Directed Evolution of a Protein Container

Wörsdörfer, Bigna; Woycechowsky, Kenneth J.; Hilvert, Donald

doi:10.1126/science.1199081

Cited by 285 publications

(283 citation statements)

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“…6 The high loading accuracy, combined with the monodispersity, stability, and robust nature of bacterial encapsulins, makes them highly promising candidates for future applications in nanotechnology. While there are examples reported of designed protein cages that promote cargo packaging during in vivo selfassembly, 13 bacterial encapsulins are the first example of a nonviral protein cage which contains a naturally encoded selfsorting peptidic sequence. Moreover, since the entire packaging and assembly occurs in vivo in the presence of ≥2600 cytosolic E. coli proteins, 12 this work demonstrates the self-sorting nature of encapsulins by the selective packaging of cargo bearing the Cterminal docking sequence.…”

Section: Journal Of the American Chemical Societymentioning

confidence: 99%

Predicting the Loading of Virus-Like Particles with Fluorescent Proteins

et al. 2014

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Nature uses bottom-up approaches for the controlled assembly of highly ordered hierarchical structures with defined functionality, such as organelles, molecular motors, and transmembrane pumps. The field of bionanotechnology draws inspiration from nature by utilizing biomolecular building blocks such as DNA, proteins, and lipids, for the (self-) assembly of new structures for applications in biomedicine, optics, or electronics. Among the toolbox of available building blocks, proteins that form cagelike structures, such as viruses and virus-like particles, have been of particular interest since they form highly symmetrical assemblies and can be readily modified genetically or chemically both on the outer or inner surface. Bacterial encapsulins are a class of nonviral protein cages that self-assemble in vivo into stable icosahedral structures. Using teal fluorescent proteins (TFP) engineered with a specific native C-terminal docking sequence, we report the molecular self-sorting and selective packaging of TFP cargo into bacterial encapsulins during in vivo assembly. Using native mass spectrometry techniques, we show that loading of either monomeric or dimeric TFP cargo occurs with unprecedented high fidelity and exceptional loading accuracy. Such selfassembling systems equipped with self-sorting capabilities would open up exciting opportunities in nanotechnology, for example, as artificial (molecular storage or detoxification) organelles or as artificial cell factories for in situ biocatalysis.

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Section: Journal Of the American Chemical Societymentioning

confidence: 99%

Predicting the Loading of Virus-Like Particles with Fluorescent Proteins

et al. 2014

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“…1,2 Moreover, protein-protein interactions are also integral to the generation of cellular selfassembled nano-structures, and establishing how they control self-assembly could lead not only to fundamental understanding but also to the eventual rational design of novel structures for a myriad of applications such as the templation of inorganic nano-materials and for encapsulated reaction chemistry. [3][4][5][6][7][8] Although protein-protein interactions are intriguing medicinal targets, they have only recently been pursued for drug development studies somewhat owing to the discovery that although they often involve large, buried surface area, they can be inhibited using low molecular weight small molecules that target ''hot spot'' residues where the binding energy is concentrated. 9,10 Alanine shaving, where individual side changes are conceptually shaved to a methyl residuum and where the stabilities of the resulting mutants are determined with respect to wild type, is the most common method to identify hot spot residues.…”

Section: Introductionmentioning

confidence: 99%

Rational disruption of the oligomerization of the mini‐ferritin E. coli DPS through protein‐protein interface mutation

