A novel, modular and straightforward strategy to build active protein-based nanomaterials is designed by fusing soft amyloid cores (SAC) to globular proteins.

In many disease-related and functional amyloids, the amyloid-forming regions of proteins are flanked by globular domains. When located in close vicinity of the amyloid regions along the chain, the globular domains can prevent the formation of amyloids because of the steric repulsion. Experimental tests of this effect are few in number and non-systematic, and their interpretation is hampered by polymorphism of amyloid structures. In this situation, modeling approaches that use such a clear-cut criterion as the steric tension can give us highly trustworthy results. In this work, we evaluated this steric effect by using molecular modeling and dynamics. As an example, we tested hybrid proteins containing an amyloid-forming fragment of Aβ peptide (17-42) linked to one or two globular domains of GFP. Searching for the shortest possible linker, we constructed models with pseudo-helical arrangements of the densely packed GFPs around the Aβ amyloid core. The molecular modeling showed that linkers of 7 and more residues allow fibrillogenesis of the Aβ-peptide flanked by GFP on one side and 18 and more residues when Aβ-peptide is flanked by GFPs on both sides. Furthermore, we were able to establish a more general relationship between the size of the globular domains and the length of the linkers by using analytical expressions and rigid body simulations. Our results will find use in planning and interpretation of experiments, improvement of the prediction of amyloidogenic regions in proteins, and design of new functional amyloids carrying globular domains.

Natural selection has endorsed proteins with amazing structures and functionalities that cannot be matched by synthetic means, explaining the exponential interest in developing protein-based materials. Protein self-assembly allows fabricating complex supramolecular structures from relatively simple building blocks, a bottom-up strategy naturally employed by amyloid fibrils. However, the design of amyloid-inspired materials with biological activity is inherently difficult. Here, we exploit a modular procedure to generate functional amyloid nanostructures with tight control of their mesoscopic properties. The soft amyloid core of a yeast prion was fused to dihydrofolate reductase through flexible linkers of different sizes. This enabled us to produce, for the first time, biocompatible protein-only amyloid-like oligomeric nanoparticles with defined dimensions in which the embedded enzyme remained highly active, as assessed by biophysical and enzymatic assays. The modular design allowed one to obtain multifunctional nanoparticles by incorporating the antibody-binding Zdomain to the protein fusion. We show how these assemblies can be exploited for antibody-directed targeting of specific cell types and the localized delivery of methotrexate, resulting in the intracellular uptake of the drug by cancer cells and their death. Overall, the novel protein particles we describe in this work might find applications in areas as diverse as biocatalysis, bioimaging, or targeted therapies.disordered and low-complexity domains of yeast prions. 8 They 49 differ from the classical amyloid cores of pathogenic proteins in 50 that they are slightly longer and more polar. 9,10 This results in 51 a weaker and more diffuse amyloid propensity but still 52 sufficient to nucleate the self-assembly reaction. The SAC is 53 fused through a Gly/Ser soluble and flexible linker to the 54 globular protein of interest. The resulting fusion proteins are 55 produced recombinantly at high yield and in a soluble manner; 56 still, their ordered aggregation can be induced under defined 57 conditions, rendering functional synthetic amyloid fibrils in 58 which the SAC forms the amyloid spine and the globular 59 domains hang from it in an active conformation, as 60 demonstrated for fluorescent proteins and enzymes. 7 61 We use the SAC (residues 100−118) of the Sup35 yeast 62 prion (Sup35-SAC) as the default assembling module. Its small 63 size allows one to use a connecting linker of only eight residues 64 and obtain amyloid fibrils in which the fused protein is active 65 without major steric impediments and with a reduced entropy 66 penalization for the assembly reaction. This is in contrast with 67 natural amyloid proteins displaying a similar architecture, like