The design of novel proteins that self-assemble into supramolecular complexes is an important step in the development of synthetic biology and nanotechnology. Recently, we described the three-dimensional structure of WA20, a de novo protein that forms an intermolecularly folded dimeric 4-helix bundle (PDB code 3VJF ). To harness the unusual intertwined structure of WA20 for the self-assembly of supramolecular nanostructures, we created a protein nanobuilding block (PN-Block), called WA20-foldon, by fusing the dimeric structure of WA20 to the trimeric foldon domain of fibritin from bacteriophage T4. The WA20-foldon fusion protein was expressed in the soluble fraction in Escherichia coli, purified, and shown to form several homooligomeric forms. The stable oligomeric forms were further purified and characterized by a range of biophysical techniques. Size exclusion chromatography, multiangle light scattering, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) analyses indicate that the small (S form), middle (M form), and large (L form) forms of the WA20-foldon oligomers exist as hexamer (6-mer), dodecamer (12-mer), and octadecamer (18-mer), respectively. These findings suggest that the oligomers in multiples of 6-mer are stably formed by fusing the interdigitated dimer of WA20 with the trimer of foldon domain. Pair-distance distribution functions obtained from the Fourier inversion of the SAXS data suggest that the S and M forms have barrel- and tetrahedron-like shapes, respectively. These results demonstrate that the de novo WA20-foldon is an effective building block for the creation of self-assembling artificial nanoarchitectures.

To probe the potential for activity in unevolved amino acid sequence space, we created a third generation combinatorial library of de novo four-helix bundle proteins. The "artificial superfamily" of helical bundles was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. WA20, picked from the library, is one of the most stable proteins in the superfamily, and has rudimentary activities such as esterase and lipase. Here we report the crystal structure of WA20, determined by the multiwavelength anomalous dispersion method. Unexpectedly, the WA20 crystal structure is not a monomeric four-helix bundle, but a dimeric four-helix bundle. Each monomer comprises two long α-helices that intertwist with the helices of the other monomer. The two monomers together form a 3D domain-swapped four-helix bundle dimer. In addition, there are two hydrophobic pockets, which may potentially provide substrate binding sites. Small-angle X-ray scattering shows that the molecular weight of WA20 is ~25 kDa and the shape is rod-like (the maximum length, D(max) = ~8 nm), indicating that WA20 forms a dimeric four-helix bundle in solution. These results demonstrate that our de novo protein library contains not only simple monomeric proteins, but also stable and functional multimeric proteins.

Pancreatic islet transplantation is a promising treatment for diabetes but still faces several challenges. Poor islet isolation efficiency and poor long-term insulin independence are currently two major issues, although donor shortage and the need for immunosuppressants also need to be addressed. We established the Kyoto islet isolation method (KIIM), which has enabled us to isolate and transplant islets even from non-heart-beating donors. KIIM involves 1) cooling the donor pancreas in situ, 2) preserving the ducts with modified Kyoto solution, 3) using a modified two-layer pancreas preservation method, and 4) adjusting the density of the density gradient centrifugation and using an iodixanol-based solution for purification. KIIM has enabled us to transplant 17 islet preparations out of 21 isolations (an 81% success rate). All transplanted islets functioned, and all transplanted patients had improved glycemic control without hypoglycemic unawareness. Recently, we used KIIM for islet isolation from a brain-dead donor at Baylor, which resulted in a very high islet yield (789,984 IE) with high viability (100% by fluorescein diacetate/propidium iodide staining and a stimulation index of 4.7). This preliminary evidence suggests that KIIM may also be promising for islet isolation from brain-dead donors. In addition, to assess engrafted islet mass, we developed a secretory unit of islet transplant objects (SUITO) index: fasting C-peptide (ng/dL) / [fasting blood glucose (mg/dL) - 63] x 1500. This simple index has enabled us to monitor the engrafted islet mass. This index should be useful when deciding whether to perform additional islet transplantations to maintain insulin independence. Poor islet isolation efficacy and poor long-term results could be resolved with ongoing research.

