Biology subjects

Crawford, T.

Publications and source records attributed to Crawford, T..

3 recordsLinked to original sources

bioRxiv2022

Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation

Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery--either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Such enzymatic methods require careful optimisation of cyclisation over polymerisation. Here, we describe the engineering of self-cyclising "autocyclase" proteins, where an intramolecular rearrangement can be triggered to yield a monomeric cyclic product in high yields. We characterise the self-cyclisation reaction mechanism and demonstrate how the unimolecular reaction path can circumvent existing challenges of enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple and scalable way to access a vast diversity of macrocyclic biomolecules.

biochemistry↗

bioRxiv2022

Arabidopsis mutants representing each of the four Mediator modules reveal unique functions in the transcriptional response to salt stress

Changes in growth environment trigger stress responses in most organisms. The mechanisms mediating these responses are only partly understood and involve signaling pathways and transcription factors. Mediator is a conserved co-regulator complex required for transcriptional regulation of all eukaryotic protein-encoding genes. However, its function in abiotic stress responses is elusive. We here describe global gene expression changes triggered by salt stress in Arabidopsis. To explore the involvement of Mediator in salt stress response we characterized med9, med16, med18, and cdk8 mutants representing each of the four modules of Mediator. Our transcriptome data revealed enrichment of shared and specific cis-elements corresponding to unique transcription factors in promoters of mis-regulated genes for each mutant. We show that individual Mediator subunits interact with specific transcription factors to generate a transcriptional stress response and that the mutant phenotypes support the transcriptome data. med16 and med18, and to some extent cdk8, display defects in abscisic acid and anthocyanin metabolism and we identify signal molecules, transcription factors and target genes involved in these pathways as dysregulated in the Mediator mutants. Our results reveal how signals from different stress response pathways are dependent on and integrated by Mediator subunits to coordinate a functional response to salt stress.

genomics↗

bioRxiv2022

ScrepYard: an online resource for disulfide-stabilised tandem repeat peptides

Receptor avidity through multivalency is a highly sought-after property of ligands. While readily available in nature in the form of bivalent antibodies, this property remains challenging to engineer in synthetic molecules. The discovery of several bivalent venom peptides containing two homologous and independently folded domains (in a tandem repeat arrangement) has provided a unique opportunity to better understand the underpinning design of multivalency in multimeric biomolecules, as well as how naturally occurring multivalent ligands can be identified. In previous work we classified these molecules as a larger class termed secreted cysteine-rich repeat-proteins (SCREPs). Here, we present an online resource; ScrepYard, designed to assist researchers in identification of SCREP sequences of interest and to aid in characterizing this emerging class of biomolecules. Analysis of sequences within the ScrepYard reveals that two-domain tandem repeats constitute the most abundant SCREP domain architecture, while the interdomain "linker" regions connecting the ordered domains are found to be abundant in amino acids with short or polar sidechains and contain an unusually high abundance of proline residues. Finally, we demonstrate the utility of ScrepYard as a virtual screening tool for discovery of putatively multivalent peptides, by using it as a resource to identify a previously uncharacterised serine protease inhibitor and confirm its predicated activity using an enzyme assay.

bioinformatics↗