The influence of metals in biology has become more and more apparent within the past century. Metal ions perform essential roles as critical scaffolds for structure and as catalysts in reactions. Speciation is a key concept that assists researchers in investigating processes that involve metal ions. However, translation of the essential area across scientific fields has been plagued by language discrepancies. To rectify this, the IUPAC Commission provided a framework in which speciation is defined as the distribution of species. Despite these attempts, contributions from inorganic chemists to the area of speciation have not fully materialized in part because the past decade's contributions focused on technological advances, which are not yet to the stage of measuring speciation distribution in biological solutions. In the following, we describe how speciation influences the area of metals in medicine and how speciation distribution has been characterized so far. We provide two case studies as an illustration, namely, vanadium and iron. Vanadium both has therapeutic importance and is known as a cofactor for metalloenzymes. In addition to being a cation, vanadium(V) has analogy with phosphorus and as such is a potent inhibitor for phosphorylases. Because speciation can change the metal's existence in cationic or anionic forms, speciation has profound effects on biological systems. We also highlight how speciation impacts iron metabolism, focusing on the rather low abundance of biologically relevant iron cation that actually exists in biological fluids. fluids. Furthermore, we point to recent investigations into the mechanism of Fenton chemistry, and that the emerging results show pH dependence. The studies suggest formation of Fe(IV)-intermediates and that the generally accepted mechanism may only apply at low pH. With broader recognition toward biological speciation, we are confident that future investigations on metal-based systems will progress faster and with significant results. Studying metal complexes to explore the properties of a potential "active species" and further uncovering the details associated with their specific composition and geometry are likely to be important to the action.

Ca2+-release channels are giant membrane proteins that control the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. The two members, ryanodine receptors (RyRs) and inositol-1,4,5-trisphosphate Receptors (IP3Rs), are evolutionarily related and are both activated by cytosolic Ca2+. They share a common architecture, but RyRs have evolved additional modules in the cytosolic region. Their massive size allows for the regulation by tens of proteins and small molecules, which can affect the opening and closing of the channels. In addition to Ca2+, other major triggers include IP3 for the IP3Rs, and depolarization of the plasma membrane for a particular RyR subtype. Their size has made them popular targets for study via electron microscopic methods, with current structures culminating near 3Å. The available structures have provided many new mechanistic insights int the binding of auxiliary proteins and small molecules, how these can regulate channel opening, and the mechanisms of disease-associated mutations. They also help scrutinize previously proposed binding sites, as some of these are now incompatible with the structures. Many questions remain around the structural effects of post-translational modifications, additional binding partners, and the higher-order complexes these channels can make in situ. This review summarizes our current knowledge about the structures of Ca2+-release channels and how this informs on their function.

Propofol is a widely used intravenous general anesthetic. We synthesized 2-fluoro-1,3-diisopropylbenzene, a compound that we call “fropofol”, to directly assess the significance of the propofol 1-hydroxyl for pharmacologically relevant molecular recognition in vitro and for anesthetic efficacy in vivo. Compared to propofol, fropofol had a similar molecular volume and only a small increase in hydrophobicity. Isothermal titration calorimetry and competition assays revealed that fropofol had higher affinity for a protein site governed largely by van der Waals interactions. Within another protein model containing hydrogen bond interactions, propofol demonstrated higher affinity. In vivo, fropofol demonstrated no anesthetic efficacy, but at high concentrations produced excitatory activity in tadpoles and mice; fropofol also antagonized propofol-induced hypnosis. In a propofol protein target that contributes to hypnosis, α1β2γ2L GABAA receptors, fropofol demonstrated no significant effect alone or on propofol positive allosteric modulation of the ion channel, suggesting an additional requirement for the 1-hydroxyl within synaptic GABAA receptor site(s). However, fropofol caused similar adverse cardiovascular effects as propofol by a dose-dependent depression of myocardial contractility. Our results directly implicate the propofol 1-hydroxyl as contributing to molecular recognition within protein targets leading to hypnosis, but not necessarily within protein targets leading to side effects of the drug.

Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity.