their different oligomerization states within a Physicochemical characterization range from 25 to 90°C, center of spectral mass (CSM) was cal- A3C8-GFP-H6 and EM1-GFP-H6, at their different oligomer- culated as previously described [39] for analysis and compar- ization states, were analyzed by SDS-PAGE and western blot isons. CSM is related with the relative exposure of the based on an anti-His (Santa Cruz Biotechnology, USA) to assess tryptophan (Trp) to the protein solvent. The maximum red-shift protein purity and integrity. The same molar amount was used in the CSM of the Trp is compatible with a large solvent for each protein. For characterization experiments, proteins accessibility. On the contrary, the blue shift is related to a highly were set to 0.2 mg mL−1 and pH 8.0. Dynamic light scattering hydrophobic environment for Trp. (DLS) was used to determine the volume size distribution of A3C8-GFP-H6 and EM1-GFP-H6 at their different oligomer- ization states. Measurements were carried out in triplicate at Functional characterization 25°C in a Zetasizer Advanced Pro Blue (Malvern Instruments CXCR4+ human cervical adenocarcinoma cells (Hela, ATCC, Limited, Malvern, Worcestershire, UK) at 633 nm. CCL-2) were used for functional characterization experiments. Fluorescence emission spectra of all protein variants were HeLa cells were maintained in Mem-Alpha (Gibco) media analyzed with a Varian Cary Eclipse Fluorescence Spectro- supplemented with fetal bovine serum (Gibco) at 5% CO2 in a photometer (Agilent Technologies). For tryptophan fluores- humidified atmosphere. HeLa cells were used to test the neu- cence, the excitation wavelength was set at 295 nm and the tralization capacity of A3C8-GFP-H6 at its different oligomer- excitation and emission slits at 5 nm. Measurements were car- ization states. They were seeded in a 96-well plate at 3.5 × 103 ried out by triplicate at 20°C. For GFP fluorescence, the exci- cells/well and the proteins were added after 24 h. A3C8-GFP-H6 tation wavelength was set at 488 nm and the emission peak was was pre-incubated for 1 h with T22-mRTA-H6 (to allow achieved at 511 nm. Measurements were carried out by triplicate potential binding and neutralization before exposure to cells), a at 20 and 37°C and the excitation and emission slits were set at recombinant modular protein containing the CXCR4-targeting 5 nm. peptide T22 and the active site of ricin toxin, at the three dif- Circular dichroism (CD) measurements were made with a ferent molar ratios of A3C8:ricin (1:10, 1:1 and 10:1). T22- JASCO J-715 spectropolarimeter (JASCO, USA) using a 0.2-mm mRTA-H6 concentration was set at 50 nmol L−1 in all cases. path length quartz cell. Two spectra were acquired for each Protein mixtures were added at a final volume of 100 μL for 48 h protein species. Each spectrum was an average of ten scans. The and the Cell-Titer Glo Luminescent Cell Viability Assay (Pro- scan speed was set at 50 nm min−1 with a 1 s response time. mega, USA) Protocol was followed. Viabilities of cells without Measurements were obtained as ellipticity in millidegrees protein incubation and cells treated only with 50 nmol L−1 T22- (mdeg) in the 190–260 nm region. The secondary structure mRTA-H6 were used as controls. A Victor3 (PerkinElmer, USA) content of each protein format was analyzed using the Dicroweb microplate reader was used for the measurements. Experiments platform [38]. were performed in triplicate. Ultrastructural characterization Statistical analysis Ultrastructural morphology (size and shape) of A3C8-GFP-H6 Data is represented as mean ± standard error of the mean. For and EM1-GFP-H6 NPs with and without Zn2+ was visualized the neutralization assay data, a Shapiro-Wilk test was performed with two rapid high-resolution electron microscopy techniques. to ensure normality. Significant differences among means were Drops of 10 μL of the four samples resuspended in its buffer identified through a One-Way ANOVA test. GraphPad Prism were deposited in silicon wafers (Ted Pella Inc.) for 1 min, air was used for statistical tests. 