Stephen J. Watt

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

An Escherichia coli cell‐free transcription/translation system was used to explore the high‐level incorporation of l‐3,4‐dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI‐MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15N‐HSQC NMR spectra. A redox‐staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell‐free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome‐mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High‐yield cell‐free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

Binding studies of nNOS-active amphibian peptides and Ca2+ calmodulin, using negative ion electrospray ionisation mass spectrometry

Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin/peptide/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ pump C20W (LRRGQILWFRGLNRIQTQIK-OH) with CaM has been shown by previous two-dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C-terminal end of CaM. Under identical conditions to those used for the amphibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied amphibian peptides requires Ca2+CaM to contain its full complement of four Ca2+ ions, this indicates that the amphibian peptides require both ends of the CaM to effect complex formation. Charge-state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca2+CaM undergoing a conformational change to a more compact structure.

Proteomic dissection of DNA polymerization

DNA polymerases replicate the genome by associating with a range of other proteins that enable rapid, high-fidelity copying of DNA. This complex of proteins and nucleic acids is termed the replisome. Proteins of the replisome must interact with other networks of proteins, such as those involved in DNA repair. Many of the proteins involved in DNA polymerization and the accessory proteins are known, but the array of proteins they interact with, and the spatial and temporal arrangement of these interactions, are current research topics. Mass spectrometry is a technique that can be used to identify the sites of these interactions and to determine the precise stoichiometries of binding partners in a functional complex. A complete understanding of the macromolecular interactions involved in DNA replication and repair may lead to discovery of new targets for antibiotics against bacteria and biomarkers for diagnosis of diseases, such as cancer, in humans.