Zhang

Chee

et al. 2011

Protein Science

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DNA-binding protein from starved cells (DPS), a mini-ferritin capable of self-assembling into a 12-meric nano-cage, was chosen as the basis for an alanine-shaving mutagenesis study to investigate the importance of key amino acid residues, located at symmetry-related protein-protein interfaces, in controlling protein stability and self-assembly. Nine mutants were designed through simple inspection, synthesized, and subjected to transmission electron microscopy, circular dichroism, size exclusion chromatography, and ''virtual alanine scanning'' computational analysis. The data indicate that many of these residues may be hot spot residues. Most remarkably, two residues, R83 and R133, were observed to shift the oligomerization state to~50% dimer. Based on the hypothesis that these two residues constitute a ''hot strip,'' located at the ferritin-like threefold axis, the double mutant was generated which completely shuts down detectable formation of 12-mer in solution, favoring a cooperatively folded dimer. The fact that this effect logically builds upon the single mutants emphasizes that complex self-assembly has the potential to be manipulated rationally. This study should have an impact on the fundamental understanding of the assembly of DPS protein cages specifically and protein quaternary structure in general. In addition, as there is much interest in applying these and similar systems to the templation of nano-materials and drug delivery, the ability to control this ferritin's oligomerization state and stability could prove especially valuable.

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“…Generally, oppositely-charged proteins interact through simple electrostativ interactions. 32 Previously, this propensity has been exploited in artificial biosystems to engineer binary protein crystals from naturally oppositely charged proteins 33,34 as well as and artificial encapsulation [35][36][37] and Matryoshka-like cages 38 from naturally assembling proteins. Here, we suspected that shape and physicochemical features would favor assembly of oppositely charged protein pairs along particular interfaces to produce defined oligomeric assemblies (Fig.…”

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confidence: 99%

Supercharging enables organized assembly of synthetic biomolecules

Simon

Ramasubramani

Gläser

et al. 2018

Preprint

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Abstract:There are few methods for the assembly of defined protein oligomers and higher order structures that could serve as novel biomaterials. Using fluorescent proteins as a model system, we have engineered novel oligomerization states by combining oppositely supercharged variants. A well-defined, highly symmetrical 16-mer (two stacked, circular octamers) can be formed from alternating charged proteins; higher order structures then form in a hierarchical fashion from this discrete protomer. During SUpercharged PRotein Assembly (SuPrA), electrostatic attraction between oppositely charged variants drives interaction, while shape and patchy physicochemical interactions lead to spatial organization along specific interfaces, ultimately resulting in protein assemblies never before seen in nature.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/323261 doi: bioRxiv preprint first posted online May. 16, 2018; 2 The assembly of genetically-encoded molecules into symmetric, supramolecular materials enables complex functions in natural biosystems and would likewise prove valuable in the development of bionanotechnologies including drug delivery, energy transport, and biological information storage. [1][2][3][4][5][6][7] Repeated, symmetric interactions between molecular building blocks enable the formation of complex, defined, assemblies from a small total number of components as well as dynamic propagation of conformational change. [3][4][5][6][7][8] However, while de novo engineering of artificial symmetrical biomolecular complexes has begun to be explored, 9-15 these efforts generally rely upon precise design of molecular interfaces. 16,17 In contrast, studies of simple synthetic colloids suggests that higher order structures can be derived based solely on packing and energetic and considerations. Computational and experimental studies of polyhedral or spherical colloids indicate that complementary particle shapes 18-24 and simple attractive "patches" [24][25][26][27][28][29] alone can enable formation of complex symmetrical assemblies. Shape complementarity has likewise been exploited to arrange synthetic linear biomolecules, i.e., double-stranded DNA strands, into distinct structures, demonstrating its utility for for the higher order arrangement of synthetic linear biomolecules, suggesting a utility beyond inorganic systems. 30,31Herein we demonstrate a simple, robust strategy to assemble normally monomeric proteins into well defined, oligomeric quaternary structures and micron-scale particles. This strategy centers on driving protein interactions by engineering oppositely supercharged variants. Generally, oppositely-charged proteins interact through simple electrostativ interactions. 32 Previously, this propensity has been exploited in artificial biosystems to engineer binary protein crystals from naturally oppos...

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Directed Evolution of a Protein Container

Cited by 285 publications

References 30 publications

Predicting the Loading of Virus-Like Particles with Fluorescent Proteins

Predicting the Loading of Virus-Like Particles with Fluorescent Proteins

Rational disruption of the oligomerization of the mini‐ferritin E. coli DPS through protein‐protein interface mutation

Supercharging enables organized assembly of synthetic biomolecules

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