Construction of protein-loadable protein cages using the hybrid proteins of the oleosin hydrophobic domain and hydrophilic dimeric coiled-coil

2021 ◽  
Vol 40 ◽  
pp. 100352
Author(s):  
Taiki Nishiyama ◽  
Kento Sugiura ◽  
Kouta Sugikawa ◽  
Atsushi Ikeda ◽  
Toshihisa Mizuno
Keyword(s):  
Coiled Coil ◽  
Hydrophobic Domain ◽  
Protein Cages ◽  
Hybrid Proteins

scholarly journals Conservation and Identity Selection of Cationic Residues Flanking the Hydrophobic Regions in Intermediate Filament Superfamily

2021 ◽  
Vol 9 ◽  
Author(s):  
Wenbo Zhang ◽  
Mingwei Liu ◽  
Robert L. Dupont ◽  
Kai Huang ◽  
Lanlan Yu ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Hydrophobic Interactions ◽  
Coiled Coil ◽  
Separation Distance ◽  
Hydrophobic Domain ◽  
Protein Architecture ◽  
Correlated Mutations ◽  
Cationic Residues ◽  
Positively Charged ◽  
Selection Of

The interplay between the hydrophobic interactions generated by the nonpolar region and the proximal functional groups within nanometers of the nonpolar region offers a promising strategy to manipulate the intermolecular hydrophobic attractions in an artificial molecule system, but the outcomes of such modulations in the building of a native protein architecture remain unclear. Here we focus on the intermediate filament (IF) coiled-coil superfamily to assess the conservation of positively charged residue identity via a biostatistical approach. By screening the disease-correlated mutations throughout the IF superfamily, 10 distinct hotspots where a cation-to-cation substitution is associated with a pathogenic syndrome have been identified. The analysis of the local chemical context surrounding the hotspots revealed that the cationic diversity depends on their separation distance to the hydrophobic domain. The nearby cationic residues flanking the hydrophobic domain of a helix (separation <1 nm) are relatively conserved in evolution. In contrast, the cationic residues that are not adjacent to the hydrophobic domain (separation >1 nm) tolerate higher levels of variation and replaceability. We attribute this bias in the conservation degree of the cationic residue identity to reflect the interplay between the proximal cations and the hydrophobic interactions.

scholarly journals Reductive power of the archaea right-handed coiled coil nanotube (RHCC-NT) and incorporation of mercury clusters inside protein cages

2018 ◽  
Vol 203 (3) ◽  
pp. 281-287 ◽  
Author(s):  
Matthew McDougall ◽  
Kevin McEleney ◽  
Olga Francisco ◽  
Benchmen Trieu ◽  
Efehi Kelly Ogbomo ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Protein Cages

scholarly journals Self-assembly and regulation of protein cages from pre-organised coiled-coil modules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fabio Lapenta ◽  
Jana Aupič ◽  
Marco Vezzoli ◽  
Žiga Strmšek ◽  
Stefano Da Vela ◽  
...  
Keyword(s):  
Self Assembly ◽  
De Novo ◽  
Coiled Coil ◽  
Dna Nanotechnology ◽  
De Novo Design ◽  
Single Chain ◽  
Sequential Order ◽  
Conformational Switch ◽  
Protein Cages ◽  
Protease Cleavage

AbstractCoiled-coil protein origami (CCPO) is a modular strategy for the de novo design of polypeptide nanostructures. CCPO folds are defined by the sequential order of concatenated orthogonal coiled-coil (CC) dimer-forming peptides, where a single-chain protein is programmed to fold into a polyhedral cage. Self-assembly of CC-based nanostructures from several chains, similarly as in DNA nanotechnology, could facilitate the design of more complex assemblies and the introduction of functionalities. Here, we show the design of a de novo triangular bipyramid fold comprising 18 CC-forming segments and define the strategy for the two-chain self-assembly of the bipyramidal cage from asymmetric and pseudo-symmetric pre-organised structural modules. In addition, by introducing a protease cleavage site and masking the interfacial CC-forming segments in the two-chain bipyramidal cage, we devise a proteolysis-mediated conformational switch. This strategy could be extended to other modular protein folds, facilitating the construction of dynamic multi-chain CC-based complexes.

scholarly journals Author Correction: Self-assembly and regulation of protein cages from pre-organised coiled-coil modules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fabio Lapenta ◽  
Jana Aupič ◽  
Marco Vezzoli ◽  
Žiga Strmšek ◽  
Stefano Da Vela ◽  
...  
Keyword(s):  
Self Assembly ◽  
Coiled Coil ◽  
Protein Cages

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21969-9

scholarly journals Preferential association of syntaxin 8 with the early endosome

2000 ◽  
Vol 113 (6) ◽  
pp. 997-1008 ◽  
Author(s):  
V.N. Subramaniam ◽  
E. Loh ◽  
H. Horstmann ◽  
A. Habermann ◽  
Y. Xu ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Early Endosome ◽  
Rat Tissues ◽  
Attachment Protein ◽  
Hydrophobic Domain ◽  
Vesicle Docking ◽  
Indirect Immunofluorescence Microscopy ◽  
Sensitive Factor ◽  
Carboxyl Terminal ◽  
Golgi Network

Members of the syntaxin family play a fundamental role in vesicle docking and fusion of diverse transport events. We have molecularly characterized syntaxin 8, a novel member of the syntaxin family. The nucleotide sequence of cloned rat cDNA predicts a polypeptide of 236 residues with a carboxyl-terminal 18-residue hydrophobic domain that may function as a membrane anchor. Characteristic of syntaxins, syntaxin 8 also contain regions that have the potential to form coiled-coil structures. Among the known syntaxins, syntaxin 8 is most homologous to syntaxin 6 which is predominantly associated with the trans-Golgi network (TGN). The syntaxin 8 transcript is detected in all rat tissues examined by northern blot. Antibodies against recombinant syntaxin 8 recognize a 27 kDa protein that is enriched in membrane fractions containing the Golgi apparatus and the endosomal/lysosomal compartments. Syntaxin 8 in membrane extract could be incorporated into a 20S protein complex in a way that is dependent on the soluble N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein ((alpha)-SNAP), suggesting that syntaxin 8 is indeed a SNAP receptor (SNARE). Indirect immunofluorescence microscopy reveals that the majority of syntaxin 8 is localized to the early endosome marked by Rab5. This is corroborated by immunogold labeling experiments showing enrichment of syntaxin 8 in the early endosome and its co-labeling with Rab5.

scholarly journals A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures

2021 ◽  
Vol 118 (17) ◽  
pp. e2021899118
Author(s):  
Andreja Majerle ◽  
San Hadži ◽  
Jana Aupič ◽  
Tadej Satler ◽  
Fabio Lapenta ◽  
...  
Keyword(s):  
Protein Design ◽  
Self Assembly ◽  
De Novo ◽  
Protein Structures ◽  
Coiled Coil ◽  
Triangular Prism ◽  
Single Chain ◽  
Protein Cages ◽  
X Ray Scattering ◽  
Structure Relationship

Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence–structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that determine the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong positive cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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