Electron microscopic visualization of asymmetric precursor translocation intermediates: SecA functions as a dimer

Many functions in living cells are governed by macromolecular complexes. To fully describe the underlying mechanisms, they have to be understood at atomic level. The present study combines data obtained by X-ray crystallography and cryo-electron microscopy (cryo-EM) with molecular dynamics (MD) simulations. Two functions of macromolecular complexes, the downregulation of neurotransmitter release by the SNARE protein complex under oxidative stress and the translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis, were investigated. First, the hypothesis that oxidation of two cysteines on linker of the SNARE protein SNAP-25B and consequent disulfide bond formation shortens this linker sufficiently to hinder complex formation was tested. For this purpose, MD simulations of the SNARE complex with and without the disulfide bond were compared. Disulfide bond formation lead to conformational changes of the linker and of three central hydrophobic layers necessary to form the SNARE complex. Previously, mutations of residues contributing to these layers have been shown to reduce neurotransmitter release, suggesting that the stability of these layers is crucial for complex formation. The results from the simulations agree with the hypothesis that disulfide bond formation leads to a destabilization of the SNARE complex thus rendering it dysfunctional. This mechanism is interpreted as a chemomechanical regulation to shut down neurotransmitter release under oxidative stress, which has been linked to neurodegenerative diseases. In a second part I investigated the ribosome, where after peptide bond formation, bound tRNAs translocate by more than 7 nm to adjacent binding sites (A and P to P and E), accompanied by large-scale conformational motions (L1-stalk, intersubunit rotation) of the ribosome. By combining existing cryo-EM reconstructions of translocation intermediates with high resolution crystal structures, we obtained 13 near-atomic resolution structures. Subsequently, MD simulations of were carried out for each intermediate state. The obtained dynamics within these states allowed to estimate transition rates between states for motions of the L1-stalk, tRNAs and intersubunit rotations. Rapid motions of the L1-stalk and the small (30S) subunit on sub-microsecond timescales were revealed, whereas tRNA motions were seen to be rate-limiting for most transitions. By calculating the interaction free energy between L1-stalk and tRNA, molecular forces were derived showing that the L1-stalk is actively pulling the tRNA from P to E binding site, thereby overcoming barriers for the tRNA motion. Further, ribosomal proteins L5 and L16 guide the tRNAs by 'sliding' and 'stepping' mechanisms involving key protein-tRNA contacts. This explains how tRNA binding affinity is kept sufficiently constant to allow rapid translocation despite large-scale displacements. Translocation is accompanied by rotations of the 30S ribosomal subunit of more than 20 degrees relative to the large (50S) subunit. For each translocation intermediate, the affinity of the two subunits with each other must be finely tuned enabling such conformational flexibility while maintaining integrity of the ribosomal complex. Analyzing the trajectories at residue level reveals two classes of intersubunit contact interactions: i) persistent residue contacts which are independent of 30S rotation and primarily located close to the axis of rotation. ii) contacts that are formed and ruptured depending on the rotation angle, seen mainly on the periphery. Strikingly, also these rotation specific contacts substantially contribute to the overall stability of the ribosomal assembly and are expected to maintain a constant interaction energy with low barriers for rotation. The simulations reveal that upon removal of tRNAs peripheral contacts are weakened and, in turn, intersubunit rotation angles decrease, in agreement with cryo-EM analysis of tRNA depleted ribosomes. The identified mechanisms for lowering free energy barriers and for fine-tuning affinities might have developed similarly in other macromolecular complexes.

Protein translocation occurs across the energy-conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation-enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super-active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.

Assessment techniques for measurement of target erosion/ redeposition in large tokamaks

