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.
The modes of binding of heat shock protein 90 with phenyl-Sepharose, myristoylated AE-cellulose, and monomyristoylated lysozyme were studied to characterize a hydrophobic region(s) on the surface of the heat shock protein 90 molecule and the following results were obtained. (1) The binding of heat shock protein 90 with phenyl-Sepharose was inhibited by the addition of 30% ethylene glycol. This indicates that the binding involves a hydrophobic interaction. (2) The binding was strengthened by the addition of 10 mM Mg2+, Ca2+, Sr2+, and Ba2+ ions, but not by K+ or Na+ ions. (3) The binding of hsp 90 with phenyl-Sepharose decreased initially and then increased as the temperature was increased from 0 to 50 degrees C, with a minimum at around 35 degrees C. (4) Lowering the pH stimulated the binding of hsp 90 with phenyl-Sepharose. (5) Heat shock protein 90 bound to myristoylated AE-cellulose, which has aliphatic hydrophobic residues, but not to acetylated AE-cellulose. (6) Heat shock protein 90 bound to monomyristoylated lysozyme, but not to control unmodified lysozyme. Based on these results, the possible function of the hydrophobic region(s) of heat shock protein 90 in the interaction with hydrophobic proteins is discussed.
As a first step m the mvesttgatton of the reconstttutton of sterotd hormone receptor systems, we studied the reconstttutton of 9 S estrogen receptor
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