Comparative Simulative Analysis and Design of Single-Chain Self-Assembled Protein Cages.

Coiled-coil protein origami (CCPO) is a modular strategy for the de novo design of polypeptide nanostructures. It represents a type of modular design based on pairwise-interacting coiled-coil (CC) units with a single-chain protein programmed to fold into a polyhedral cage. However, the mechanisms underlying the self-assembly of the protein tetrahedron are still not fully understood. In the present study, 18 CCPO cages with three different topologies were modeled in silico. Then, molecular dynamics simulations and CC parameters were calculated to characterize the dynamic properties of protein tetrahedral cages at both the local and global levels. Furthermore, a deformed CC unit was redesigned, and the stability of the new cage was significantly improved.

Designed folding pathway of modular coiled-coil-based proteins.

Natural proteins are characterised by a complex folding pathway defined uniquely for each fold. Designed coiled-coil protein origami (CCPO) cages are distinct from natural compact proteins, since their fold is prescribed by discrete long-range interactions between orthogonal pairwise-interacting coiled-coil (CC) modules within a single polypeptide chain. Here, we demonstrate that CCPO proteins fold in a stepwise sequential pathway. Molecular dynamics simulations and stopped-flow Förster resonance energy transfer (FRET) measurements reveal that CCPO folding is dominated by the effective intra-chain distance between CC modules in the primary sequence and subsequent folding intermediates, allowing identical CC modules to be employed for multiple cage edges and thus relaxing CCPO cage design requirements. The number of orthogonal modules required for constructing a CCPO tetrahedron can be reduced from six to as little as three different CC modules. The stepwise modular nature of the folding pathway offers insights into the folding of tandem repeat proteins and can be exploited for the design of modular protein structures based on a given set of orthogonal modules.

Functional self-assembled nanovesicles based on ß-cyclodextrin, liposomes and adamantyl guanidines as potential nonviral gene delivery vectors.

Multicomponent self-assembled supramolecular nanovesicles based on an amphiphilic derivative of ß-cyclodextrin and phosphatidylcholine liposomes (PC-liposomes) functionalized with four structurally different adamantyl guanidines were prepared and characterized. Incorporation efficiency of the examined adamantyl guanidines as well as size and surface charge of the prepared supramolecular nanovesicles was determined. Changes in the surface charge of the prepared nanovesicles confirmed that guanidinium groups were exposed on the surface. ITC and 1H NMR spectroscopy complemented by molecular dynamics (MD) simulations were used to elucidate the structural data and stability of the inclusion complexes of ß-cyclodextrin and adamantyl guanidines (AG1-5). The results are consistent and point to a significant contribution of the guanylhydrazone residue to the complexation process for AG1 and AG2 with ß-cyclodextrin. In order to evaluate the potential of the self-assembled supramolecular nanomaterial as a nonviral gene delivery vector, fluorescence correlation spectroscopy was used. It showed that the prepared nanovesicles functionalized with adamantyl guanidines AG1-4 effectively recognize and bind the fluorescently labelled DNA. Furthermore, gel electrophoretic assay confirmed the formation of nanoplexes of functionalized nanovesicles and plasmid DNA. These findings together suggest that the designed supramolecular nanovesicles could be successfully applied as nonviral gene delivery vectors.

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo.

Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest containing >700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.

Advances in design of protein folds and assemblies.

Conceptual and computational advances triggered an explosion of designed protein structures in the recent years. Various protein fold geometries have been robustly designed with atomic accuracy, including protein folds unseen in nature. The same principles and tools have been extended to design multi-chain assemblies. By exploiting symmetry, mega-Dalton structures have been created with exciting potential applications for synthetic biology. In this review we focus on design of single chain and multi polypeptide chain assemblies of defined size and composition. Several innovative strategies have been developed to create de novo protein assemblies, with the two main approaches to the design of multi-chain assemblies being genetic fusion of interacting modules and engineering of novel protein-protein interfaces.

Designed Protein Origami.

Proteins are highly perfected natural molecular machines, owing their properties to the complex tertiary structures with precise spatial positioning of different functional groups that have been honed through millennia of evolutionary selection. The prospects of designing new molecular machines and structural scaffolds beyond the limits of natural proteins make design of new protein folds a very attractive prospect. However, de novo design of new protein folds based on optimization of multiple cooperative interactions is very demanding. As a new alternative approach to design new protein folds unseen in nature, folds can be designed as a mathematical graph, by the self-assembly of interacting polypeptide modules within the single chain. Orthogonal coiled-coil dimers seem like an ideal building module due to their shape, adjustable length, and above all their designability. Similar to the approach of DNA nanotechnology, where complex tertiary structures are designed from complementary nucleotide segments, a polypeptide chain composed of a precisely specified sequence of coiled-coil forming segments can be designed to self-assemble into polyhedral scaffolds. This modular approach encompasses long-range interactions that define complex tertiary structures. We envision that by expansion of the toolkit of building blocks and design strategies of the folding pathways protein origami technology will be able to construct diverse molecular machines.

Fluorescence microspectroscopy as a tool to study mechanism of nanoparticles delivery into living cancer cells.

Lack of better understanding of nanoparticles targeted delivery into cancer cells calls for advanced optical microscopy methodologies. Here we present a development of fluorescence microspectroscopy (spectral imaging) based on a white light spinning disk confocal microscope with emission wavelength selection by a liquid crystal tunable filter. Spectral contrasting of images was used to localize polymer nanoparticles and cell membranes labeled with fluorophores that have substantially overlapping spectra. In addition, fluorescence microspectroscopy enabled spatially-resolved detection of small but significant effects of local molecular environment on the properties of environment-sensitive fluorescent probe. The observed spectral shift suggests that the delivery of suitably composed cancerostatic alkylphospholipid nanoparticles into living cancer cells might rely on the fusion with plasma cell membrane.