Molecular insights into the mechanisms of transport and energy coupling in membrane transport proteins

Membrane transport proteins are the main gatekeepers controlling the traffic of molecules in and out the cell. The mechanism by which they mediate selective and regulated transport across the membrane is of broad physiological and biophysical relevance. In this dissertation, several critical aspects of the transport process have been studied through molecular dynamics (MD) simulations, including ion binding and its coupling to chemical processes such as H+ transport, translocation of the transported substrate and cotransported ions, dynamics of the catalytic site, coordinated motions of the remote regions, as well as other molecular events facilitating the transport of the cargo. The first part of the dissertation covers topics on a Cl-/H+ transporter from the CLC superfamily, which catalyzes stoichiometrically coupled exchange of Cl- and H+ across biological membranes. CLC transporters exchange H+ for halides and certain polyatomic anions, but exclude cations, F-, and larger physiological anions, such as PO4^3- and SO4^2-. Despite comparable transport rates of different anions, the H+ coupling in CLC transporters varies significantly depending on the chemical nature of the transported anion. Although the molecular mechanism of exchange remains unknown, studies on bacterial ClC-ec1 transporter have revealed that Cl- binding to the central anion-binding site is crucial for the anion-coupled H+ transport. This study shows that Cl-, F-, NO3-, and SCN- display distinct binding coordinations at the central site and are hydrated in different manners. Consistent with the observation of differential bindings, ClC-ec1 exhibits markedly variable ability to support the formation of the transient water wires, which are necessary to support the connection of the two H+ transfer sites (Gluin and Gluex), in the presence of different anions. These findings provide structural details of anion binding in ClC-ec1 and reveal a putative atomic-level mechanism for the decoupling of H+ transport to the transport of anions other than Cl-. Another important question concerning the functional mechanism of CLC transporters is that no large conformational change have been detected crystallographically, even though transporters usually undergo global conformational change to alternately expose substrate-binding sites to opposite sides of the membrane. The collaborative work here demonstrates the formation of a previously uncharacterized `outward-facing open' state enrich by high H+ concentration, which involves global structural changes ~20 A away from the outer gate. This long distance conformational change highlights the coupled motions as well as the relevance of global structural changes in CLC transport cycle. The second part of the dissertation focus on a phospholipid scramblase which mediates rapid transbilayer redistribution (scrambling) of phospholipids at plasma membrane. This process dissipates lipid asymmetry in response to signals for critical cellular events like apoptosis that elevate cytoplasmic Ca^2+ concentration. The work here shows that the hydrophilic aqueduct on the surface of the fungal scramblase nhTMEM16 serves as the path for lipid translocation, and that Ca$^{2+}$ binding plays a key role in determining an open conformation of the path for lipid diffusion. The fully occupied lipid track connects the inner and outer leaflets and forms a “proteolipidic” pore, which allows ion conduction through the aqueous pathway formed between the protein and lipid headgroups under transmembrane electric potentials. Supporting this mechanism, site-specific mutagenesis experiments show that nhTMEM16 ionic currents are synergistically linked to phospholipid scrambling. To further validate the idea that ions permeate through TMEM16s via the same structural pathway taken by phospholipids, two specific residues in the pore region were pinpointed, which are able to convert TMEM16A Ca^2+-activated Cl- channel (CaCC) into robust scramblase upon point mutations. This novel view of flexible pore structure explains a number of unusual features of the TMEM16 ionic currents, especially the highly variable ionic selectivity and the ability to permeate large ions, which also provides crucial information on the functional dichotomy in TMEM16s.Ope

The effect of water dynamics on conformation changes of albumin in pre-denaturation state:photon correlation spectroscopy and simulation

Water is essential for protein three-dimensional structure, conformational dynamics, and activity. Human serum albumin (HSA) is one of major blood plasma proteins, and its functioning is fundamentally determined by the dynamics of surrounding water. The goal of this study is to link the conformational dynamics of albumin to the thermal motions in water taking place in the physiological temperature range. We report the results of photon correlation spectroscopy and molecular dynamics simulations of HSA in aqueous solution. The experimental data processing produced the temperature dependence of the HSA hydrodynamic radius and its zeta potential. Molecular dynamics reproduced the results of experiments and revealed changes in the secondary structure caused by the destruction of hydrogen bonds in the macromolecule's globule

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity

Radiolabeled Compounds for Diagnosis and Treatment of Cancer

Radiopharmaceuticals are used in the diagnosis and treatment of various diseases, especially cancer. In general, radiopharmaceuticals are either salts of radionuclides or radionuclides bound to biologically active molecules, drugs, or cells. Tremendous progress has been made in discovering, developing, and commercializing numerous radiopharmaceuticals for the imaging and therapy of cancer. Significant research is ongoing in academia and the pharmaceutical industry to develop more novel radiolabeled compounds as potential radiopharmaceuticals for unmet needs. This Special Issue aims to focus on all aspects of the design, characterization, evaluation, and development of novel radiolabeled compounds for the diagnosis and treatment of cancer and the application of new radiochemistry and methodologies for the development of novel radiolabeled compounds. Outstanding contributions presented in this Special Issue will significantly add to the field of radiopharmaceuticals

Safety and Reliability - Safe Societies in a Changing World

The contributions cover a wide range of methodologies and application areas for safety and reliability that contribute to safe societies in a changing world. These methodologies and applications include: - foundations of risk and reliability assessment and management - mathematical methods in reliability and safety - risk assessment - risk management - system reliability - uncertainty analysis - digitalization and big data - prognostics and system health management - occupational safety - accident and incident modeling - maintenance modeling and applications - simulation for safety and reliability analysis - dynamic risk and barrier management - organizational factors and safety culture - human factors and human reliability - resilience engineering - structural reliability - natural hazards - security - economic analysis in risk managemen