1664 © The Author(s) 2022. This article is published with open access at link.springer.com. June 2022 | Vol. 65 No. 6 SCIENCE CHINA Materials ARTICLES RESULTS AND DISCUSSION aggregates but that they have some extent of regular organiza- The IMAC purification of A3C8-GFP-H6 and EM1-GFP-H6 tion at the nanoscale. Importantly, only negligible traces of from cell extracts of the producing bacteria resulted, in both nucleic acids were detected associated to the protein in each of cases, in two distinguishable pure protein peaks, sequentially these samples according to the A280/A260 ratio [40] (Fig. 1e), released from the columns because of a differential affinity of the whose abundance might have accounted for the formation of proteins to the immobilized Ni2+. Both proteins showed a similar nanoscale protein-DNA complexes through cationic protein distribution in the chromatogram, with an initial peak estimated stretches. Both models, namely A3C8-GFP-H6 and EM1-GFP- to represent 86%–87% of the total protein amount and a final H6, showed a similar profile when determining all these para- peak, accounting for the remaining 13%–14% of the protein meters. population. The DLS of these samples revealed, for both pro- As expected for a H6-tagged protein [41,42], the addition of teins, that the primarily eluted protein peak presumably corre- equimolar amounts of Zn2+ to the monomeric protein versions sponded to monomers (Ms) while the protein sample released at promoted a shift in the DLS plots from 6.2–6.7 to 10.2–10.5 nm, higher imidazole concentrations corresponded to larger mate- indicative of protein assembling as NPs (Zn NPs, Fig. 2a). Also, rials (Fig. 1c). The monomer-containing samples showed a as expected, such size increase was reversed by the addition of hydrodynamic size of around 6–7 nm by DLS (Fig. 1d, e), a EDTA, since the metal chelation is able to disassemble multi- dimension compatible to those estimated in a plain modeling of meric protein materials organized through cross molecular both proteins, in which a certain degree of flexibility is expected interactions with metals (Fig. 2b) [1]. Contrarily, the pre-formed between the nanobody and the GFP modules (Fig. 1b). The protein NPs peaking at 70 nm were not disassembled by EDTA larger materials, leaked at latter elution volumes, showed a mean or by imidazole (Fig. 2c and data not shown), even at con- size slightly above 100 nm (Fig. 1e), with a mode peak size centrations higher than those used to disassemble the small NPs around 70 nm (Fig. 1d). The fact that such large entities were (Fig. 2b and data not shown). This finding was indicative that eluted from IMAC at high concentrations of imidazole was forces other than divalent cation coordination were supporting indicative of a tighter attachment to the column than the the organization of such a more complex material. For other monomeric versions (Fig. 1c). The polydispersity indexes of the types of related protein-only NPs, hydrogen bonds, van der protein in these slowly eluted fractions were relatively low Waals and especially electrostatic interactions were predicted to (Fig. 1e), indicating that the materials are not mere protein act, promoting and maintaining protein-protein contacts [23], in Figure 2 Physicochemical characterization. (a) Volume size distributions (nm) of A3C8-GFP-H6 and EM1-GFP-H6 in four different states (monomer, in grey; NP, in dark red/purple; Zn-mediated NP, in mild red/purple; and detergent-disassembled NP, in dark grey). Color legends are maintained hereafter. (b) Volume size distributions (nm) of A3C8-GFP-H6 and EM1-GFP-H6 Zn NPs upon EDTA-mediated disassembling (dashed line). Values depicted indicate the mode size of each peak. (c) Volume size distributions (nm) of A3C8-GFP-H6 and EM1-GFP-H6 NPs upon addition of EDTA (dashed line). Imidazole (400 mmol L−1) was also unable to disassemble these materials (not shown). (d) Protein gel electrophoresis image of each protein state. Numbers at left indicate the molecular weight (MW) bands of the marker in kDa. (e) His-tag immunodetection of bands shown in (c). (f) Intrinsic GFP-emission (a.u.) at 511 nm upon fluorophore excitation at 488 nm, for both proteins, at 25°C (assembly temperature) and 37°C (working temperature). Proteins were adjusted to 0.2 mg mL−1 prior to measurements in (a, b, c and f). June 2022 | Vol. 65 No. 6 © The Author(s) 2022. This article is published with open access at link.springer.com. 1665 ARTICLES SCIENCE CHINA Materials a similar way in which monomers are kept together in viral clustered by Zn showed a rather spherical organization. The capsids [43–45]. In this context, the addition of 0.5% Triton X- higher amount