Better together: building protein oligomers naturally and by design

This is the final version. Available on open access from Portland Press via the DOI in this recordProtein oligomers are more common in nature than monomers, with dimers being the most prevalent final structural state observed in known structures. From a biological perspective, this makes sense as it conserves vital molecular resources that may be wasted simply by generating larger single polypeptide units, and allows new features such as cooperativity to emerge. Taking inspiration from nature, protein designers and engineers are now building artificial oligomeric complexes using a variety of approaches to generate new and useful supramolecular protein structures. Oligomerisation is thus offering a new approach to sample structure and function space not accessible through simply tinkering with monomeric proteins

Delta Flucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi‐rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an “omega‐loop” motif (T352‐G360) significantly enhanced activity, altered kinetics, reduced Km for D‐luciferin, altered emission colors, and altered substrate specificity for redshifted analog DL‐infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω‐loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox

Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon Methanothermus fervidus

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData availability statement: The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.rcsb.org/, PDB 80RK, PDB 80RU.The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals. The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallized and its structure solved to 1.7 Å resolution by experimental phasing. It has also been crystallized in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Å resolution. The enzyme structure has two domains, the core domain shares some structural similarity with other NTP-dependent enzymes. A significant proportion of the structure, including a 127 amino acid N-terminal domain, has no structural similarity to other known enzyme structures. The structure of the complex shows a large conformational change that occurs in the enzyme during catalytic turnover. The reaction involves the transfer of the γ-phosphate group from ATP to the substrate 2,3 -diphosphoglycerate and the subsequent SN2 attack to form a phosphoanhydride. This results in the production of the unusual extremolyte cyclic 2,3 -diphosphoglycerate which has important industrial applications.European Union Horizon 2020Biotechnology and Biological Sciences Research Council (BBSRC)University of ExeterEVONIK IndustriesGerman Federal Ministry of Education and Research (BMBF

Differential roles for ACBD4 and ACBD5 in peroxisome-ER interactions and lipid metabolism

This is the author accepted manuscript. The final version is available on open access from Elsevier via the DOI in this record Data availability: The research data supporting this publication are provided within this paper, or as supplementary information.Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. As well as collaborating in lipid metabolism, e.g. of very long chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work has identified tethering complexes on the ER and peroxisome membranes which connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane proteinassociated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme Abinding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4, and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remains unclear. Here, we address these questions, using a combination of molecular cell biology, biochemical and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal β-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of β-oxidation of VLCFAs. Finally, we observe interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.Biotechnology & Biological Sciences Research Council (BBSRC)UK Research and InnovationRoyal SocietyEuropean Union Horizon 2020Medical Research Council (MRC

Unveiling professional development: A critical review of stage models

In research across professions, the development of professional skill traditionally was seen as a process of accumulation of knowledge and skills, promoted by practical experience. More recently, this view has been modified to incorporate skillful know-how that is progressively acquired by passing through developmental stages, such as novice, competent, and expert. The authors of this article critically review contemporary stage models that are typically applied across professions. Their principal critique is that a focus on stages veils or conceals more fundamental aspects of professional skill development. On the basis of their critique, the authors propose an alternative model that builds on the strengths of previous models while seeking to overcome their main limitations. Finally, the authors outline the implications of their alternative model for professional education, workplace practices, and research on professional development

Site-Specific One-to-One Click Coupling of Single Proteins to Individual Carbon Nanotubes: A Single-Molecule Approach

We report the site-specific coupling of single proteins to individual carbon nanotubes (CNTs) in solution and with singlemolecule control. Using an orthogonal Click reaction, Green Fluorescent Protein (GFP) was engineered to contain a genetically encoded azide group and then bound to CNT ends in different configurations: in close proximity or at longer distances from the GFP’s functional centre. Atomic force microscopy and fluorescence analysis in solution and on surfaces at the single-protein level confirmed the importance of bioengineering optimal protein attachment sites to achieve direct protein-nanotube communication and bridging