The heavy ion time‐of‐flight elastic recoil detection analysis (HI‐ERDA) technique was used to investigate the possibility of measuring near‐surface elemental depth profiles of light and mid‐ Z elements in thin films of plasma‐assisted molecular beam epitaxy (PAMBE)‐grown GaN and helium‐implanted titanium. The great advantage of HI‐ERDA is the ability to measure mass‐separated elemental depth profiles simultaneously. However for some materials it is not certain whether HI‐ERDA can be used successfully because significant sputtering or other beam‐induced damage may occur. The damage to the surfaces by a 77 MeV iodine beam was assessed using RBS, AFM and profilometry. The results show that for thin PAMBE‐grown polycrystalline GaN films and for titanium that has been heavily implanted with helium a significant modification of the near‐surface region is caused by the probing heavy ion beam. For the PAMBE‐grown GaN films the most significant loss trend is observed for nitrogen. Surprisingly this was not accompanied by a change in surface topology. In contrast, an almost complete removal of the heavily helium‐implanted surface layer was measured for the titanium specimens. The investigation shows that reference measurements with additional techniques such as RBS, AFM and profilometry have to be performed to ascertain sample integrity before HI‐ERDA data can be used. Copyright © 2004 John Wiley & Sons, Ltd.

Surface modification by substitution: Changing topology of conformational potential energy surfaces

In the global scenario, wind turbines and their aerodynamics are always subjected to constant research for increasing their efficiency which converts the abundant wind energy into usable electrical energy. In this research, an attempt is made to increase the efficiency through the changes in surface topology of wind turbines through computational fluid dynamics. Dimples on the other hand are very efficient in reducing air drag as is it evident from the reduction of drag and increase in lift in golf balls. The predominant factors influencing the efficiency of the wind turbines are lift and drag which are to be maximized and minimized respectively. In this research, surface of turbine blades are integrated with dimples of various sizes and arrangements and are analyzed using computational fluid dynamics to obtain an optimum combination. The analysis result shows that there is an increase in power with about 15% increase in efficiency. Hence, integration of dimples on the surface of wind turbine blades has helped in increasing the overall efficiency of the wind turbine.

Control over minivalley polarization and interlayer coupling is demonstrated in double bilayer graphene twisted with an angle of 2.37°. This intermediate angle is small enough for the minibands to form and large enough such that the charge carrier gases in the layers can be tuned independently. Using a dual-gated geometry we identify and control all possible combinations of minivalley polarization via the population of the two bilayers. An applied displacement field opens a band gap in either of the two bilayers, allowing us to even obtain full minivalley polarization. In addition, the carriers, formerly separated by their minivalley character, are mixed by tuning through a Lifshitz transition, where the Fermi surface topology changes. The high degree of control over the minivalley character of the bulk charge transport in twisted double bilayer graphene offers new opportunities for realizing valleytronics devices such as valley valves, filters, and logic gates.

The paper is dedicated to studying of the efficiency of application of the dust control agents not only for their original aims, but also for increasing of oxidation resistance of the solid fossil fuels. The complex investigation included: studying of coals quality alteration after treatment by the selected solution, scanning electron microscopy for investigation of their surface topology changes and, finally, ozonating treatment at low ozone concentrations with kinetic modeling for evaluation of oxidation resistivity. As it was expected, application of the selected solution allows no quality loss for coals. Scanning electron microscopy allowed to visualize the mechanism of working of the dust control agent. It revealed that the studied solution forms a thin smooth film around coal particles. This film tightly binds all the fine particles that were already formed and stored at the coal surface. Also, the solution tends to cover the surface thus decreasing the accessibility of the surface to active oxygen. The latter was proved by treated and untreated coals ozonating processing at low concentrations of ozone. The applied kinetic model allowed evaluation of the total coals activity to ozone decomposition. It was found that the treated coals have twice as low total activity to ozone decomposition as compared with the untreated ones. The latter parameter is closely related to coals oxidation resistivity (the lower the coals activity, the higher the coals oxidation resistivity). Therefore, it was finally concluded that application of the studied dust control agent allows for significant increase of sought-for quality indicator.

A descriptive model of wear evolution in rolling bearings

We describe the state of development of a white light interferometer to characterize the cryogenic mirrors for GW detector on operation. We include the first experimental results from the proof of concept of the metrology instrument. The instrument will characterize the topology as well as the vibration of the mirrors. This development takes place in the frame of the E-TEST project. E-TEST is one of the technology demonstrators for the future Einstein Telescope (ET). ET is dedicated to the measure and characterization of gravitational waves. The prototype built by E-TEST includes a large silicon mirror of 40 cm diameter suspended by innovative vibration isolation hanging modules. To reach the detection specification, the mirror is cooled down at cryogenic temperatures around 20 K. Nevertheless, even after the isolation, the mirror may not reach perfect stability once at cryogenic temperatures. Furthermore, the mirror may experience surface topology changes and wavefront deformation due to the extreme variations in temperature and gradient. With our metrology instrument, we can obtain on a single camera frame a set of interferogram maps of the area observed on the mirror at different optical path differences. To do this, we design an innovative phase mask for a white light low-coherence interferometer. In addition, we implement new algorithms for the white light interferogram analysis, avoiding the limitations of the conventional Phase Shifting Interferometry algorithms.