of bulk material in NPs should result from a 100 reduced the size of the materials from more than 100 nm higher number of building blocks arranged in each individual (mean) to 10 nm (Fig. 2a), a value very similar to that exhibited item. If so, we should expect a more intense reactivity of the by the small Zn NPs. Such disassembling process appeared to be nanobody moiety provided it is solvent-exposed, when com- not complete, since forms with a size compatible to that pared with Zn NPs or with Ms. Since the nanobody A3C8, as a expected for monomeric versions were not observed in presence modular GFP-containing protein, binds the plant toxin ricin of the detergent (Fig. 2a). Therefore, NPs and Zn NPs were [33], highly loaded A3C8 NPs should inactivate more efficiently clearly distinguishable materials regarding the interactions that this toxin, that has a very strong biological activity over mam- regulate oligomerization. malian cells [34,46,47]. To assess this hypothesis, the different In all these samples and under the tested assembling and versions of A3C6-GFP-H6 generated here were incubated with a disassembling conditions, the proteins remained proteolytically biologically active recombinant ricin that had been developed for stable (Fig. 2d, e), with only symptoms of minor and partial cancer therapies [34]. When incubating this ricin-based proteolysis in the NP versions of both fusion proteins. The GFP recombinant protein with equimolar or ten times molar amount fluorescence emission was similar in all the constructs, and of the nanostructured nanobody (A3C8 NPs), its toxicity was slightly lower in the case of both NP versions (Fig. 2f). The Zn- majorly abolished. This event occurred at a lesser extent when mediated oligomerization did not disturb the GFP emission and using Zn NPs and even less when using Ms (Fig. 4a). This still, Zn NPs tended to be more fluorescent than the Ms versions observation was in agreement with the afore-mentioned multi- (Fig. 2f). valence hypothesis, and it demonstrated that at least part of the To comparatively evaluate the ultrastructural morphometry of nanobody moieties are available for interaction in NPs as well as NPs and Zn NPs, both type of materials made of the two in Zn NPs. In addition, these data strongly suggested that higher alternative constructs were examined by TEM and FESEM multivalency in NPs is more efficient in neutralizing the toxin (Fig. 3). These high-resolution imaging techniques showed clear than the smaller Zn NPs and the Ms versions, what would size and shape differences between NPs and Zn NPs. When probably occur because of the clustering of a higher number of exploring different magnifications and field broadness, we toxin ligands in NPs, showing a higher density when compared observed structured materials in both cases, with sizes fully with Zn NPs or Ms. compatible to those determined by DLS. The smaller Zn NPs At that point, it was demonstrated that Zn NPs (with reg- appeared as more regular, dense structures than the larger NPs, ulatable assembling and disassembling) and NPs (spontaneously which were still regular but showed important deformability. formed, non-regulatable) were materials with distinguishable Apparently, protein building blocks in NPs might be organized biophysical properties, despite being formed by the same into toroidal NPs, with empty cores, while the modular proteins building block polypeptide. This was proved with two model Figure 3 Ultrastructural characterization. Representative high resolution electron microscopy images (top, TEM; bottom, FESEM) of A3C8-GFP-H6 and EM1-GFP-H6 Zn NPs and NPs showing round shape and stable size around 10 nm in diameter (Zn NPs) and around 80 nm in diameter (NPs). Scale bars: 200 nm. 1666 © The Author(s) 2022. This article is published with open access at link.springer.com. June 2022 | Vol. 65 No. 6 SCIENCE CHINA Materials ARTICLES Figure 4 Functional and structural comparison. (a) Neutralization assays of T22-mRTA-H6 with A3C8-GFP-H6 in their three different oligomerization states. Y-axis represents HeLa CXCR4+ cell viability in percentage (being 100% the cell viability without protein incubation). T22-mRTA-H6 at 50 nmol L−1 was pre-incubated for 1 h with A3C8-GFP-H6 at three different molar ratios (10:1, 1:1, 1:10) before being administered for 48 h to HeLa cells. Horizontal green line represents HeLa