PRION 2020, VOL. 14, NO. 1, 170–179 https://doi.org/10.1080/19336896.2020.1785659 REVIEW Prion domains as a driving force for the assembly of functional nanomaterials Weiqiang Wang and Salvador Ventura Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain ABSTRACT ARTICLE HISTORY Amyloids display a highly ordered fibrillar structure. Many of these assemblies appear associated Received 14 May 2020 with human disease. However, the controllable, stable, tunable, and robust nature of amyloid Revised 11 June 2020 fibrils can be exploited to build up remarkable nanomaterials with a wide range of applications in Accepted 12 June 2020 biomedicine and biotechnology. Functional prions constitute a particular class of amyloids. These KEYWORDS transmissible proteins exhibit a modular architecture, with a disordered prion domain responsible Prions; amyloids; self- for the assembly and one or more globular domains that account for the activity. Importantly, the assembly; prion-like original globular protein can be replaced with any protein of interest, without compromising the domains; nanomaterials; fibrillation potential. These genetic fusions form fibrils in which the globular domain remains yeast folded, rendering functional nanostructures. However, in some cases, steric hindrance restricts the activity of these fibrils. This limitation can be solved by dissecting prion domains into shorter sequences that keep their self-assembling properties while allowing better access to the active protein in the fibrillar state. In this review, we will discuss the properties of prion-like functional nanomaterials and the amazing applications of these biocompatible fibrillar arrangements. Introduction conditions, such as treatment with proteinases, chao­ tropic agents, and high temperatures[13]. These prop­ Amyloid proteins can self-assemble into ordered fibril­ erties make amyloid fibrils ideal building blocks for lar structures. These amyloid fibrils, constitute designing novel protein/peptide self-assembled nanos­ a hallmark of human amyloidosis as well as of neuro­ tructures. Indeed, amyloid fibrils have been incorpo­ degenerative diseases, including Alzheimer’s, rated as structural components in functional materials Parkinson’s, Type Ⅱ diabetes, and others [1–3]. The with different biomedical and biotechnological applica­ discovery of functional amyloids in different organisms, tions, including nanodevices [14], biomembranes [15], from bacteria to humans, assisting different physiologi­ hydrogels [16], biosensors [17], and energy conversion cal functions in living cells [4], has changed the amy­ materials [18]. However, the inherent conformational loid/pathogenesis paradigm. For instance, functional transition towards a β-sheet rich structure during fibril­ amyloids are involved in curli-mediated biofilm forma­ lation necessarily implies the loss of the globular native tion in E. coli [5], memory persistence in Drosophila fold and the subsequent inactivation of the protein. [6], hypersensitive response activation in plants [7], Therefore, designing fibrillar structures containing cor­ melanin polymerization in mammalian cells [8], and rectly folded and active proteins is still a challenging hormone storage in humans [9]. Amyloid fibrils display task. A way to bypass this limitation is by designing a remarkably similar quaternary structure, formed by a hybrid structure in which the globular proteins are the assembly of monomers into a characteristic ‘cross- chemically attached to preformed amyloid fibrils [19]. β’ structure [10], resulting from the stacking of inter­ However, this approach is restrained by a reduced pro­ molecular β-sheets arrayed perpendicular to fibrils axis, tein conjugating chemistry, undesired cross-reactivity which are stabilized by numerous hydrogen bonds between the polypeptides, and the unavoidable inacti­ between neighbouring strands [11], together with π-π vation of a fraction of the protein during conjugation. and hydrophobic interactions [12]. Such a unique spa­ Prions are proteins able to switch between two or tial structure endows amyloid fibrils with high stability more conformations, of which at least one corresponds and high resistance to extreme physicochemical to an amyloid fold [20–22]. So far, only a prion protein CONTACT Salvador Ventura salvador.ventura@uab.es Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular; Universitat Autònoma de Barcelona, Bellaterra (Barcelona) 08193, Spain This article was originally published with errors, which have now been corrected in the online version. Please see Correction ((http://dx.doi.org/10.1080/ 19336896.2020.1804218). © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. PRION 171 (PrP) has identified in mammals; PrP is well conserved Initially, the phenotypes [PSI+] and [URE3] of yeast across species and linked to different neurological were identified as non-chromosomal genetic elements. pathologies [23]. Roughly