RT Journal Article
SR Electronic
T1 Coiled Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 316331
DO 10.1101/316331
A1 Ajitha S. Cristie-David
A1 Junjie Chen
A1 Derek B. Nowak
A1 Sung I. Park
A1 Mark M. Banaszak Holl
A1 Min Su
A1 E. Neil G. Marsh
YR 2018
UL http://biorxiv.org/content/early/2018/05/07/316331.abstract
AB 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.