Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM.

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.

Identification of General Anesthetic Target Protein-Binding Sites by Photoaffinity Labeling and Mass Spectrometry.

General anesthetics are unique in that they represent a diverse range of chemical structures. Therefore, it is not surprising that the desired and undesired molecular targets, and binding sites therein, are as equally diverse and unique. Photoaffinity labeling has proven to be a valuable strategy for the identification of anesthetic molecular targets, as well as binding sites within those targets. In combination with the advances in mass spectrometry-based proteomics, along with the ability to comprehensively map posttranslational modifications, the method is likely to undergo continued improvement. Here, we provide the fundamentals for the design and development of an anesthetic photolabel. We also outline a protocol for the identification of photolabeled residues by mass spectrometry. The major steps include the photolabeling experiment, sample preparation, high-resolution mass spectrometry, and data analysis. The protocol can be used as a foundation for further optimization for the specific protein of interest and conditions of an experiment. The use of photoaffinity labeling adds an advantageous alternative and/or complementary approach to increase understanding of anesthetic molecular mechanisms.

Common general anesthetic propofol impairs kinesin processivity.

Propofol is the most widely used i.v. general anesthetic to induce and maintain anesthesia. It is now recognized that this small molecule influences ligand-gated channels, including the GABAA receptor and others. Specific propofol binding sites have been mapped using photoaffinity ligands and mutagenesis; however, their precise target interaction profiles fail to provide complete mechanistic underpinnings for the anesthetic state. These results suggest that propofol and other common anesthetics, such as etomidate and ketamine, may target additional protein networks of the CNS to contribute to the desired and undesired anesthesia end points. Some evidence for anesthetic interactions with the cytoskeleton exists, but the molecular motors have received no attention as anesthetic targets. We have recently discovered that propofol inhibits conventional kinesin-1 KIF5B and kinesin-2 KIF3AB and KIF3AC, causing a significant reduction in the distances that these processive kinesins can travel. These microtubule-based motors are highly expressed in the CNS and the major anterograde transporters of cargos, such as mitochondria, synaptic vesicle precursors, neurotransmitter receptors, cell signaling and adhesion molecules, and ciliary intraflagellar transport particles. The single-molecule results presented show that the kinesin processive stepping distance decreases 40-60% with EC50 values <100 nM propofol without an effect on velocity. The lack of a velocity effect suggests that propofol is not binding at the ATP site or allosteric sites that modulate microtubule-activated ATP turnover. Rather, we propose that a transient propofol allosteric site forms when the motor head binds to the microtubule during stepping.

Metal speciation in health and medicine represented by iron and vanadium.

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.

Gel formulation containing mixed surfactant and lipids associating with carboplatin.

The interaction of amphiphilic molecules such as lipids and surfactants with the hydrophilic drug carboplatin was investigated to identify suitable self-assembling components for a potential gel-based delivery formulation. (1) H-NMR Studies in sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT, AOT)-based reverse micelles show that carboplatin associates and at least partially penetrates the surfactant interface. Langmuir monolayers formed by dipalmitoyl(phosphatidyl)choline are penetrated by carboplatin. Carboplatin was found to also penetrate the more rigid monolayers containing cholesterol. A combined mixed surfactant gel formulation containing carboplatin and cholesterol for lymphatic tissue targeting was investigated for the intracavitary treatment of cancer. This formulation consists of a blend of the surfactants lecithin and AOT (1 : 3 ratio), an oil phase of isopropyl myristate, and an aqueous component. The phases of the system were defined within a pseudo-ternary phase diagram. At low oil content, this formulation produces a gel-like system over a wide range of H(2) O content. The carboplatin release from the formulation displays a prolonged discharge with a rate three to five times slower than that of the control. Rheological properties of the formulation exhibit pseudoplastic behavior. Microemulsion and Langmuir monolayer studies support the interactions between carboplatin and amphiphilic components used in this formulation. To target delivery of carboplatin, two formulations containing cholesterol were characterized. These two formulations with cholesterol showed that, although cholesterol does little to alter the phases in the pseudo-ternary system or to increase the initial release of the drug, it contributes significantly to the structure of the formulation under physiological temperature, as well as increases the rate of steady-state discharge of carboplatin.