ten prion proteins have Later on, Aigle and Lacroute reported that ure2 nuclear been characterized in the yeast Saccharomyces cerevi­ gene mutant strains were unable to propagate the siae. They are usually referred to as functional prions [URE3] element but manifested the same phenotype since, in contrast to ‘toxic’ mammalian prions, they do as [URE3] cells, namely de-repression of nitrogen cat­ not seem to have a significant impact on viability, but abolism genes[37]; however, they did not realize that instead, they play physiological roles, especially in the [URE3] could be a prion of Ure2p. It was Reed adaptation to changing extracellular environments B. Wickner, who, based on these pieces of evidence [24,25]. They usually consist of an intrinsically unstruc­ and his own experiments, recognized that the relation­ tured region of low complexity enriched in asparagine ship between ure2 and [URE3] was not that expected (N) and glutamine (Q) residues, known as the prion for a chromosomal gene necessary for the propagation domain (PrD), accompanied by one or more globular of a nucleic acid replicon, but instead that [URE3] was domains [26,27]. The PrD is both necessary and suffi­ an inactive form of Ure2p able to inactivate the normal cient for self-assembly, whereas the globular domain protein form, acting thus as a prion [38,39]. Then his accounts for the protein functionality [28]. Yeast prions group inferred that this could also be true for the [PSI are thus modular and, in principle, the original globular +] phenotype [38], and concluded that previous studies domain can be replaced with any desired functional [40–42] supported [PSI+] being a prion of Sup35p. protein without impacting the fibrillation propensity Moreover, they established a series of genetic criteria significantly. Domains with properties resembling that identify yeast prions [43]: i) a reversible curability, those of PrDs have been identified in the proteins of ii) the phenotype appearance is induced by overexpres­ different organisms, including humans, and are named sion of the prion protein, and iii) the phenotype resem­ prion-like domains (PrLDs). bles that of a prion protein gene recessive mutation. In contrast to the fast and challenging to control aggre­ These genetic criteria were useful for the discovery gation reactions of pathogenic amyloids, prion and prion- of a range of new prions including [PIN+] [44], [SWI+] like proteins generally display slow and tunable aggregation [45], [MOT+] [46] and [MOD+] [24]. The formation of kinetics, which are considered optimal for the design of amyloid in vitro by Sup35p [47], and Ure2p [48], the self-assembling materials [29]. Actually, different enzymes protease resistance of Ure2p in [URE3] strains [49], such as alkaline phosphatase (AP) [30], carbonic anhydrase together with the seeding properties of cellular extracts (CA) [31], methyl-parathion hydrolase (MPH) [32] or in [PSI+] [50], all converged to indicate that the prion other globular proteins like protein G and the Z domain form of these proteins was an amyloid. This assump­ [33] have been genetically fused to the PrDs of yeast prion tion was further confirmed after observing that in vitro Sup35 or Ure2p to generate functionalized nanomaterials formed Sup35p amyloids transmit the [PSI+] trait [51]. in which the PrDs constitute the spine of the fibrils, The inheritance of phenotypic traits due to prion whereas the globular domains hang from them in formation involves a structural conversion, which in a folded and functional conformation. These materials a majority of cases is driven by a Q/N rich PrD [52]. are biocompatible, degradable, and environmentally PrDs proved to be sufficient to induce and propagate friendly and can be employed in applications encompass­ the prion state. They become integrated into the core of ing from catalytic nanowires [30] to nanosensors [32]. the amyloid structure, which is thought to be arranged Here, we summarize the features of functional prions in as an in-register parallel β-sheet architecture [10], yeast and review the use of PrDs and PrLDs to generate according to nuclear magnetic resonance (NMR) and genetically encoded functionalized nanomaterials and dis­ electron microscopy studies of the amyloids of Sup35p cuss their potential applications. and Ure2p PrDs (Figure 1) [53,54]. The globular domains appended to the PrDs might retain their native structure in the amyloid filaments (Figure 1), Functional prions in yeast although the activity of these domains was found to The concept of infectious proteins was initially pro­ be somehow reduced, likely because of steric impedi­ posed to lie behind different neurological diseases, ments [31]. Such folded, in-register parallel β-sheet including Creutzfeldt-Jacob disease in humans, sheep architecture perfectly explains the mechanism of pro­ scrapie and bovine spongiform encephalopathy, all tein templating in prion conformational