Robust fully controlled nanometer liquid layers for high resolution liquid-cell electron microscopy.

Liquid cell electron microscopy (LCEM) has long suffered from irreproducibility and its inability to confer high-quality images over a wide field of view. LCEM demands the encapsulation of the in-liquid sample between two ultrathin membranes (windows). In the vacuum environment of the electron microscope, the windows bulge, drastically reducing the achievable resolution and the usable viewing region. Herein, we introduce a shape-engineered nanofluidic cell architecture and an air-free drop-casting sample loading technique, which combined, provide robust bulgeless imaging conditions. We demonstrate the capabilities of our stationary approach through the study of in-liquid model samples and quantitative measurements of the liquid layer thickness. The presented LCEM method confers high throughput, lattice resolution across the complete viewing window, and sufficient contrast for the observation of unstained liposomes, paving the way to high-resolution movies of biospecimens in their near native environment.

A plastic feedthrough suitable for high-voltage DC femtosecond electron diffractometers.

Highly energetic ultrashort electron bunches have the potential to reveal the ultrafast structural dynamics in relatively thicker in-liquid samples. However, direct current voltages higher than 100 kV are exponentially difficult to attain as surface and vacuum breakdown become an important problem as the electric field increases. One of the most demanding components in the design of a high-energy electrostatic ultrafast electron source is the high voltage feedthrough (HVFT), which must keep the electron gun from discharging against ground. Electrical discharges can cause irreversible component damage, while voltage instabilities render the instrument inoperative. We report the design, manufacturing, and conditioning process for a new HVFT that utilizes ultra-high molecular weight polyethylene as the insulating material. Our HVFT is highly customizable and inexpensive and has proven to be effective in high voltage applications. After a couple of weeks of gas and voltage conditioning, we achieved a maximum voltage of 180 kV with a progressively improved vacuum level of 1.8 × 10-8 Torr.

Trapping a Photoelectron behind a Repulsive Coulomb Barrier in Solution.

Multiply charged anions (MCAs) display unique photophysics and solvent-stabilizing effects. Well-known aqueous species such as SO42- and PO43- experience spontaneous electron detachment or charge-separation fragmentation in the gas phase owing to the strong Coulomb repulsion arising from the excess of negative charge. Thus, anions often present low photodetachment thresholds and the ability to quickly eject electrons into the solvent via charge-transfer-to-solvent (CTTS) states. Here, we report spectroscopic evidence for the existence of a repulsive Coulomb barrier (RCB) that blocks the ejection of "CTTS-like" electrons of the aqueous B12F122- dianion. Our spectroscopic experimental and theoretical studies indicate that despite the exerted Coulomb repulsion by the nascent radical monoanion B12F12-•aq, the photoexcited electron remains about the B12F12-• core. The RCB is an established feature of the potential energy landscape of MCAs in vacuo, which seems to extend to the liquid phase highlighting recent observations about the dielectric behavior of confined water.

High flow rate nanofluidics for in-liquid electron microscopy and diffraction.

We introduce a nanofluidic platform that can be used to carry out femtosecond electron diffraction (FED) and transmission electron microscopy (TEM) measurements in liquid samples or in-liquid specimens, respectively. The nanofluidic cell (NFC) system presented herein has been designed to withstand high sample refreshing rates (over one kilohertz), a prerequisite to succeed with FED experiments in our lab. Short beam paths, below 1 µm, in combination with ultrathin membranes (less than 100 nm thick) are necessary conditions for in-liquid FED and TEM studies due to the strongly interacting nature of electrons. Depending on the application, the beam path in our NFC can be tuned between 50 nm and 10 µm with ultrathin stoichiometric silicon nitride (Si3N4) windows as thin as 20 nm. Stoichiometric Si3N4 has been selected to reduce membrane bulging owing to its higher tensile stress and transparency in the UV-vis-NIR region to allow for laser excitation in FED experiments. Key design parameters and improvements made over previous NFC systems are discussed, and some preliminary electron images obtained by 200 kV scanning TEM are presented.

An aligned octahedral core in a nanocage: synthesis, plasmonic, and catalytic properties.

Plasmonic metal nanostructures with complex morphologies provide an important route to tunable optical responses and local electric field enhancement at the nanoscale for a variety of applications including sensing, imaging, and catalysis. Here we report a high-concentration synthesis of gold core-cage nanoparticles with a tethered and structurally aligned octahedral core and examine their plasmonic and catalytic properties. The obtained nanostructures exhibit a double band extinction in the visible-near infrared range and a large area electric field enhancement due to the unique structural features, as demonstrated using finite difference time domain (FDTD) simulations and confirmed experimentally using surface enhanced Raman scattering (SERS) tests. In addition, the obtained structures had a photoelectrochemical response useful for catalyzing the CO2 electroreduction reaction. Our work demonstrates the next generation of complex plasmonic nanostructures attainable via bottom-up synthesis and offers a variety of potential applications ranging from sensing to catalysis.

Tertiary and quaternary structural basis of oxygen affinity in human hemoglobin as revealed by multiscale simulations.

