Combining experiment and energy landscapes to explore anaerobic heme breakdown in multifunctional hemoproteins.

To survive, many pathogens extract heme from their host organism and break down the porphyrin scaffold to sequester the Fe2+ ion via a heme oxygenase. Recent studies have revealed that certain pathogens can anaerobically degrade heme. Our own research has shown that one such pathway proceeds via NADH-dependent heme degradation, which has been identified in a family of hemoproteins from a range of bacteria. HemS, from Yersinia enterocolitica, is the main focus of this work, along with HmuS (Yersinia pestis), ChuS (Escherichia coli) and ShuS (Shigella dysenteriae). We combine experiments, Energy Landscape Theory, and a bioinformatic investigation to place these homologues within a wider phylogenetic context. A subset of these hemoproteins are known to bind certain DNA promoter regions, suggesting not only that they can catalytically degrade heme, but that they are also involved in transcriptional modulation responding to heme flux. Many of the bacterial species responsible for these hemoproteins (including those that produce HemS, ChuS and ShuS) are known to specifically target oxygen-depleted regions of the gastrointestinal tract. A deeper understanding of anaerobic heme breakdown processes exploited by these pathogens could therefore prove useful in the development of future strategies for disease prevention.

Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation without Magnetic Beads.

Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilization onto beads, risks losses during wash steps, and exhibits losses and greater costs at higher protein inputs. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced protein aggregates by brief centrifugation rather than magnetism─with optional low-cost inert glass beads to simplify handling. SP4 recovered equivalent or greater protein yields for 1-5000 µg preparations and improved reproducibility (median protein R2 0.99 (SP4) vs 0.97 (SP3)). Deep proteome profiling revealed that SP4 yielded a greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80 vs 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs across eight sample types and five lysis buffers─all confirming equivalent or improved proteome characterization vs SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein cleanup and identifies that magnetic capture risks losses, especially at higher protein concentrations and among more hydrophobic proteins. SP4 offers a minimalistic approach to protein cleanup that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications─all while retaining the speed and compatibility of SP3.

Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation.

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII (η6 -arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19 F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.

Targeting the Shc-EGFR interaction with indomethacin inhibits MAP kinase pathway signalling.

Receptor tyrosine kinase (RTK)-mediated hyperactivation of the MAPK/Erk pathway is responsible for a large number of pathogenic outcomes including many cancers. Considerable effort has been directed at targeting this pathway with varying degrees of long term therapeutic success. Under non-stimulated conditions Erk is bound to the adaptor protein Shc preventing aberrant signalling by sequestering Erk from activation by Mek. Activated RTK recruits Shc, via its phosphotyrosine binding (PTB) domain (ShcPTB), precipitating the release of Erk to engage in a signalling response. Here we describe a novel approach to inhibition of MAP kinase signal transduction through attempting to preserve the Shc-Erk complex under conditions of activated receptor. A library of existing drug molecules was computationally screened for hits that would bind to the ShcPTB and block its interaction with the RTKs EGFR and ErbB2. The primary hit from the screen was indomethacin, a non-steroidal anti-inflammatory drug. Validation of this molecule in vitro and in cellular efficacy studies in cancer cells provides proof of principle of the approach to pathway down-regulation and a potential optimizable lead compound.

Use of a fluorinated probe to quantitatively monitor amino acid binding preferences of ruthenium(ii) arene complexes.

In order to address outstanding questions about ruthenium complexes in complex biological solutions, 19F NMR spectroscopy was used to follow the binding preferences between fluorinated RuII(η6-arene)(bipyridine) complexes and protected amino acids and glutathione. Reporting what ruthenium compounds bind to in complex environments has so far been restricted to relatively qualitative methods, such as mass spectrometry and X-ray spectroscopic methods; however, quantitative information on the species present in the solution phase cannot be inferred from these techniques. Furthermore, using 1H NMR, in water, to distinguish and monitor a number of different complex RuII(η6-arene) adducts forming is challenging. Incorporating an NMR active heteroatom into ruthenium organometallic complexes provides a quantitative, diagnostic 'fingerprint' to track solution-phase behaviour and allow for unambiguous assignment of any given adduct. The resulting 19F NMR spectra show for the first time the varied, dynamic behaviour of organoruthenium compounds when exposed to simple biomolecules in complex mixtures. The rates of formation of the different observed species are dramatically influenced by the electronic properties at the metal, even in a closely related series of complexes in which only the electron-donating properties of the arene ligand are altered. Preference for cysteine binding is absolute: the first quantitative solution-phase evidence of such behaviour.