conversion caused by the amyloid state of the prion protein (PrP) (Figure 1) [55]. In particular, the favourable interac­ [34–36]. Thus, prions were assumed to be pathogenic tions between hydrophobic or hydrophilic side chains agents without any beneficial functional implication. along the long axis of the filament maintains the in- 172 W. WANG AND S. VENTURA Figure 1. Typical prions [URE3] and [PSI+] of S. cerevisiae. These prions rely on self-propagating amyloids of Ure2p and Sup35p, respectively. The insert is the proposed mechanism of conformational templating by the prion domain. Energetically favourable interactions between identical side chains enforce the in-register architecture of these amyloids. A new monomer being added to the end of the filament must assume the same conformation as that of molecules previously incorporated into the filament. (adapted with permission).[43] register β-sheet structure. The same interactions force the prion phenotype during cell division is generally the new monomer to join the end of the filament to accomplished by the action of the chaperone system, adopt the same conformation as the molecules already which fragments existing fibrils, generating seeds that in the fibril, just as nucleic acids template their initiate the polymerization reaction in daughter cells sequences [39]. An exception to this rule is the HET-s [60,61]. prion, from the filamentous fungi Podospora anserine, The first and most studied of yeast prion proteins involved in heterokaryon incompatibility, protecting are Sup35p and Ure2p (Figure 1), involved in transla­ cells from fungal viruses [56]. The amyloid formed by tion termination and nitrogen catabolism regulation, the PrD of HET-s displays a β-helix structure, in which respectively [38]. Sup35p, a subunit of the translation each monomer contributes two turns to the β-helix termination factor, has a clear three-domain structure. [57]. Recently, cryo-EM and computational studies sug­ The N-terminal residues 1–123 (Sup35 N) are sufficient gest that PrPSc would adopt a similar β-solenoid struc­ to induce and propagate the [PSI+] prion phenotype ture [58,59]. In fungal prions, the self-propagation of and thus constitute the PrD. The C-terminal residues 254–685 (Sup35 C), fold into a globular domain, PRION 173 responsible for the translation termination function [62,63]. The charged middle domain (Sup35 M), corre­ sponding to residues 124 to 253, is important but not required for [PSI+] appearance and maintenance [64,65]. Sup35 N drives self-assembly of Sup35p into an amyloid, which results in a substantial reduction of its translation termination function and leads to an increase of nonsense codon readthrough. The PrD of Sup35p has itself a non-prion function, facilitating mRNA turnover. Ure2p has an architecture similar to that of Sup35p, including an N-terminal PrD (residues 1–65) responsible for [URE3] formation [66] and a C-terminal domain (Ure2 C) structurally similar to glutathione transferase, but involved in repression of Figure 2. The general genetic fusion approach for functiona­ lized nanomaterials. (a) Domain organization of the prion pro­ nitrogen catabolism genes by binding to the transcrip­ tein: soft amyloid core (SAC) (red) within the prion domain tion factor Gln3p, an interaction that is significantly (PrD) (green) and the respective functional domains (purple) impeded in the amyloid state [67]. in a prion protein are shown. (b) Cartoon of the prion domain The modular structure of yeast prions allows to fused to molecule A, which represents a globular functional dissect them in order to assess the prionogenic poten­ protein. (c) Soluble fusion protein self-assembly into nanofibrils, tial of other proteins and, in this way, identify new which preserve the function of molecule A. prions. This is performed by replacing the PrD or globular domain, via genetic engineering, with any desired polypeptide or reporter protein, such as the a protein of interest and its recombinant production in green fluorescent protein (GFP), to study the ability microbial cell factories (Figure 2). The purified soluble of the fusion protein to induce [PSI+]-like appearance fusion protein can self-assemble spontaneously into or to visualize protein inclusions formation in cells nanofibrils, which exhibit the function of the appended [68]. These studies demonstrated that yeast prions globular domain, under appropriate conditions of tem­ could be easily engineered, immediately suggesting perature, pH, and agitation, in some instances, requir­ that they can be exploited in the design of self- ing the presence of detergents. assembling functionalized materials. Biosensors Prion-like nanomaterials obtained via genetic The first prion-like nanomaterial obtained via genetic protein fusion protein fusion was intended to work as a biosensor. It has been reported that the yeast prion Ure2p exhibits Dong Men et al. [32] generated