survival when exposed to T22-mRTA-H6 for 48 h at 50 nmol L−1. Significant differences are shown as * (p < 0.05), in black for differences with non-exposed cells (100%), in green for differences with cells exposed to T22-mRTA-H6 alone and in dark red for differences with cells exposed to (T22-mRTA-H6:A3C8-GFP-H6 NPs) at the same molar ratio. (b) Fluorescence emission spectra of A3C8-GFP-H6 (left) and EM1-GFP-H6 (right) at their different oligomerization states, upon excitation at 295 nm. Vertical dashed lines indicate the position of the peaks for each spectrum. (c) CD spectra of A3C8-GFP-H6 (left) and EM1-GFP-H6 (right) at their different oligomerization states. (d) Secondary structure data according to measurements in (c). NRMSD: normalized root mean square error. Proteins were adjusted to 0.2 mg mL−1 prior to measurements in (b) and (c). nanobody-containing proteins, but such a variability had been (Fig. 5). While both types of oligomers remained stable during internally observed in the laboratory when developing other the whole tested temperature range, the monomeric versions of protein building blocks (not shown). Since these materials are both proteins suffered a conformational conversion (more evi- formed by recombinant proteins bioproduced in bacteria, a dent for A3C8-GFP-H6) between 60 and 80°C (Fig. 5a, b). Then, conformational variability in these proteins could account for irrespective of the precise conformational states of the building dissimilar building block populations, even having the same blocks in the NPs, resulting in disparate morphologies and primary structure. In fact, the conformational variability of interactivities (Figs 3 and 4a), the oligomeric organization recombinant proteins is a well-recognized event in multiple proves to be protective in front of thermal stress. models and production systems [48–55], but the impact of such Such stabilizing property, which is linked to the formation of diversity over self-assembling as protein materials has remained supramolecular complexes, prompts the development of trans- so far unexplored. In this context, intrinsic fluorescence analysis versal assembling platforms that, like those studied here, would demonstrated that Tryptophan residues sense a high hydro- allow the organization of functional proteins with therapeutic phobic environment in both types of NPs as the fluorescence potential into nanoscale multimeric materials. In particular, the peaks move to lower wavelengths respect to Ms (Fig. 4b). This use of histidine-rich peptides as architectonic agents benefits result confirmed the oligomerization states of both types of from the simplicity of the engineering methods that is based on oligomers. On the other hand, the CD spectra of the whole set of the addition of an H6 or related peptide to the protein of A3C8-GFP-H6 and EM1-GFP-H6 variants (Ms, Zn NPs and interest. Since H6 also allows simple, one-step purification of NPs) suggested structural similarities between Ms and Zn NPs any protein by IMAC [2,3,60,61], the dual role of the peptide and also common differences between this pair and NPs shows benefits from both the bioproduction and purification (Fig. 4c). A higher content of β-sheet secondary structure was side, and the nanofabrication process itself, through self- observed in Ms and the derived Zn NPs in comparison with NPs assembling. In contrast to other more refined protein-specific (Fig. 4d). These data, concomitantly with the different interac- engineering approaches [17–20,62–64], the protein self-assem- tion types involved in the disassembling (Fig. 2a, b), strongly bling process promoted by H6 tails can be universally applied to supported again the concept that NPs and Zn NPs derive from any protein of clinical interest. The data shown here indicate structurally different building blocks resulting from bacterial that the recombinant production of distinct His-tagged nano- production, which, despite having the same amino acid body-GFP fusions result in at least two distinguishable con- sequence, fold into alternative conformations. In this same formers of the protein that assemble into two categories of NPs. context, the mild proteolysis observed in these proteins (Fig. 2d) One of the conformers, enriched in β-sheet secondary structure, again discriminated the sensitivity between the protein forming is found in a monomeric form upon