A Set of <i>de Novo</i> Designed Parallel Heterodimeric Coiled Coils with Quantified Dissociation Constants in the Micromolar to Sub-nanomolar Regime

Thomas, Franziska; Boyle, Aimee L.; Burton, Antony J.; Woolfson, Derek N.

doi:10.1021/ja312310g

Cited by 157 publications

(244 citation statements)

References 47 publications

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“…Coiled coils are among the simplest and best-understood protein-protein interactions (33). As such, there are a large number of well-characterized designs available as "off-the-shelf" components for use in protein engineering applications, including dimeric, trimeric, tetrameric, pentameric, and hexameric designs in both parallel and antiparallel forms (34)(35)(36). A further advantage is that the strength of the coiled-coil interaction can easily be manipulated by varying the number of heptad repeats.…”

Section: Resultsmentioning

confidence: 99%

Flexible, symmetry-directed approach to assembling protein cages

Sciore

Koldewey

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

135

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The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C 3 -symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C 4 -symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C 4 and C 3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.coiled coils | protein design | native mass spectrometry | analytical ultracentrifugation | cryoelectron microscopy

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Section: Resultsmentioning

confidence: 99%

Flexible, symmetry-directed approach to assembling protein cages

Sciore

Koldewey

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

135

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“…To expand possibilities in this area, we sought a general and modular method for producing PNTs from chemically accessible and standard building blocks, which could be altered predictably to access a range of PNTs with channels of predetermined internal diameter and chemistry. dimers and a heterotrimer [19][20][21] . Recently, we have expanded the toolkit to include more-complex coiled coils, including pentamers, hexamers and a heptamer, which are the lowest-order α-helical barrels (Fig.…”

Section: Introductionmentioning

confidence: 99%

Modular Design of Self-Assembling Peptide-Based Nanotubes

et al. 2015

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An ability to design peptide-based nanotubes (PNTs) rationally with defined and mutable internal channels would advance understanding of peptide self-assembly, and present new biomaterials for nanotechnology and medicine. PNTs have been made from Fmoc dipeptides, cyclic peptides, and lock-washer helical bundles. Here we show that blunt-ended α-helical barrelsi.e., pre-assembled bundles of α-helices with central channels-can be used as building blocks for PNTs. This approach is general and systematic, and uses a set of de novo helical bundles as standards. One of these bundles, a hexameric α-helical barrel, assembles into highly ordered PNTs, for which we have determined a structure by combining cryo-transmission electron microscopy, Xray fiber diffraction and model building. The structure reveals that the overall symmetry of the peptide module plays a critical role in ripening and ordering of the supramolecular assembly. PNTs based on pentameric, hexameric and heptameric α-helical barrels sequester hydrophobic dye within their lumens.

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“…2,16,18,19 This is also exploited in coiled-coil design: judicious placement of complementary charges-e.g., Lys-Glu pairs at e and g-can be used to pattern sequences to control homotypic or heterotypic assembly of peptides otherwise possessing the same core a & d residues. [19][20][21][22] Together, such heuristics have been used to design a suite of coiled-coil components, 8,[23][24][25] which have been used as modular building blocks in various synthetic-biology applications. [26][27][28][29][30] The above said, the a/d interfaces are not the exclusive province of hydrophobic residues; indeed, ≈1/4 of residues at these interfaces of structurally defined coiled coils are polar.…”

Section: Introductionmentioning

confidence: 99%

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

Fletcher¹,

Bartlett²,

Boyle³

et al. 2017

ACS Chem. Biol.

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The α-helical coiled coil is one of the best-studied protein–protein interaction motifs. As a result, sequence-to-structure relationships are available for the prediction of natural coiled-coil sequences and the de novo design of new ones. However, coiled coils adopt a wide range of oligomeric states and topologies, and our understanding of the specification of these and the discrimination between them remains incomplete. Gaps in our knowledge assume more importance as coiled coils are used increasingly to construct biomimetic systems of higher complexity; for this, coiled-coil components need to be robust, orthogonal, and transferable between contexts. Here, we explore how the polar side chain asparagine (Asn, N) is tolerated within otherwise hydrophobic helix–helix interfaces of coiled coils. The long-held view is that Asn placed at certain sites of the coiled-coil sequence repeat selects one oligomer state over others, which is rationalized by the ability of the side chain to make hydrogen bonds, or interactions with chelated ions within the coiled-coil interior of the favored state. We test this with experiments on de novo peptide sequences traditionally considered as directing parallel dimers and trimers, and more widely through bioinformatics analysis of natural coiled-coil sequences and structures. We find that when located centrally, rather than near the termini of such coiled-coil sequences, Asn does exert the anticipated oligomer-specifying influence. However, outside of these bounds, Asn is observed less frequently in the natural sequences, and the synthetic peptides are hyperthermostable and lose oligomer-state specificity. These findings highlight that not all regions of coiled-coil repeat sequences are equivalent, and that care is needed when designing coiled-coil interfaces.

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A Set of de Novo Designed Parallel Heterodimeric Coiled Coils with Quantified Dissociation Constants in the Micromolar to Sub-nanomolar Regime

Cited by 157 publications

References 47 publications

Flexible, symmetry-directed approach to assembling protein cages

Flexible, symmetry-directed approach to assembling protein cages

Modular Design of Self-Assembling Peptide-Based Nanotubes

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

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