bifunctional protein glutathione peroxidase activity in both the native and nanowires (bFPNw) that displayed two different glob­ fibrillar forms [69]. Besides, exogenous globular ular proteins: protein G and methyl-parathion hydro­ domains fused to the Ure2p PrD retain a native-like lase (MPH), which provide antibody binding and fold and show catalytic activity within in vitro formed catalytic activities, respectively. They aimed to obtain amyloid filaments [31]. This ability to combine amyloid fibrils with a high enzyme to protein G ratio. Towards and globular folds in the aggregated state seems to be this objective, both protein G and MPH were fused generic of yeast prions since the analysis of Sup35p individually to the N-terminus of the Sup35p PrD amyloid filaments by cryo-EM, STEM [70], and solid (residues 1–61) and expressed as soluble monomers in state NMR [71], revealed that filaments of full length E.coli. The fibrils formed by the self-assembly of the Sup35p indicated a thin fibril backbone surrounded by Sup351−61-protein G fusion were broken into small globular C-domains. The formation of a spatially fragments to be used as seeds. The bFPNw exhibiting defined fibrillar backbone and the segregation of folded two different functions with different ratios were globular domains are central for the design of prion- obtained by mixing these seeds and soluble Sup351−61- like self-assembling functionalized nanomaterials, with MPH in different proportions; this also allowed to tune functions that include enzymatic catalysis [30], biosen­ the length of the fibrils. The biological activity of these sing [32], and protein binding [33]. The general strat­ bFPNw was tested by a typical ELISA assay, imple­ egy to obtain the building blocks for such materials menting them instead of the classical enzyme- consists of the genetic fusion of a yeast prion PrD to conjugated secondary antibody. As a general trend, 174 W. WANG AND S. VENTURA the longer the nanowires were, the higher the signal a concentration as low as 1pmol mL−1, which was amplification they provided. In that work, optimized about 10,000-fold that of the soluble fusion protein nanowires of 500 nm in length (ratio of protein G to biosensor E2GFP-MPH and 107 -fold higher than the MPH was 1:8) showed a sensitivity for the detection of one of an equimolar mixture of free E2GFP and MPH the F1 protein from Yersinia pestis 1000-fold higher moieties. These nanostructures might become tools than that of a protein G-MPH fusion and 100-fold for the detection of pesticides in the food industry. higher than that of the traditional HRP-conjugated antibody-based ELISA. Enzyme immobilization In subsequent work, the same group designed auto- biotinylated bFPNw by introducing a biotin avidin Immobilization can enhance the value and usage of binding system, which can efficiently attach any avidin- commercial enzymes, providing advantages such as an labelled commercial enzyme, such as HRP or AP, to the easy separation of the product and the reuse of the nanowires backbone. This skips the need to produce enzyme. The lab of Sarah Perrett has recently and purify a different fusion for each intended applica­ described PrD-based enzyme immobilization systems tion[72]. In particular, the biotin acceptor peptide tag [30]. On this occasion, it was the PrD of Ure2p that (BAP) and the IgG-binding domain, C1, of protein was exploited to drive the self-assembly, and two G from Streptococcus (SPG) were individually fused to different enzymes, AP and HRP, were individually the Sup35p PrD segment described above. The con­ fused to it. Both fusion proteins formed active nano­ struct Sup351−61-BAP was co-expressed in E.coli with fibrils, where the enzymes become immobilized. another construct encoding for the biotin holoenzyme Although the fibrils exhibited slightly lower activity synthetase (BirA). In this way, the BAP tag was bioti­ than that of the soluble wild type proteins, they could nylated by BirA during the expression of the Sup351−61- be recycled both in a conventional enzymatic setup BAP fusion. Instead of inducing the formation of and in the context of a continuous microreactor. In bifunctional nanowires by seeding, which they found a follow up study, the authors encapsulated the solu­ reduced the IgG binding activity of protein G because ble PrD-AP protein fusion into a uniform droplet of the sonication processes, on this occasion, they just using microfluidic techniques [74]. The encapsulated mixed the pre-formed biotinylated Sup351−61-BAP soluble protein spontaneously self-assembled into fibrils with the Sup351−61-SPG monomer, which was a three-dimensional fibrillar network within the dro­ templated and incorporated at the end of the fibrils. plet, immobilizing AP molecules in their active form. The function of the bFPNw was evaluated by perform­ As a result, a catalytically active microgel with ing a typical ELISA in the presence of Streptavidin- a porous