IMAC protein purification. Zn NPs and NPs, especially in the case of the EM1 fusion. Its assembly is finely controlled by externally added Zn2+ ions Importantly, protein conformation is a well-known determinant and its disassembly by the addition of a divalent cation chelator of proteolytic susceptibility [56–59]. such as EDTA (Fig. 2). On the other hand, the alternative The DLS and CSM analyses of the proteins revealed that both conformer with low β-sheet secondary structure spontaneously NPs and Zn NPs were structurally more robust than plain Ms assembles during elution from IMAC columns into larger NPs, June 2022 | Vol. 65 No. 6 © The Author(s) 2022. This article is published with open access at link.springer.com. 1667 ARTICLES SCIENCE CHINA Materials Figure 5 Conformational stability. (a) Volume size distributions (nm) of A3C8-GFP-H6 and EM1-GFP-H6 in their three different oligomerization states upon heating from 25 to 90°C. (b) CSM study of A3C8-GFP-H6 and EM1-GFP-H6 in their three different oligomerization states upon heating from 25 to 90°C. (c) Scheme of possible oligomerization states for His-tagged nanobody-GFP containing proteins. (d) Schematic illustration of the folding energy landscape reflecting the oligomerization states presented in (c). Proteins were adjusted to 0.2 mg mL−1 prior to measurements in (a) and (b). with regular toroid-like morphology with important extent of resulting materials linked to the conformational spectrum of the mechanical flexibility. Such NP version, over the Zn NP, shows recombinant proteins, specific of the type of soft material that an enhanced capability to neutralize a target ricin (Fig. 4a). This they generate. This concept, irrelevant in the use of more rigid observation demonstrates the full functionality of the nano- building blocks for material design, might be highly relevant in bodies and also the reaching of local ligand concentrations in the emerging field of protein-based materials for clinical appli- NPs higher than in Zn NPs, which favors the neutralization of cations, which is seen as a broad and promising technology for a the toxin. Importantly, the ligand is active in both nanomaterials diversity of therapeutic applications [6,17–21,62]. Being this fact as well as in the monomeric form. an issue with regulatory implications, the awareness of such The origin of both populations (monomers and spontaneously potentially disparate assembling makes possible a proper quality formed NPs) is determined by the genetic cell background and control of a clinically oriented product by a simple separation of culture conditions of the recombinant production in E. coli cells relevant peaks during the chromatographic step of the produc- and in a differential folding pattern followed by each protein tion process. fraction, rendering different conformers. The structural analysis of the NPs and the unassembled monomer (Fig. 4) strongly Received 8 October 2021; accepted 30 November 2021; suggests that the larger NPs are formed by a conformer that published online 17 January 2022 might come from an unstable monomeric intermediate in the 1 López-Laguna H, Sánchez J, Unzueta U, et al. 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Protein Sci, 1994, 3: 757–768 of the experiments; Vázquez E and Villaverde A conceived and supervised 60 Li S, Yang K, Liu L, et al. Surface sieving coordinated IMAC material the whole study; Villaverde A prepared the first draft of the manuscript. All for purification of His-tagged proteins. Anal Chim Acta, 2018, 997: 9– authors contributed to the general discussion and approved the manuscript. 15 61 Spriestersbach A, Kubicek J, Schafer F, et al. Purification of His-tagged Conflict of interest The authors declare that they have no conflict of proteins. In: Lorsch JR (ed). Methods in Enzymology. San diego: interest. Academic Press, 2015, 1–15 62 Hamley IW. Protein assemblies: Nature-inspired and designed nano- structures. Biomacromolecules, 2019, 20: 1829–1848 Supplementary information The original data presented in this paper can 63 Wang Y, Katyal P, Montclare JK. Protein-engineered functional ma- be found at: https://ddd.uab.cat/record/248706. terials. Adv Healthcare Mater, 2019, 8: 1801374 64 Zou Q, Yan X. Amino acid coordinated self-assembly. Chem Eur J, Open Access This article is licensed under a Creative Commons Attribu- 2018, 24: 755–761 tion 