245 research outputs found
woolfson-group/isambard: 2017.0.1a
No full text<p>ISAMBARD (Intelligent System for Analysis, Model Building And Rational Design), is a Python-based framework for structural analysis and rational design of biomolecules. It is developed and maintained by members of the Woolfson group, University of Bristol.</p>
<p>The first release of 2017, this addresses a range of bug and reimplements the some of the code for testing the availability of dependancies.</p>CWW was funded by Biotechnology and Biological Sciences Research Council (BBSRC) studentship (1228976
Conformational Dynamics of Asparagine at Coiled-Coil Interfaces
No full textCoiled coils (CCs) are among the best-understood protein folds. Nonetheless, there are gaps in our knowledge of CCs. Notably, CCs are likely to be structurally more dynamic than often considered. Here, we explore this in an abundant class of CCs, parallel dimers, focusing on polar asparagine (Asn) residues in the hydrophobic interface. It is well documented that such inclusions discriminate between different CC oligomers, which has been rationalized in terms of whether the Asn can make side-chain hydrogen bonds. Analysis of parallel CC dimers in the Protein Data Bank reveals a variety of Asn side-chain conformations, but not all of these make the expected inter-side-chain hydrogen bond. We probe the structure and dynamics of a de novo-designed coiled-coil homodimer, CC-Di, by multidimensional nuclear magnetic resonance spectroscopy, including model-free dynamical analysis and relaxation-dispersion experiments. We find dynamic exchange on the millisecond time scale between Asn conformers with the side chains pointing into and out of the core. We perform molecular dynamics simulations that are consistent with this, revealing that the side chains are highly dynamic, exchanging between hydrogen-bonded-paired conformations in picoseconds to nanoseconds. Combined, our data present a more dynamic view for Asn at CC interfaces. Although inter-side-chain hydrogen bonding states are the most abundant, Asn is not always buried or engaged in such interactions. Because interfacial Asn residues are key design features for modulating CC stability and recognition, these further insights into how they are accommodated within CC structures will aid their predictive modeling, engineering, and design
Controlling the assembly of coiled-coil peptide nanotubes
No full textAn ability to control the assembly of peptide nanotubes (PNTs) would provide biomaterials for applications in nanotechnology and synthetic biology. Recently, we presented a modular design for PNTs using α-helical barrels with tunable internal cavities as building blocks. These first-generation designs thicken beyond single PNTs. Herein we describe strategies for controlling this lateral association, and also for the longitudinal assembly. We show that PNT thickening is pH sensitive, and can be reversed under acidic conditions. Based on this, repulsive charge interactions are engineered into the building blocks leading to the assembly of single PNTs at neutral pH. The building blocks are modified further to produce covalently linked PNTs via native chemical ligation, rendering ca. 100 nm-long nanotubes. Finally, we show that small molecules can be sequestered within the interior lumens of single PNTs. Thick to thin: The assembly in coiled-coil peptide nanotubes (PNTs) can be controlled. Arrays of hexameric coiled-coil PNTs can be reversibly disassembled by acidification. Accordingly, repulsive-charge interactions engineered into the coiled-coil units result in the formation of single PNTs at neutral pH. Non-covalent or covalent linkage by native chemical ligation can be used to vary the stability of, and small-molecule encapsulation by, the resulting PNTs.</p
Guiding Biomolecular Interactions in Cells Using de Novo Protein - Protein Interfaces
No full textAn improved ability to direct and control biomolecular interactions in living cells would have an impact on synthetic biology. A key issue is the need to introduce interacting components that act orthogonally to endogenous proteomes and interactomes. Here, we show that low-complexity, de novo designed protein–protein interaction (PPI) domains can substitute for natural PPIs and guide engineered protein–DNA interactions in Escherichia coli. Specifically, we use de novo homo- and heterodimeric coiled coils to reconstitute a cytoplasmic split adenylate cyclase, recruit RNA polymerase to a promoter and activate gene expression, and oligomerize both natural and designed DNA-binding domains to repress transcription. Moreover, the stabilities of the heterodimeric coiled coils can be modulated by rational design and, thus, adjust the levels of gene activation and repression in vivo. These experiments demonstrate the possibilities for using designed proteins and interactions to control biomolecular systems such as enzyme cascades and circuits in cells
The evolution and structure prediction of coiled coils across all genomes
No full textCoiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available, but key issues remain: oligomeric state and protein–protein interface prediction and extension to all genomes. We present SpiriCoil (http://supfam.org/SUPERFAMILY/spiricoil), which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds of hidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils help. SpiriCoil performs at least as well as existing methods at detecting coiled coils and significantly advances the state of the art for oligomer state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (more than 1200), and a resulting Web interface supplies data downloads, alignments, scores, oligomeric state classifications, three-dimensional homology models and visualisation. This has allowed, for the first time, a genomewide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times, and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life. The vast majority of occasions that individual coiled coils have arisen de novo were before the last universal common ancestor of life; we do, however, observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein.Coiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available, but key issues remain: oligomeric state and protein–protein interface prediction and extension to all genomes. We present SpiriCoil (http://supfam.org/SUPERFAMILY/spiricoil), which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds of hidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils help. SpiriCoil performs at least as well as existing methods at detecting coiled coils and significantly advances the state of the art for oligomer state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (more than 1200), and a resulting Web interface supplies data downloads, alignments, scores, oligomeric state classifications, three-dimensional homology models and visualisation. This has allowed, for the first time, a genomewide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times, and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life. The vast majority of occasions that individual coiled coils have arisen de novo were before the last universal common ancestor of life; we do, however, observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein
Fragment-linking peptide design yields a high-affinity ligand for microtubule-based transport
No full textSynthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides which make improved interactions with the target. Here, we devise a fragment-linking strategy - ‘mash-up’ design - to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolaraffinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.<br/
Magic-wand: a single, designed peptide that assembles to stable, ordered helical fibres
No full textWe describe a straightforward single-peptide design that self-assembles into extended and thickened nano-to-mesoscale fibers of remarkable stability and order. The basic chassis of the design is the well-understood dimeric ?-helical coiled-coil motif. As such, the peptide has a heptad sequence repeat, abcdefg, with isoleucine and leucine residues at the a and d sites to ensure dimerization. In addition, to direct staggered assembly of peptides and to foster fibrillogenesisthat is, as opposed to blunt-ended discrete speciesthe terminal quarters of the peptide are cationic and the central half anionic with lysine and glutamate, respectively, at core-flanking e and g positions. This +,?,?,+ arrangement gives the peptide its name, MagicWand (MW). As judged by circular dichroism (CD) spectra, MW assembles to ?-helical structures in the sub-micromolar range and above. The thermal unfolding of MW is reversible with a melting temperature >70 °C at 100 ?M peptide concentration. Negative-stain transmission electron microscopy (TEM) of MW assemblies reveals stiff, straight, fibrous rods that extended for tens of microns. Moreover, different stains highlight considerable order both perpendicular and parallel to the fiber long axis. The dimensions of these features are consistent with bundles of long, straight coiled ?-helical coiled coils with their axes aligned parallel to the long axis of the fibers. The fiber thickening indicates inter-coiled-coil interactions. Mutagenesis of the outer surface of the peptidei.e., at the b and f positionscombined with stability and microscopy measurements, highlights the role of electrostatic and cation?? interactions in driving fiber formation, stability and thickening. These findings are discussed in the context of the growing number of self-assembling peptide-based fibrous systems
De Novo-Designed α-Helical Barrels as Receptors for Small Molecules
No full textWe describe de novo-designed α-helical barrels (αHBs) that bind and discriminate between lipophilic biologically active molecules. αHBs have five or more α-helices arranged around central hydrophobic channels the diameters of which scale with oligomer state. We show that pentameric, hexameric, and heptameric αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene (DPH) in the micromolar range and fluoresce. Displacement of the dye is used to report the binding of nonfluorescent molecules: palmitic acid and retinol bind to all three αHBs with submicromolar inhibitor constants; farnesol binds the hexamer and heptamer; but β-carotene binds only the heptamer. A co-crystal structure of the hexamer with farnesol reveals oriented binding in the center of the hydrophobic channel. Charged side chains engineered into the lumen of the heptamer facilitate binding of polar ligands: a glutamate variant binds a cationic variant of DPH, and introducing lysine allows binding of the biosynthetically important farnesol diphosphate.</p
De novo coiled-coil peptides as scaffolds for disrupting protein-protein interactions
No full textProtein-protein interactions (PPIs) play pivotal roles in the majority of biological processes. Therefore, improved approaches to target and disrupt PPIs would provide tools for chemical biology and leads for therapeutic development. PPIs with α-helical components are appealing targets given that the secondary structure is well understood and can be mimicked or stabilised to render small-molecule and constrained-peptide-based inhibitors. Here we present a strategy to target α-helix-mediated PPIs that exploits de novo coiled-coil assemblies and test this using the MCL-1/NOXA-B PPI. First, computational alanine scanning is used to identify key α-helical residues from NOXA-B that contribute to the interface. Next, these residues are grafted onto the exposed surfaces of de novo designed homodimeric or heterodimeric coiled-coil peptides. The resulting synthetic peptides selectively inhibit a cognate MCL-1/BID complex in the mid-nM range. Furthermore, the heterodimeric system affords control as inhibition occurs only when both the grafted peptide and its designed partner are present. This establishes proof of concept for exploiting peptides stabilised in de novo coiled coils as inhibitors of PPIs. This dependence on supramolecular assembly introduces new possibilities for regulation and control
Coiled-Coil Design:Updated and Upgraded
No full textα-Helical coiled coils are ubiquitous protein-folding and protein-interaction domains in which two or more α-helical chains come together to form bundles. Through a combination of bioinformatics analysis of many thousands of natural coiled-coil sequences and structures, plus empirical protein engineering and design studies, there is now a deep understanding of the sequence-to-structure relationships for this class of protein architecture. This has led to considerable success in rational design and what might be termed in biro de novo design of simple coiled coils, which include homo- and hetero-meric parallel dimers, trimers and tetramers. In turn, these provide a toolkit for directing the assembly of both natural proteins and more-complex designs in protein engineering, materials science and synthetic biology. Moving on, the increased and improved use of computational design is allowing access to coiled-coil structures that are rare or even not observed in nature, for example α-helical barrels, which comprise five or more α-helices and have central channels into which different functions may be ported. This chapter reviews all of these advances, outlining improvements in our knowledge of the fundamentals of coiled-coil folding and assembly, and highlighting new coiled coil-based materials and applications that this new understanding is opening up. Despite considerable progress, however, challenges remain in coiled-coil design, and the next decade promises to be as productive and exciting as the last
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