architecture was obtained. These hydrated HRP instead of the typical secondary IgG-HRP conju­ functional nanoparticles, when combined with micro­ gate. For the detection of Y. pestis F1 antigen, the auto- fluidics, are ideal entities to implement microscale biotinylated bFPNw showed higher signal intensity, analytical assays. lower background noise, and better signal stability than that of previous seeding promoted bFPNw. More Bioelectrodes importantly, they showed 2000- to 4000-fold higher sensitivity than that of a conventional ELISA. Vincent Forge and colleagues designed a protein-only The lessons learned in these two previous works redox biofilm inspired by the architecture of bacterial were exploited to construct a highly sensitive fluores­ electroactive biofilms [75]. Briefly, the formation of the cent molecular biosensor. First, an F64 L/S65 T/ nanowires was induced by the self-assembly of the PrD T203Y/L231 H green fluorescent protein mutant of HET-s P. anserine prion, which was genetically fused (E2GFP) was fused to MPH [73], E2GFP shows dis­ to rubredoxin (Rd). This redox protein acts as an elec­ tinct excitation and emission spectra depending on tron carrier. The formation of the fibrillar structures the pH, which makes it an effective ratiometric pH allowed the alignment of Rd along the fibril axis in an indicator. The chimeric E2 GFP-MPH protein allowed array, which facilitates the transportation of electrons the detection of the pesticide methyl parathion (MP) through the network resulting in an exceptional con­ by sensing the H+ ions released during enzymatic ductivity. As a proof of concept, such redox active reaction. Interestingly enough, when this chimeric fibrils were successfully applied to mediate the electron protein was further fused to the Sup35p PrD and transfer towards the multi-cooper enzyme laccase for incorporated into fibrils through its self-assembly, catalytic oxygen reduction. Therefore, these protein the obtained nanowires showed dramatic enhance­ fibrils are expected to find applications as nanometric ment of sensitivity and allowed detection of MP at bioelectrodes. PRION 175 Antibody purification Importantly, the separation of the fibrillar material from the cell culture required only centrifugation and their The increasing demand of antibodies for diagnostic and resuspension in a saline buffer. The high yield, high homo­ therapeutic purposes requests cost efficient means for their geneity, and high stability of the purified material, together purification. In recent work, Torleif Härd and co-workers with minimal equipment requirement and the low hands exploited the small size and high affinity to IgG of the on time of this strategy, allows foreseeing, for the first time, Z-domain from staphylococcal protein A to build up anti­ the possibility of producing functional prion-like fibrils at body capturing fibrils. A Z-domain dimer (ZZ) was fused an industrial scale. to the PrD of either Sup35p (Sup35-ZZ) or Ure2p (ZZ- Ure2). Both of them promoted the fibrillation of the chi­ meric protein [33]. These fibrils bind to IgG, but their Biocatalytic cascades loading capacity was limited by steric hindrance. The PrDs of Sup35p and Ure2p were then co-fibrillated with As discussed above, the chemistry-free covalent enzyme chimeric Sup35-ZZ and ZZ-Ure2 proteins, respectively, to immobilization and the high surface to volume ratio of maximize the accessibility of antibodies to ZZ. This fibril prion-based fibrils make of these nanostructures opti­ doping strategy resulted in co-fibrils in which ZZ globular mal recyclable catalysers. However, many industrially domains are more spaced in the fibrillar structure, thus relevant processes require two or more coupled reac­ maximizing the fibrils binding capacity (Figure 3). tions, and, in most cases, only single-enzyme functio­ Different doping frequencies were assayed, and in the nalized fibrils have been characterized. Mats Sandgren optimal PrD:protein fusion ratio, the resulting fibrils and co-workers addressed this issue by building up yielded a capture capacity of 1.8 mg of antibody per mg a catalytic cascade by genetically fusing xylanase A, β- fibrils, which is 20-fold higher than the one of commercial xylosidase, and aldose sugar dehydrogenase to the protein A agarose. Sup35 PrD to create three different Sup35–enzyme Despite the exceptional binding capacity of ZZ functio­ chimeras [77]. As in the case of the Z-domain, the nalized fibrils, this material should be produced and engi­ fibrils were doped with the PrD alone to introduce neered at large scale before it can commercially compete enough space between the functional enzyme molecules with the present well positioned methods for purification to eliminate steric restrictions. The objective was to of antibodies. Towards this objective, the Härd lab inte­ convert beechwood xylan to xylonolactone, a valuable grated both the Sup35-PrD and the chimeric