4.0 International License, which permits use, sharing, adaptation, dis- 65 López-Laguna H, Cubarsi R, Unzueta U, et al. Endosomal escape of tribution and reproduction in any medium or format, as long as you give protein nanoparticles engineered through humanized histidine-rich appropriate credit to the original author(s) and the source, provide a link to peptides. Sci China Mater, 2020, 63: 644–653 the Creative Commons licence, and indicate if changes were made. 66 López-Laguna H, Sánchez-García L, Serna N, et al. Engineering protein The images or other third party material in this article are included in the nanoparticles out from components of the human microbiome. Small, article’s Creative Commons licence, unless indicated otherwise in a credit 2020, 16: 2001885 line to the material. If material is not included in the article’s Creative 67 Falgàs A, Pallarès V, Serna N, et al. Selective delivery of T22-PE24-H6 Commons licence and your intended use is not permitted by statutory to CXCR4+ diffuse large B-cell lymphoma cells leads to wide ther- regulation or exceeds the permitted use, you will need to obtain permission apeutic index in a disseminated mouse model. Theranostics, 2020, 10: directly from the copyright holder. 5169–5180 To view a copy of this licence, visit http://creativecommons.org/licenses/ Acknowledgements We are indebted to the Agencia Estatal de In- by/4.0/. vestigación (AEI) and to the Fondo Europeo de Desarrollo Regional (FED- ER) (PID2020-116174RB-I00, AEI/FEDER, UE). The AGAUR (2017SGR- Eric Voltà-Durán graduated in biotechnology in 2018 and 229) and the CIBER-BBN (project NANOPROTHER) are granted to Villa- pursued Master studies in advanced nanoscience and na- notechnology (2019). He is a young, early-stage researcher verde A. Vázquez E received support from the AEI (PID2019-105416RB-I00/ at the Nanobiotechnology group, a team led by Prof. AEI/10.13039/501100011033 and CIBER-BBN (project NANOREMOTE). Villaverde at the Autonomous University of Barcelona We also appreciate the support from the Javna Agencija za Raziskovalno (UAB). He is working on his PhD studies, in which he dejavnost Republike Slovenije (ARRS/N4-0046 and ARRS/J4-9322) to de focuses on the design and production of proteins with Marco A. Sánchez-García L was supported by a predoctoral fellowship from biomedical interest, specially in the context of targeted the AGAUR (2018FI_B2_00051), Voltà-Durán E by a predoctoral fellowship therapies. from Ministerio de Ciencia, Innovacion y Universidades (FPU18/04615), Unzueta U was supported by Miguel Servet contract (CP19/00028) from ISCIII co-funded by European Social Fund (ESF investing in your future) and by an ISCIII project (PI20/00400) co-funding FEDER (A way to make Europe). López-Laguna H was supported by a predoctoral fellowship from the AGAUR (2019FI_B00352). Villaverde A received an ICREA ACADEMIA Antonio Villaverde graduated in biological sciences in award. Protein production was partially performed by the ICTS ‘‘NAN- 1982 and got his PhD degree in 1985. He has been sci- BIOSIS”, more specifically by the Protein Production Platform of CIBER in entifically formed in Barcelona, Madrid, London, Lau- Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/ IBB, at the sanne and Braunschweig. Since 1987, he has been UAB (http://www.nanbiosis.es/portfolio/u1-protein-production-platform- professor of microbiology at the Universitat Autònoma de Barcelona, Spain, where he got a full professorship in 2002. ppp/). Cell culture experiments were performed at SCAC facilities in the He leads the Nanobiotechnology group in this university UAB. Molecular graphics and analyses were performed with UCSF Chimera, and in the CIBER-BBN, focusing on the design of protein- developed by the Resource for Biocomputing, Visualization, and Informatics based materials for biomedical applications. He founded at the University of California, San Francisco, with support from NIH P41- the journal Microbial Cell Factories in 2002, being its GM103311. Electron microscopy studies were performed by the Servei de Editor-in-Chief for 15 years. Microscòpia in the UAB. Funding note Open Access Funding provided by Universitat Autonoma de Barcelona. Author contributions Voltà-Durán E performed most of the experiments 1670 © The Author(s) 2022. This article is published with open access at link.springer.com. June 2022 | Vol. 65 No. 6