Rationally seeded computational protein design of ɑ-helical barrels

Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by 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. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression in Escherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy

Studies on protein folds and folding

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Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a Gram-negative bacterium.

Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in Gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for Gram-negative Qnr proteins. The AhQnr crystal structure, the first for any Gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of Gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A ‘tower’ and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in Gram-negative bacteria

De novo designed peptides for cellular delivery and subcellular localisation

Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes

Construction and Characterization of Kilobasepair Densely Labeled Peptide-DNA

Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (similar to 1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (similar to 1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology