Protein–protein
interactions are ubiquitous to all domains of life and have gained
recent interest as drug targets. However, many current methods to
study protein–protein interactions can be costly and are low-throughput.
Here, we demonstrate a solvatochromic tool based on the natural post-translational
modification of the Escherichia coli acyl carrier
protein (EcACP) used to visualize protein–protein interactions
between EcACP and 13 different partner enzymes from several biosynthetic
pathways. We use this tool to confirm proposed interactions between
EcACP and both catalytic and regulatory proteins. We also show the
utility of this method toward detecting allosteric changes to partner
enzyme structure and the validation of active site inhibitors. We
anticipate the future adaptation of this assay into a high-throughput
screen for antibiotic discovery.
We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase–polymer–nucleotide complexes bound to clonal copies of DNA targets. These polymer–nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates. Avidity sequencing achieves high accuracy, with 96.2% and 85.4% of base calls having an average of one error per 1,000 and 10,000 base pairs, respectively. We show that the average error rate of avidity sequencing remained stable following a long homopolymer.
While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using a fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and non-ribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.
All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well.
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