Human hemoglobin (Hb) is a benchmark protein of structural biology that shaped our view of allosterism over 60 years ago, with the introduction of the MWC model based on Perutz structures of the oxy(R) and deoxy(T) states and the more recent Tertiary Two-State model that proposed the existence of individual subunit states -"r" and "t"-, whose structure is yet unknown. Cooperative oxygen binding is essential for Hb function, and despite decades of research there are still open questions related to how tertiary and quaternary changes regulate oxygen affinity. In the present work, we have determined the free energy profiles of oxygen migration and for HisE7 gate opening, with QM/MM calculations of the oxygen binding energy in order to address the influence of tertiary differences in the control of oxygen affinity. Our results show that in the α subunit the low to high affinity transition is achieved by a proximal effect that mostly affects oxygen dissociation and is the driving force of the allosteric transition, while in the ß subunit the affinity change results from a complex interplay of proximal and distal effects, including an increase in the HE7 gate opening, that as shown by free energy profiles promotes oxygen uptake.

Tyrosine oxidation and nitration in transmembrane peptides is connected to lipid peroxidation.

Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.

Shaped cathodes for the production of ultra-short multi-electron pulses.

An electrostatic electron source design capable of producing sub-20 femtoseconds (rms) multi-electron pulses is presented. The photoelectron gun concept builds upon geometrical electric field enhancement at the cathode surface. Particle tracer simulations indicate the generation of extremely short bunches even beyond 40 cm of propagation. Comparisons with compact electron sources commonly used for femtosecond electron diffraction are made.

Mapping the protein-binding sites for iridium(iii)-based CO-releasing molecules.

A combination of mass spectrometry, Raman microspectroscopy, circular dichroism and X-ray crystallography has been used to obtain detailed information on the reaction of an iridium-based CO-releasing molecule (Ir-CORM), Cs2IrCl5CO, with a model protein, bovine pancreatic ribonuclease. The results show that Ir-compound fragments bind to the N-terminal amine and close to histidine and methionine side chains, and the CO ligand is retained for a long time. The data provide helpful information for identifying protein targets for Ir-CORMs and for studying the mechanism that allows them to exhibit their interesting biological properties.

Coarse-Grained Simulations of Heme Proteins: Validation and Study of Large Conformational Transitions.

Heme proteins are ubiquitous in nature and perform many diverse functions in all kingdoms of life. Many of these functions are related to large-scale conformational transitions and allosteric processes. Sampling of these large conformational changes is computationally very challenging. In this context, coarse-grain simulations emerge as an efficient approach to explore the conformational landscape. In this work, we present a coarse-grained model of the heme group and thoroughly validate this model in different benchmark examples, which include the monomeric heme proteins myoglobin and neuroglobin and the tetrameric human hemoglobin where we evaluated the method's ability to explore conformational changes (as the formation of hexacoordinated species) and allosteric transitions (as the well-known R → T transition). The obtained results are compared with atomistic molecular dynamics simulations. Overall, the results indicate that this approach conserves the essential dynamical information on different allosteric processes.

WATCLUST: a tool for improving the design of drugs based on protein-water interactions.

MOTIVATION: Water molecules are key players for protein folding and function. On the protein surface, water is not placed randomly, but display instead a particular structure evidenced by the presence of specific water sites (WS). These WS can be derived and characterized using explicit water Molecular Dynamics simulations, providing useful information for ligand binding prediction and design. Here we present WATCLUST, a WS determination and analysis tool running on the VMD platform. The tool also allows direct transfer of the WS information to Autodock program to perform biased docking. AVAILABILITY AND IMPLEMENTATION: The WATCLUST plugin and documentation are freely available at http://sbg.qb.fcen.uba.ar/watclust/. CONTACT: marcelo@qi.fcen.uba.ar, adrian@qi.fcen.uba.ar.

Fusion of raft-like lipid bilayers operated by a membranotropic domain of the HSV-type I glycoprotein gH occurs through a cholesterol-dependent mechanism.

A wealth of evidence indicates that lipid rafts are involved in the fusion of the viral lipid envelope with the target cell membrane. However, the interplay between these sterol- and sphingolipid-enriched ordered domains and viral fusion glycoproteins has not yet been clarified. In this work we investigate the molecular mechanism by which a membranotropic fragment of the glycoprotein gH of the Herpes Simplex Virus (HSV) type I (gH625) drives fusion of lipid bilayers formed by palmitoyl oleoyl phosphatidylcholine (POPC)-sphingomyelin (SM)-cholesterol (CHOL) (1 : 1 : 1 wt/wt/wt), focusing on the role played by each component. The comparative analysis of the liposome fusion assays, Dynamic Light Scattering (DLS), spectrofluorimetry, Neutron Reflectivity (NR) and Electron Spin Resonance (ESR) experiments, and Molecular Dynamics (MD) simulations shows that CHOL is fundamental for liposome fusion to occur. In detail, CHOL stabilizes the gH625-bilayer association by specific interactions with the peptide Trp residue. The interaction with gH625 causes an increased order of the lipid acyl chains, whose local rotational motion is significantly hampered. SM plays only a minor role in the process, favoring the propagation of lipid perturbation to the bilayer inner core. The stiffening of the peptide-interacting bilayer leaflet results in an asymmetric perturbation of the membrane, which is locally destabilized thus favoring fusion events. Our results show that viral fusion glycoproteins are optimally suited to exert a high fusogenic activity on lipid rafts and support the relevance of cholesterol as a key player of membrane-related processes.

