Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks

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.

Better together: building protein oligomers naturally and by design

“…Designing and producing artificial self-assembling protein complexes is currently of great interest to protein engineering, and a variety of inventive techniques [5,16,17] have been used to mediate this, including fusion proteins/domains [18] (e.g. Nanohedra [19] and protein nanobuilding blocks [20,21]), split proteins/domains (e.g. Spycatcher [22] and split luciferase domains [23]), helix-helix interactions [24–26], metal ion bridging [27–29], cofactor bridging [30], and disulfide bridging [7] — to name a few [31–34].…”

Yeast-Based Biosensors: Current Applications and New Developments

“…Moreover, synthetic structural biology methods that allow the design and construction of such chimera are constantly improving and becoming easier thanks to computer-assisted rational and computational approaches [94,95]. Such an approach allowed the design of a new synthetic biosensor capable of sensing and reporting the intracellular level of 4-hydroxybenzoic acid (pHBA) an important industrial precursor of muconic acid in S. cerevisiae [96].…”

Lectin engineering: the possible and the actual

“…In the case of galectins, their multivalent features toward bGal-bindingcontaining conjugates are based on their different protein architectures: the 'proto' type exists as dimers, and thus cross-link with homologous counterpart molecules potentially on other cells, the 'chimera' type forms pentameric assemblies via their N-terminal non-lectin domain, and the 'tandemrepeat' type recognizes significantly different glycan ligands [66]. In addition, a recent trend in protein engineering aiming at artificial multimeric protein complexes is based on the idea of composing artificial fusion proteins assembled to form larger symmetric architectures [67] using nanohedra [68] and protein nano-building blocks [69,70]. For example, nanostructures of a barrel-shaped hexamer and a tetrahedron-like dodecamer self-assembled from protein nano-building blocks were reported by Kobayashi et al [69].…”