The deazaflavin cofactor F420 is involved in a range of important redox reactions in bacteria and archaea. However, there are aspects of the F420 biosynthetic pathway that remain unclear. This work presents a revised biosynthetic pathway for F420, showing that phosphoenolpyruvate, rather than 2-phospho-L-lactate, is the key intermediate during the biosynthesis of F420. A range of techniques, including Mass Spec and X-ray crystallography were used to gain a better understanding of the enzymes and intermediates in this pathway.
Importantly, this information allowed us to heterologously express a functional F420 biosynthetic pathway in E. coli at levels comparable to native F420-producing organisms.
This collaborative piece of work was published in Nature Communications. Well done to James, Ghader and all those involved!
See it here.
Rational protein engineering efforts normally focus on altering parts of proteins that are directly involved in function – e.g. active sites or ligand binding sites. But changes to residues that are remote from these sites can have a large impact on protein structure, dynamics and function. In this review, we discuss recent literature that reports, and rationalises, the successful engineering of proteins at remote sites. As the use of protein technologies expands, exploiting the potential improvements made possible through modifying remote regions will become vital if we are to realise the full potential of protein engineering and design.
Great work Matt Wilding, Nansook and Matt Spence!
We recently collaborated on this piece of work from members of Max Cryle’s group (Monash/EMBL Australia). Using a range of techniques including protein X-ray crystallography, isothermal titration calorimetry and activity assays, this work characterises the structure and function of the C-terminal domain of the nonribosomal peptide synthetase Ebony. Results show that this C-terminal region encodes a eukaryotic example of an alternative type of nonribosomal peptide synethetase condensation domain, and experiments identified how this protein is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates.
You can find the paper here: https://www.pnas.org/content/116/8/2913.abstract
Well done to everyone that worked on this paper!
New eLIFE paper available online now! https://elifesciences.org/articles/40789
In this work, a collaboration with members of the Tokuriki (UBC) and Kamerlin (Upsalla University) groups, we performed directed evolution of four orthogous metallo-beta-lactamases towards a new function and found that different starting genotypes led to distinct evolutionary outcomes. We used a range of techniques, including directed evolution, enzyme kinetics, stability assays, X-ray crystallography and molecular dynamics to explore the differences in the structural and functional changes that occurred along each of these evolutionary pathways.
This work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution. The results from this work suggest that it may be effective to explore diverse initial genotypes when attempting to engineer or evolve new protein functions.
Congratulations to Nansook and all others that worked on this paper!
Our latest Current Opinion in Structural Biology review on the structural and evolutionary approaches to the design of fluorescence-based small-molecule biosensors is available online now (https://authors.elsevier.com/c/1YeAO_,2BdUwRJk).
In this paper, we outline current and emerging approaches for designing and optimizing genetically encoded small-molecule biosensors: using naturally-occurring sensory proteins as scaffolds for building sensors recognition domains, strategies for engineering and optimizing ligand specificity, creating novel biosensor architecture, and engineering fluorescent proteins with desirable properties to optimize biosensor output. We also discuss the importance of linker design in the overall process, and explore how computational and high-throughput methods are aiding biosensor design. Throughout the review we highlight several outstanding recent research articles that have used a combination of these techniques to produce novel genetically encoded small-molecule biosensors for applications in research, medicine and neuroscience.
A massive well done to Cassidy, who graduated last year with First Class Honours. We also found out that she can add “good at throwing mortarboards” to her long list of skills.
Well done Cassidy!
A massive congratulations to Dr Brendon Lee and Dr Eleanor Campbell, who celebrated their graduations at the end of 2018. They looked very nice and happy in their floppy hats. Smiles all around!
Brendon has taken up a post-doctoral position in the Jackson group and Eleanor has been working as a post-doc in the Hollfelder group at the University of Cambridge.
You can find some of Brendon’s work here: https://scholar.google.com/citations?hl=en&user=HiRpH44AAAAJ&view_op=list_works&sortby=pubdate
Find a list of Eleanor’s publications here: https://scholar.google.com.au/citations?user=py1VAe8AAAAJ&hl=en
In this work, recently published in the Journal of Biological Chemistry, we use a number of biochemical and structural analyses to show that a previously uncharacterised protein from Mycobacterium smegmatis acts as a flavin-sequestering protein that is required for survival during hypoxia. We show that this protein is a member of the flavin- and deazaflavin-dependent oxidoreductases (FDORs) and is distributed across mycobacterial species. X-ray crystallography revealed how FAD binds and showed no other substrate-binding cavities – consistent with its role as a flavin-sequestering protein. These findings present a new paradigm in mycobacterial adaptation to hypoxia.
This work was led by members of Greg Cook’s group (University of Otago/University of Auckland) and a collaboration with Trevor Rapson (CSIRO) and Chris Greening (Monash). Congratulations to all involved with this work.
Find the paper here.
Understanding how proteins gain new functions following gene duplication (i.e. neofunctionalisation) via structural changes is a key research theme in the Jackson group. In this paper, published recently in Insect Biochemistry and Molecular Biology, we use phylogenetic analysis, biochemical comparisons, and structural analysis to explore the evolutionary trajectories that link two Drosophila esterases.
This work was done in collaboration with members of John Oakeshott group (CSIRO). Congratulations to Davis and all those involved in this work!
Read the paper here
Read the paper here!
Cataracts, the clouding of the eye lens, is a leading cause of blindness and visual impairments worldwide. Cataracts form when oxidative stress in the lens causes lens proteins, such as crystallin, to destabilise and aggregate. The molecular basis for the oxidation-induced aggregation of these proteins, however, has remained elusive. In this paper, recently published in the Journal of Molecular Biology, we use X-ray crystallography and small-angle X-ray light scattering to describe the structure of a disulfide-linked dimer of human gammaS-crystallin. This disulfide-linked dimer is prone to forming aggregates and would likely be prevalent in aging eyes. These findings provide insight into how oxidative modification of crystallins contributes to cataract formation.
This work was a collaboration with, and led by, members of the Carver Group at the Australian National University. Congratulations to all involved!