A thermostable enzyme PET2, found in a metagenome library, has been engineered to improve its hydrolytic activity against polyethylene terephthalate (PET). The PET2 wild-type (WT) showed a melting temperature of 69.0 °C and produced water-soluble reaction products at a rate of 0.40 min–1 (2.4 μM products from 0.1 μM enzyme after 60 min reaction) from an amorphous PET film at 60 °C. Mutations for surface charge modification, backbone stabilization, and formation of additional disulfide bond were introduced into the PET2 WT, and the best mutant (PET2 7M) showed a melting temperature of 75.7 °C and hydrolytic activity of 1.3 min–1 (7.8 μM products from 0.1 μM enzyme after 60 min reaction at 60 °C). X-ray crystal structures of PET2 mutants showed that introduced arginine and lysine residues oriented to the solvent, similar to a PET hydrolase from Ideonella sakaiensis 201-F6. Single-molecule fluorescence imaging revealed that these positively charged surface residues increased binding rate constant of PET2 7M to PET surface 2.7 times, compared with PET2 WT, and resulted in higher activity. Optimal temperature for amorphous PET hydrolysis by PET2 7M (68 °C) was 8 °C higher than that by PET2 WT (60 °C), and hydrolytic activity of PET2 7M at the optimal temperature (2.7 min–1, 16.2 μM products from 0.1 μM enzyme after 60 min reaction) was 6.8 times higher than that of PET2 WT (0.40 min–1). Furthermore, PET2 7M generated reaction products with a constant rate for at least 24 h at 68 °C, indicating long-term thermal stability at the optimal temperature.

No abstract

The design of novel proteins that self-assemble into supramolecular complexes is important for development in nanobiotechnology and synthetic biology. Recently, we designed and created a protein nanobuilding block (PN-Block), WA20-foldon, by fusing an intermolecularly folded dimeric de novo WA20 protein and a trimeric foldon domain of T4 phage fibritin (Kobayashi et al., J. Am. Chem. Soc. 2015, 137, 11285). WA20-foldon formed several types of self-assembling nanoarchitectures in multiples of 6-mers, including a barrel-like hexamer and a tetrahedron-like dodecamer. In this study, to construct chain-like polymeric nanostructures, we designed de novo extender protein nanobuilding blocks (ePN-Blocks) by tandemly fusing two de novo binary-patterned WA20 proteins with various linkers. The ePN-Blocks with long helical linkers or flexible linkers were expressed in soluble fractions of Escherichia coli, and the purified ePN-Blocks were analyzed by native PAGE, size exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS), and transmission electron microscopy. These results suggest formation of various structural homo-oligomers. Subsequently, we reconstructed hetero-oligomeric complexes from extender and stopper PN-Blocks by denaturation and refolding. The present SEC-MALS and SAXS analyses show that extender and stopper PN-Block (esPN-Block) heterocomplexes formed different types of extended chain-like conformations depending on their linker types. Moreover, atomic force microscopy imaging in liquid suggests that the esPN-Block heterocomplexes with metal ions further self-assembled into supramolecular nanostructures on mica surfaces. Taken together, the present data demonstrate that the design and construction of self-assembling PN-Blocks using de novo proteins is a useful strategy for building polymeric nanoarchitectures of supramolecular protein complexes.

In our first case of living donor islet transplantation, both the donor and the recipient have been maintaining excellent glycemic control with no untreatable complications for more than one year.

Background:IseA from Bacillus subtilis is an inhibitor protein against DL-endopeptidases including synthetic lethal autolysins. Results: We solved the solution structure of IseA using NMR. Titration with LytF DL-endopeptidase indicated interaction sites around the loop region. Conclusion: The IseA structure revealed a novel "hacksaw"-like fold with a characteristic inhibitory loop. Significance: The results suggest a new inhibition mechanism of IseA with a unique loop.