Interaction between proteins and Ir based CO releasing molecules: mechanism of adduct formation and CO release.

Carbon monoxide releasing molecules (CORMs) have important bactericidal, anti-inflammatory, neuroprotective, and antiapoptotic effects and can be used as tools for CO physiology experiments, including studies on vasodilation. In this context, a new class of CO releasing molecules, based on pentachlorocarbonyliridate(III) derivative have been recently reported. Although there is a growing interest in the characterization of protein-CORMs interactions, only limited structural information on CORM binding to protein and CO release has been available to date. Here, we report six different crystal structures describing events ranging from CORM entrance into the protein crystal up to the CO release and a biophysical characterization by isothermal titration calorimetry, Raman microspectroscopy, and molecular dynamics simulations of the complex between a pentachlorocarbonyliridate(III) derivative and hen egg white lysozyme, a model protein. Altogether, the data indicate the formation of a complex in which the ligand can bind to different sites of the protein surface and provide clues on the mechanism of adduct formation and CO release.

Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: role of the CD loop and pre-A motif in electron cycling.

Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 µM/min(-1), which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly(48) may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10(-5) eV, which is lower than the value obtained for E. coli flavoHb (8.0 10(-4) eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function.

Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi iron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electron transfer.

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.

QM/MM study of the C-C coupling reaction mechanism of CYP121, an essential cytochrome p450 of Mycobacterium tuberculosis.

Among 20 p450s of Mycobacterium tuberculosis (Mt), CYP121 has received an outstanding interest, not only due to its essentiality for bacterial viability but also because it catalyzes an unusual carbon-carbon coupling reaction. Based on the structure of the substrate bound enzyme, several reaction mechanisms were proposed involving first Tyr radical formation, second Tyr radical formation, and C-C coupling. Key and unknown features, being the nature of the species that generate the first and second radicals, and the role played by the protein scaffold each step. In the present work we have used classical and quantum based computer simulation methods to study in detail its reaction mechanism. Our results show that substrate binding promotes formation of the initial oxy complex, Compound I is the responsible for first Tyr radical formation, and that the second Tyr radical is formed subsequently, through a PCET reaction, promoted by the presence of key residue Arg386. The final C-C coupling reaction possibly occurs in bulk solution, thus yielding the product in one oxygen reduction cycle. Our results thus contribute to a better comprehension of MtCYP121 reaction mechanism, with direct implications for inhibitor design, and also contribute to our general understanding of these type of enzymes.

The structure of the CD3 ζζ transmembrane dimer in POPC and raft-like lipid bilayer: a molecular dynamics study.

Plasma membrane lipids significantly affect assembly and activity of many signaling networks. The present work is aimed at analyzing, by molecular dynamics simulations, the structure and dynamics of the CD3 ζζ dimer in palmitoyl-oleoyl-phosphatidylcholine bilayer (POPC) and in POPC/cholesterol/sphingomyelin bilayer, which resembles the raft membrane microdomain supposed to be the site of the signal transducing machinery. Both POPC and raft-like environment produce significant alterations in structure and flexibility of the CD3 ζζ with respect to nuclear magnetic resonance (NMR) model: the dimer is more compact, its secondary structure is slightly less ordered, the arrangement of the Asp6 pair, which is important for binding to the Arg residue in the alpha chain of the T cell receptor (TCR), is stabilized by water molecules. Different interactions of charged residues with lipids at the lipid-cytoplasm boundary occur when the two environments are compared. Furthermore, in contrast to what is observed in POPC, in the raft-like environment correlated motions between transmembrane and cytoplasmic regions are observed. Altogether the data suggest that when the TCR complex resides in the raft domains, the CD3 ζζ dimer assumes a specific conformation probably necessary to the correct signal transduction.

Molecular basis of the NO trans influence in quaternary T-state human hemoglobin: a computational study.

NO binding to the T-state of human hemoglobin (HbA) induces the cleavage of the proximal His bonds to the heme iron in the α-chains, whereas it leaves the ß-hemes hexacoordinated. The structure of the nitrosylated T-state of the W37Eß mutant (W37E) shows that the Fe-His87α bond remains intact. Exactly how mutation affects NO binding and why tension is apparent only in HbA α-heme remains to be elucidated. By means of density functional theory electronic structure calculations and classical molecular dynamics simulations we provide an explanation for the poorly understood NO binding properties of HbA and its W37E mutant. The data suggest an interplay between electronic effects, tertiary structure and hydration site modifications in determining the tension in the NO-ligated T-state HbA α-chain.