Dataset Information

A Single Tool to Monitor Multiple Protein-Protein Interactions of the Escherichia coli Acyl Carrier Protein.

ABSTRACT: 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.

SUBMITTER: Charov K

PROVIDER: S-EPMC7607843 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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A Single Tool to Monitor Multiple Protein-Protein Interactions of the <i>Escherichia coli</i> Acyl Carrier Protein.

Charov Katherine K Burkart Michael D MD

ACS infectious diseases 20190730 9

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 <i>Escherichia coli</i> acyl carrier protein (EcACP) used to visualize protein-protein interactions between EcACP and 13 different partner enzymes from several biosynthetic pa ...[more]

PMID: 31317739

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Project description:Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.

Dataset Information

A Single Tool to Monitor Multiple Protein-Protein Interactions of the Escherichia coli Acyl Carrier Protein.

Publications

A Single Tool to Monitor Multiple Protein-Protein Interactions of the <i>Escherichia coli</i> Acyl Carrier Protein.

Similar Datasets

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