654 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
A critical edition of Derek Walcott's Omeros
No full textThe thesis is a Critical Edition of Derek Walcott’s Omeros, consisting of a Critical
Introduction and Annotations. The Critical Introduction analyses:
- Narrative
- Settings
- Metaphor and Paronomasia
- Symbolism
- Historiography
- Intertexts
- Dualism
- Autobiography
- Dialects
- Prosody.
The Annotations comment on more than 1000 references that may be obscure and on
specifics of narrative, language and prosody.
This study presents new conclusions about some aspects of Omeros:
- It challenges the prevailing view that the work is written substantially in a
variation of terza rima and shows that regular quatrains predominate.
- It demonstrates ways in which the metrics follow the sense of the narrative and
takes a more balanced position on the use of Caribbean as opposed to classical
metrics than that put forward previously.
- It identifies a paragraphic structure to the verse.
- It proposes a new prosodic structure for the significant Chapter XXX/iii.
- It extends Walcott’s recognised use of numerology into word counting the
names of characters.
- It develops the idea of Walcott’s dualism and his use of pairing and
contradiction as a dialectical method.
- It defines his wide use of paronomasia and shows that many of the puns have a
metaphorical aspect beyond mere word-play.
- It analyses some of Walcott’s symbolism.
- It identifies intertextual links to his earlier works and to some thirty other
writers, and suggests homage to Hemingway and possibly Heaney.
- It provides the first complete analysis of Walcott’s rhyme types in Omeros.
In its analysis of Omeros and in the Annotations it has included commentary from
across the critical literature, to provide some sense of other views on Walcott’s
writing, and has included as many as possible of Walcott’s own comments on Omeros
and on the writer’s task, as a background to understanding the poem
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
Derek Price’s Puzzles: Numerical Metaphors for the Operation of Science
No full textTwo of Derek Price’s scientometric models are examined-one showing bibliographic references snrong papers in the self-contsined speciafty dealing with N-rays, the nther showing exponential growth of the scientific literature, The author compares his own observations on the aging of the literature with those of %Ice, concluding that the rapid aging of scientific knowledge may he a relatively recent phenomenon, Also examined are the effects of the world wars on scientific growth
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