Biology subjects

Dawson, W. M.

Publications and source records attributed to Dawson, W. M..

7 recordsLinked to original sources

bioRxiv2025

De novo design of semisynthetic protein nanopores

Protein nanopores are essential components of single-molecule oligonucleotide sequencing and sensing devices. Here, we demonstrate that installing additional de novo subunits enables large-scale architectural changes of nanopore complexes. We design de novo proteins that integrate seamlessly with the CsgG pore to form 18-subunit, 315-kilodalton complexes with precisely sculpted pore architectures and tailored ion conduction, opening new possibilities for engineering enhanced nanopores with customized structural and functional properties.

biochemistry↗

bioRxiv2024

Exchange, promiscuity, and orthogonality in de novo designed coiled-coil peptide assemblies

De novo protein design is delivering new peptide and protein structures at a rapid pace. Many of these synthetic polypeptides form well-defined and hyperthermal-stable structures. Generally, however, less is known about the dynamic properties of the de novo designed structures. Here, we explore one aspect of dynamics in a series of de novo coiled-coil peptide assemblies: namely, peptide exchange within and between different oligomers from dimers through to heptamers. First, we develop a fluorescence-based reporter assay for peptide exchange that is straightforward to implement, and, thus, would be useful to others examining similar systems. We apply this assay to explore both homotypic exchange within single species, and heterotypic exchange between coiled coils of different coiled-coil oligomer states. For the former, we provide detailed study for the dimeric coiled coil CC-Di finding a half-life for exchange of 4.2 {+/-} 0.3 minutes when the concentration of CC-Di is 200 {micro}M. Interestingly, more broadly when assessing exchange across all of the oligomeric states, we find that some of the designs are faithful and only undergo homotypic strand exchange, whereas others are promiscuous and exchange to form unexpected hetero-oligomers. Finally, we develop two design strategies to improve the orthogonality of the different oligomers: (i) using alternate positioning of salt bridge interactions; and (ii) incorporating of non-canonical repeats into the designed sequences. In so doing, we reconcile the promiscuity and deliver a set of faithful homo-oligomeric de novo coiled-coil peptides. Our findings have implications for the application of these and other coiled coils as modules in chemical and synthetic biology.

systems biology↗

bioRxiv2023

Allosteric regulation of a molecular motor through de novo protein design

Many enzymes are allosterically regulated. Our ability to manipulate these structural changes is limited. Here we install an allosteric switch into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed auto-inhibited states. The equilibrium between these centres on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with a de novo designed peptide. The alternative states are modelled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities. One Sentence SummaryDe novo peptide and protein design are used to engineer an allosteric switch into kinesin-1 motors in vitro and directly in cells.

synthetic biology↗

bioRxiv2023

Rationally seeded computational protein design

Computational protein design is advancing rapidly. Here we describe efficient routes to two families of -helical-barrel proteins with central channels that bind small molecules. The designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides. Adjacent helices are connected using computational loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix-turn-helix-turn-helix motifs that are packed onto the barrels computationally. Throughout these pipelines, residues that define open states of the barrels are maintained. This minimises sequence sampling and accelerates routes to successful designs. For each of 6 targets, just 2 - 6 synthetic genes are made for expression in E. coli. On average, 80% express to give soluble monomeric proteins that are characterized fully, including high-resolution structures for most targets that match the seed structures and design models with high accuracy.

synthetic biology↗

bioRxiv2023

CC + : A Searchable Database of Validated Coiled coils in PDB Structures and AlphaFold2 Models

-Helical coiled coils are common tertiary and quaternary elements of protein structure. In coiled coils, two or more helices wrapped around each other to form bundles. This apparently simple structural motif can generate many architectures and topologies. Understanding the variety of and limits on coiled-coil assemblies and their sequence-to-structure relationships impacts on protein structure, design, and engineering. Coiled coil-forming sequences can be predicted from heptad repeats of hydrophobic and polar residues, hpphppp, although this is not always reliable. Alternatively, coiled-coil structures can be identified using the program SOCKET, which finds knobs-into-holes (KIH) packing between side chains of neighboring helices. SOCKET also classifies coiled-coil architecture and topology, thus allowing sequence-to-structure relationships to be garnered. In 2009, we used SOCKET to create a relational database of coiled-coil structures, CC+, from the RCSB Protein Data Bank (PDB). Here we report an update of CC+ following the recent explosion of structural data and the success of AlphaFold2 in predicting protein structures from genome sequences. With the most-stringent SOCKET parameters, CC+ contains {approx}12,000 coiled-coil assemblies from experimentally determined structures, and {approx}120,000 potential coiled-coil structures within single-chain models predicted by AlphaFold2 across 48 proteomes. CC+ allows these and other less-stringently defined coiled coils to be searched at various levels of structure, sequence, and side-chain interactions. The identified coiled coils can be viewed directly from CC+ using the Socket2 application, and their associated data can be downloaded for further analyses. CC+ is available freely at http://coiledcoils.chm.bris.ac.uk/CCPlus/Home.html. It will be regularly updated automatically. FOR THE BROADER AUDIENCEProtein assemblies and protein-protein interactions are key to all biological processes. -Helical coiled coils are one of the most common modes of directing and stabilising these interfaces. Here, we report an updated CC+ database of structurally validated coiled coils from experimental protein structures and AlphaFold2 models. CC+ contains many thousands of coiled-coil structures and models, associated parameters, and sequences. It enables the compilation of rich datasets for advancing protein structure, design, and engineering research.

bioinformatics↗

bioRxiv2021

How coiled-coil assemblies accommodate multiple aromatic residues

Rational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely the ;-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. However, current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic helices. The consequences of using aromatic residues--which would be useful for introducing structural probes, and binding and catalytic functionalities--into these interfaces is not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In the other more-complex structure, the residues are buried and form an extended hydrogen-bond network.

biochemistry↗

bioRxiv2021

Coiled coils 9-to-5: Rational de novo design of α-helical barrels with tunable oligomeric states

The rational design of linear peptides that assemble controllably and predictably in water is challenging. Sequences must encode unique target structures and avoid alternative states. However, the stabilizing and discriminating non-covalent forces available are weak in water. Nonetheless, for -helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger -helical barrels. Here, we show that pentamers to nonamers are achieved simply by varying the residue at one of these h sites. In L/I-K-E-I-A-x-Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting -helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies.

biochemistry↗