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Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Genetic code expansion in Shewanella oneidensis MR-1 allows site-specific incorporation of bioorthogonal functional groups into a c-type cytochrome Colin W. J. Lockwood , Benjamin W. Nash , Simone E. Newton-Payne , Jessica H. Van Wonderen , Keir P. S. Whiting , Abigail Connolly , Alexander L. Sutton-Cook , Archie Crook , Advait R. Aithal , Marcus J. Edwards , Thomas A. Clarke , Amit Sachdeva , Julea N. Butt Acs Synthetic Biology DOI: 10.1021/acssynbio.4c00248 Diamond Proposal Number(s): [25108, 32728] Open Access Show Abstract ▼ Abstract: Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow the understanding and modulation of biological systems and engineer new biotechnology. Here, we report the development of efficient methods for site-specific incorporation of structurally diverse noncanonical amino acids (ncAAs) into proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the biosynthetic machinery for ncAA incorporation is compatible and orthogonal to the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed the efficient synthesis of a c-type cytochrome, MtrC, containing site-specifically incorporated ncAA in S. oneidensis MR-1 cells. We demonstrate that site-specific replacement of surface residues in MtrC with ncAAs does not influence its three-dimensional structure and redox properties. We also demonstrate that site-specifically incorporated bioorthogonal functional groups could be used for efficient site-selective labeling of MtrC with fluorophores. These synthetic biology developments pave the way to expand the chemical repertoire of designer proteins expressed in S. oneidensis MR-1.	Aug 2024
I04-Macromolecular Crystallography	Design and selection of heterodimerizing helical hairpins for synthetic biology Abigail J. Smith , Elise A. Naudin , Caitlin L. Edgell , Emily G. Baker , Bram Mylemans , Laura Fitzpatrick , Andrew Herman , Helen M. Rice , David M. Andrews , Natalie Tigue , Derek N. Woolfson , Nigel J. Savery Acs Synthetic Biology DOI: 10.1021/acssynbio.3c00231 Diamond Proposal Number(s): [23269] Open Access Show Abstract ▼ Abstract: Synthetic biology applications would benefit from protein modules of reduced complexity that function orthogonally to cellular components. As many subcellular processes depend on peptide–protein or protein–protein interactions, de novo designed polypeptides that can bring together other proteins controllably are particularly useful. Thanks to established sequence-to-structure relationships, helical bundles provide good starting points for such designs. Typically, however, such designs are tested in vitro and function in cells is not guaranteed. Here, we describe the design, characterization, and application of de novo helical hairpins that heterodimerize to form 4-helix bundles in cells. Starting from a rationally designed homodimer, we construct a library of helical hairpins and identify complementary pairs using bimolecular fluorescence complementation in E. coli. We characterize some of the pairs using biophysics and X-ray crystallography to confirm heterodimeric 4-helix bundles. Finally, we demonstrate the function of an exemplar pair in regulating transcription in both E. coli and mammalian cells.	May 2023
I04-1-Macromolecular Crystallography (fixed wavelength)	De novo-designed α-helical barrels as receptors for small molecules Franziska Thomas , William M. Dawson , Eric J. M. Lang , Antony J. Burton , Gail J. Bartlett , Guto G. Rhys , Adrian J. Mulholland , Derek N. Woolfson Acs Synthetic Biology DOI: 10.1021/acssynbio.8b00225 Show Abstract ▼ Abstract: We 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.	Jul 2018
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology Jordan Fletcher , Aimee L. Boyle , Marc Bruning , Gail J. Bartlett , Thomas L. Vincent , Nathan Zaccai , Craig T. Armstrong , Elizabeth H. C. Bromley , Paula J. Booth , Leo Brady , Andrew Thomson , Derek N. Woolfson Acs Synthetic Biology 1 (6), 240 - 250 DOI: 10.1021/sb300028q PMID: 23651206 Diamond Proposal Number(s): [7131] Show Abstract ▼ Abstract: Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.	Jun 2012

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