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Structure-function investigation of substrate binding proteins from Synechococcus sp.
thesisposted on 2022-03-28, 16:47 authored by Benjamin Alexander Ford
Synechococcus is a ubiquitous genus of photosynthetic marine bacteria, and vital for primary production and nutrient cycling in global oceans. Genomic annotation for this genus has progressed faster than the functional characterisation of predicted proteins. Consequently, the veracity of predicted functions for many genes is questionable. This is particularly so for the substrate-binding protein (SBP) family, integral to nutrient import via ATP-binding cassettes, which display unusually low sequence homology (despite a highly conserved structural fold). Lack of validation concerning the natural ligands of SBP proteins impacts on our depth of understanding of the function and utility of cellular import machinery in these highly prevalent marine microorganisms. A panel of nineteen SBPs annotated to bind saccharides across representative Synechococcus species has been selected for in vitro characterisation. Two alternative formats for high-throughput production were trialled, and the yield and percentage of soluble protein products compared. Recombinant forms of three SBPs have been produced in sufficient quantities to enable ligand screening using differential scanning fluorimetry and biophysical characterisation in solution. The approach undertaken has demonstrated potential to complement genomic data for previously uncharacterised SBPs