Presecretory proteins are translocated across biological membranes through protein-conducting channels such as Sec61 (eukaryotes) and SecYEG (bacteria). SecA, a translocation ATPase, pushes preproteins out with dynamic structural changes through SecYEG. SecG, a subunit of the SecYEG channel possessing two transmembrane stretches (TMs), undergoes topology inversion coupled with SecA-dependent translocation. Recently, we characterized membrane protein integrase (MPIase), a glycolipozyme involved in not only protein integration into membranes but also preprotein translocation. We report here that SecG inversion occurs only when MPIase associates with SecYEG. We also found that MPIase modulates the dimer orientation of SecYEG. Cysteine-scanning mutagenesis mapped SecG TM 2 to a relatively hydrophilic environment. The dimer formation of SecG, crosslinked at TM 2, was not observed on SecG inversion, indicating that SecYEG undergoes a dynamic structural change during preprotein translocation.

Axisymmetric free-surface flow simulation using the moving surface mesh particle method and application to drop formation

Differential scanning calorimetry and 2H nuclear magnetic resonance and Fourier transform infrared spectroscopy studies of the effects of transmembrane α-helical peptides on the organization of phosphatidylcholine bilayers

The long-term stability of plasma-sprayed hydroxyapatite coatings is influenced by the dissolution behavior of the coating in in vivo conditions. Plasma-spraying generates a mixture of phases and this study has focused on how the balance of phases affects the in vitro dissolution behavior of the coatings in double distilled-deionized water and in tris-buffer solutions. The pH changes in double distilled-deionized water were monitored, whilst the pH value was maintained at 7.25 for the tris-buffer solution at 37 degrees C with 5% CO2 atmosphere. The phosphate and calcium ions released were measured using UV-Visible Spectrophotometer and Atomic Absorption Spectroscopy respectively. Changes in crystal and surface topology were also studied. The results indicate that the dissolution behavior of the coatings depends on several factors. The rate of release of phosphate ions was found to increase significantly for the tris-buffer solution compared to the deionized water, indicating that the presence of electrolyte constituents affects the dissolution behavior of the coatings. The Ca/P ratio in the tris-buffer solution is approximately three. Increases in the level of crystallinity of the coatings significantly decreased the dissolution rate and hence, the amount of phosphate ions released. The higher the percentage of crystallinity, the higher the stability of the coating under in vitro conditions.

Marked hydration changes occur during the self-assembly of the melittin protein tetramer in water. Hydrophobicity induces a drying transition in the gap between simple sufficiently large (more than 1 nm(2)) strongly hydrophobic surfaces as they approach each other, resulting in the subsequent collapse of the system, as well as a depletion of water next to single surfaces. Here we investigate whether the hydrophobic induced collapse of multidomain proteins or the formation of protein oligimers exhibits a similar drying transition. We performed computer simulations to study the collapse of the tetramer of melittin in water, and observed a marked water drying transition inside a nanoscale channel of the tetramer (with a channel size of up to two or three water-molecule diameters). This transition, although occurring on a microscopic length scale, is analogous to a first-order phase transition from liquid to vapour. We find that this drying is very sensitive to single mutations of the three isoleucines to less hydrophobic residues and that such mutations in the right locations can switch the channel from being dry to being wet. Thus, quite subtle changes in hydrophobic surface topology can profoundly influence the drying transition. We show that, even in the presence of the polar protein backbone, sufficiently hydrophobic protein surfaces can induce a liquid-vapour transition providing an enormous driving force towards further collapse. This behaviour was unexpected because of the absence of drying in the collapse of the multidomain protein 2,3-dihydroxybiphenyl dioxygenase (BphC).