Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins

In Vivo Tumor Targeting and Imaging with Engineered Trivalent Antibody Fragments Containing Collagen-Derived Sequences

There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed “trimerbody”, comprises a single-chain antibody (scFv) fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA), a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting

Experimental determination of the complete spin structure for p¯p→ Λ¯Λ at p{p¯}= 1.637 GeV/c

The reaction p.p → ^^ → .pπ+pπ− has been measured with high statistics at a beam momentum of pp. = 1.637 GeV/c. The use of a transversely polarized frozen-spin target combined with the self-analyzing property of ^/^ decay allows access to unprecedented information on the spin structure of the interaction. The most general spin-scattering matrix can be written in terms of 11 real parameters for each bin of scattering angle; each of these parameters is determined with reasonable precision. From these results, all conceivable spin correlations are determined with inherent self-consistency. Good agreement is found with the few previously existing measurements of spin observables in p.p → ^^ near this energy. Existing theoretical models do not give good predictions for those spin observables that had not been previously measured

A quantitative comparison of cytosolic delivery via different protein uptake systems

Contains fulltext : 180524.pdf (publisher's version ) (Open Access)Over many years, a variety of delivery systems have been investigated that have the capacity to shuttle macromolecular cargoes, especially proteins, into the cytosol. Due to the lack of an objective way to quantify cytosolic delivery, relative delivery efficiencies of the various transport systems have remained unclear. Here, we demonstrate the use of the biotin ligase assay for a quantitative comparison of protein transport to the cytosol via cell-penetrating peptides, supercharged proteins and bacterial toxins in four different cell lines. The data illustrate large differences in both the total cellular internalization, which denotes any intracellular location including endosomes, and in the cytosolic uptake of the transport systems, with little correlation between the two. Also, we found significant differences between the cell lines. In general, protein transport systems based on cell-penetrating peptides show a modest total uptake, and mostly do not deliver cargo to the cytosol. Systems based on bacterial toxins show a modest receptor-mediated internalization but an efficient delivery to the cytosol. Supercharged proteins, on the contrary, are not receptor-specific and lead to massive total internalization into endosomes, but only low amounts end up in the cytosol

Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting

The complementary determining regions (CDRs) from the fluorescein-binding antibody 4-4-20, which yields almost no soluble protein in periplasmic expression in Escherichia coli, were transplanted to the framework of the humanized antibody 4D5. The resulting single-chain Fv fragment (scFv) 4D5Flu showed both a dramatic improvement in soluble expression, even at 37 degrees C, and an improved thermodynamic stability. Antigen affinity was maintained upon this engineering by paying attention to crucial framework-CDR contacts. This demonstrates that the use of superior frameworks is a robust strategy to improve the physical properties of scFv fragments. We also report that the grafted version was selected in phage display over several competing variants of the same antibody with identical binding constant but poorer folding or stability properties. The selection required four panning rounds and a temperature of 37 degrees C and we show that the underlying reason for this selection is a higher fraction of phages carrying functional scFv molecule

Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment

By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface. We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out. The replacement of one of the residues (V84D in VH) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20. With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased. Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein. Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domain