DE NOVODesign of Protein Cages to Accommodate Metal Cofactors

2013 ◽  
pp. 43-85 ◽  
Author(s):  
Flavia Nastri ◽  
Rosa Bruni ◽  
Ornella Maglio ◽  
Angela Lombardi
Keyword(s):  
Protein Cages ◽  
Metal Cofactors

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.

scholarly journals Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets

Science ◽  
2021 ◽  
pp. eabi5224
Author(s):  
Nunziata Maio ◽  
Bernard A. P. Lafont ◽  
Debangsu Sil ◽  
Yan Li ◽  
J. Martin Bollinger ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Metal Binding ◽  
Rna Dependent Rna Polymerase ◽  
Iron Sulfur Clusters ◽  
Metal Binding Sites ◽  
Iron Sulfur ◽  
Cryo Electron Microscopy ◽  
Metal Cofactors ◽  
Antiviral Targets ◽  
Viral Helicase

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.

scholarly journals Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways

2014 ◽  
Vol 137 (1) ◽  
pp. 390-398 ◽  
Author(s):  
James N. Vranish ◽  
William K. Russell ◽  
Lusa E. Yu ◽  
Rachael M. Cox ◽  
David H. Russell ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Reaction Pathways ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Metal Cofactors

scholarly journals Introduction of Surface Loops as a Tool for Encapsulin Functionalization.

2021 ◽  
Author(s):  
Sandra Michel-Souzy ◽  
Naomi M. Hamelmann ◽  
Sara Zarzuela-Pura ◽  
Jos M. J. Paulusse ◽  
Jeroen J. L. M. Cornelissen
Keyword(s):  
Biomedical Applications ◽  
Large Scale ◽  
Thermotoga Maritima ◽  
Scale Production ◽  
Structure Property ◽  
Protein Subunit ◽  
Protein Cages ◽  
Notable Increase ◽  
The Stability ◽  
Surface Loops

Encapsulin based protein cages are nanoparticles with different biomedical applications, such as targeted drug delivery or imaging agents. These particles are biocompatible and can be produced in bacteria, allowing large scale production and protein engineering. In order to use these bacterial nanocages in different applications, it is important to further explore the potential of their surface modification and optimize their production. In this study we design and show new surface modifications of the Thermotoga maritima (Tm) and Brevibacterium linens (Bl) encapsulins. Two new loops on Tm encapsulin with a His-tag insertion after the residue 64 and the residue 127, and the modification of the C-terminal on Bl encapsulin, are reported. The multi-modification of the Tm encapsulin enables up to 240 different functionalities on the cage surface, resulting from 4 potential modifications per protein subunit. We furthermore report an improved protocol giving a better stability and providing a notable increase of the production yield of the cages. Finally, we tested the stability of different encapsulin variants over a year and the results show a difference in stability arising from the tag insertion position. These first insights in the structure-property relationship of encapsulins, with respect to the position of a function loop, allow for further study of the use of these protein nanocages in biomedical applications.

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

scholarly journals Laboratory evolution of virus-like nucleocapsids from nonviral protein cages

2018 ◽  
Vol 115 (21) ◽  
pp. 5432-5437 ◽  
Author(s):  
Naohiro Terasaka ◽  
Yusuke Azuma ◽  
Donald Hilvert
Keyword(s):  
Aquifex Aeolicus ◽  
Cellular Functions ◽  
Laboratory Evolution ◽  
Virion Assembly ◽  
Protein Cages ◽  
Protein Cage ◽  
Spherical Structures ◽  
Rna Genome ◽  
Biological Environment

Viruses are remarkable nanomachines that efficiently hijack cellular functions to replicate and self-assemble their components within a complex biological environment. As all steps of the viral life cycle depend on formation of a protective proteinaceous shell that packages the DNA or RNA genome, bottom-up construction of virus-like nucleocapsids from nonviral materials could provide valuable insights into virion assembly and evolution. Such constructs could also serve as safe alternatives to natural viruses for diverse nano- and biotechnological applications. Here we show that artificial virus-like nucleocapsids can be generated—rapidly and surprisingly easily—by engineering and laboratory evolution of a nonviral protein cage formed by Aquifex aeolicus lumazine synthase (AaLS) and its encoding mRNA. Cationic peptides were appended to the engineered capsid proteins to enable specific recognition of packaging signals on cognate mRNAs, and subsequent evolutionary optimization afforded nucleocapsids with expanded spherical structures that encapsulate their own full-length RNA genome in vivo and protect the cargo molecules from nucleases. These findings provide strong experimental support for the hypothesis that subcellular protein-bounded compartments may have facilitated the emergence of ancient viruses.

Synthesis of Nanoscale Iron Oxide Structures Using Protein Cages and Polysaccharide Networks

1994 ◽  
pp. 49-56 ◽  
Author(s):  
T. G. St. Pierre ◽  
P. Sipos ◽  
P. Chan ◽  
W. Chua-Anusorn ◽  
K. R. Bauchspiess ◽  
...  
Keyword(s):  
Iron Oxide ◽  
Protein Cages
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