A Set of Computationally Designed Orthogonal Antiparallel Homodimers that Expands the Synthetic Coiled-Coil Toolkit

Negron, Christopher; Keating, Amy E.

doi:10.1021/ja507847t

Cited by 58 publications

(67 citation statements)

References 67 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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“…14 To promote the formation of well-defined oligomers, we incorporated hydrophobic residues into positions R 8 and R 11 . The first part—the current paper—determines how the two peptides assemble in aqueous solution.…”

Section: Introductionmentioning

confidence: 99%

Assembly of Peptides Derived from β-Sheet Regions of β-Amyloid

Truex

Nowick

2016

J. Am. Chem. Soc.

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In Alzheimer’s disease, aggregation of the β-amyloid peptide (Aβ) results in the formation of oligomers and fibrils that are associated with neurodegeneration. Aggregation of Aβ occurs through interactions between different regions of the peptide. This paper and the accompanying paper constitute a two-part investigation of two key regions of Aβ: the central region and the C-terminal region. These two regions promote aggregation and adopt β-sheet structure in the fibrils, and may also do so in the oligomers. In this paper, we study the assembly of macrocyclic β-sheet peptides that contain residues 17–23 (LVFFAED) from the central region and residues 30–36 (AIIGLMV) from the C-terminal region. These peptides assemble to form tetramers. Each tetramer consists of two hydrogen-bonded dimers that pack through hydrophobic interactions in a sandwich-like fashion. Incorporation of a single 15N isotopic label into each peptide provides a spectroscopic probe with which to elucidate the β-sheet assembly and interaction: 1H,15N HSQC studies facilitate the identification of the monomers and tetramers; 15N-edited NOESY studies corroborate the pairing of the dimers within the tetramers. In the following paper, J. Am. Chem. Soc.2016, DOI: 10.1021/jacs.6b06001, we will extend these studies to elucidate the coassembly of the peptides to form heterotetramers.

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“…Recently, an improved model for scoring anti-parallel coiled-coil interactions was developed that could be used to augment the design protocol used in this work. 17 …”

Section: Resultsmentioning

confidence: 99%

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy

et al. 2015

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Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8×8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

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A Set of Computationally Designed Orthogonal Antiparallel Homodimers that Expands the Synthetic Coiled-Coil Toolkit

Cited by 58 publications

References 67 publications

Flexible, symmetry-directed approach to assembling protein cages

Flexible, symmetry-directed approach to assembling protein cages

Assembly of Peptides Derived from β-Sheet Regions of β-Amyloid

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy

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