Total Synthesis of Dansylated Park's Nucleotide for High-Throughput MraY Assays.

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence-based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high-throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high-throughput MraY assays.

Molecular anatomy of a trafficking organelle.

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.