Coiled Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability
| Autor: | Ajitha S. Cristie-David, Junjie Chen, Derek Nowak, Sung Park, Min Su, Mark M. Banaszak Holl, E. Neil G. Marsh |
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| Rok vydání: | 2018 |
| Předmět: |
Coiled coil
0303 health sciences Cryo-electron microscopy Icosahedral symmetry 010402 general chemistry 01 natural sciences 0104 chemical sciences 03 medical and health sciences Crystallography chemistry.chemical_compound chemistry Dynamic light scattering Denaturation (biochemistry) Chemical stability Guanidine Linker 030304 developmental biology |
| DOI: | 10.1101/316331 |
| Popis: | The organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Here we describe the symmetry-directed design of a hollow protein cage with dimensions similar to those of many icosahedral viruses. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain and cryo EM, and by atomic force and IR-photo-induced force microscopy established that Ico8 assembles into a flexible hollow cage with comprising 60-subunits with overall icosahedral geometry. Unexpectedly, the cages were found to encapsulate DNA, even though neither protein component binds nucleic acids on its own. Notably, the cages formed by Ico8 proved to be extremely stable towards thermal and chemical denaturation: whereas TriEst was unfolded by heating (Tm ~75 °C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 °C or in 8 M guanidine hydrochloride. The encapsulation of DNA and increased stability of the cages are new properties that emerge from the higher order structure of the protein cage, rather than being intrinsic to the components from which it is constructed. |
| Databáze: | OpenAIRE |
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