Sup35-ZZ chemical for versatile applications, in three discrete genes in the genome of Komagataella pastoris [76]. This enzymatic steps. Each type of functional fibril was yeast continuously expressed and secreted both proteins formed individually, and then they were mixed in dif­ into the extracellular medium, where they co-fibrillated ferent proportions to maximize the catalytic efficiency. into ready-to-use functionalized nanofibrils, with an These investigations revealed that a sequential cascade impressive production yield of 35 mg/L culture. in which fibrils of xylanase A and β-xylosidase were Figure 3. Schematic illustration of co-fibrillation to immobilize globular domains into amyloid fibrils with defined doping frequencies. The chimeric protein consists of a prion-like domain (red) and a Z domain (green, antibody binding domain from Protein A, PDB 1Q2 N), linked via a flexible linker (blue). The chimeric protein is fibrillated with the carrier protein (the prion-like domain alone), in order to allow enough space between globular domains in the fibrils. This reduces steric constraints and maximizes the functionality. 176 W. WANG AND S. VENTURA first mixed, the resulting product of the coupled reac­ SACs differ from the classical short amyloid cores of tion removed and then incubated with fibrils of aldose pathogenic proteins, which hold a high aggregation sugar dehydrogenase was more productive than propensity and are typically enriched in hydrophobic a mixture containing all the three kinds of fibrils residues. SACs are slightly longer and more polar, together since the immobilized enzymes displayed resulting in a less concentrated aggregation potential. hardly compatible stabilities. Despite its limitations, This allows the PrD to remain soluble under most this work constitutes of nice proof of concept for prion- physiological conditions, but also to hold a cryptic inspired biocatalytic cascades. aggregation propensity that might facilitate its efficient self-assembly in response to cellular changes [78]. The SAC from Sup35p PrD consists of 21-residues and self- Soft amyloid cores in PrDs and their use in assembles spontaneously into highly ordered amyloid nanomaterials fibrils. These fibrils can seed the amyloid formation of The absence of highly amyloidogenic sequences allows the complete PrD in vitro. Also, when the in vitro PrD to transit between soluble and aggregated states formed SAC fibrils are added to yeast cells, they pro­ [78]. The aggregation of prions seems to depend on the mote the aggregation of the endogenous full-length establishment of a large number of weak interactions Sup35p and the subsequent emergence of a prionic distributed along the complete PrD [79]. Accordingly, phenotype [83]. Moreover, this region is necessary for natural PrDs often consist of > 100 residues and 60 the induction, propagation, and inheritance of the residues was traditionally considered to be the mini­ prion state of Sup35p in mammalian cytosol [84]. All mum length required to attain an efficient PrD self- these data converge to suggest that this short sequence assembly at moderate protein concentrations [46]. stretch might have prion-like properties, which lead us With this size, the PrD might constitute a significant to hypothesize that SACs can substitute the role of fraction of the fusion protein, especially when it is fused complete PrDs and recapitulate the self-assembly prop­ to small globular domains, like the Z-domain. This erties of the larger domain in the context of modular results in steric restrictions in the fibrillar state since fusion proteins. the amyloid spine may have a larger transversal dimen­ In recent work, we genetically fused the Sup35p SAC sion that the adjacent protein, a property that has been (Sup35SAC, Figure 4) to three globular proteins displaying reported to reduce the diffusion of substrates and limit different structures, namely the all-α FF domain (FF), the the functionality of the fibril[70]. Besides, large PrDs all-β green fluorescent protein (GFP) and the α/β carbonic often compromise the solubility and stability of the anhydrase enzyme (CA) [85]. As expected, the low aggre­ appended globular domains in the monomeric fusions gation propensity of the SAC allowed to express and purify state, impacting the recombinant production of the the three fusions at high yields in a soluble and monomeric chimeric proteins and their storage after purification. form, while still allowing to induce their self-assembly into We have recently proposed that inner weak amyloi­ amyloid fibrils under controlled conditions. As for com­ dogenic sequence stretches within PrD might contri­ plete PrDs, the globular proteins adjacent to the SAC bute to nucleate the conformational conversion of retained their native structures and functions in the fibrillar prionic proteins into a cross-β structure [80]. These state. 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