Position of helical kinks in membrane protein crystal structures and the accuracy of computational prediction

The structural features of helical transmembrane (TM) proteins, such as helical kinks, tilts, and rotational orientations are important in modulation of their function and these structural features give rise to functional diversity in membrane proteins with similar topology. In particular, the helical kinks caused by breaking of the backbone hydrogen bonds lead to hinge bending flexibility in these helices. Therefore it is important to understand the nature of the helical kinks and to be able to reproduce these kinks in structural models of membrane proteins. We have analyzed the position and extent of helical kinks in the transmembrane helices of all the crystal structures of membrane proteins taken from the MPtopo database, which are about 405 individual helices of length between 19 and 35 residues. 44% of the crystal structures of TM helices showed a significant helical kink, and 35% of these kinks are caused by prolines. Many of the non-proline helical kinks are caused by other residues like Ser and Gly that are located at the center of helical kinks. The side chain of Ser makes a hydrogen bond with the main chain carbonyl of the i − 4th or i + 4th residue thus making a kink. We have also studied how well molecular dynamics (MD) simulations on isolated helices can reproduce the position of the helical kinks in TM helices. Such a method is useful for structure prediction of membrane proteins. We performed MD simulations, starting from a canonical helix for the 405 TM helices. 1 ns of MD simulation results show that we can reproduce about 79% of the proline kinks, only 59% of the vestigial proline kinks and 18% of the non-proline helical kinks. We found that similar results can be obtained from choosing the lowest potential energy structure from the MD simulation. 4–14% more of the vestigial prolines were reproduced by replacing them with prolines before performing MD simulations, and changing the amino acid back to proline after the MD simulations. From these results we conclude that the position of the helical kinks is dependent on the TM sequence. However the extent of helical kinking may depend on the packing of the rest of the protein and the lipid bilayer.     

A novel microreactor with 3D rotating flow to boost fluid reaction and mixing of viscous fluids

Based on a concept encompassing splitting and recombination (SAR) and chaotic advection, an efficient microreactor, called a SAR μ-reactor, has been designed to mix fluids at Reynolds numbers from 0.01 to 100 and to be suitable for mixing fluids with viscosity over a wide range (μ = 0.000855–0.186 kg m⁻¹ s⁻¹). This SAR μ-reactor was compared, numerically and experimentally, with a slanted-groove micromixer (SGM) for reaction or mixing of fluids. Results of simulations characterized the designed structure with inducing a 3D rotating flow involving a strong lateral component to stretch intensely the contact interface in the SAR μ-reactor. Chemical colorimetry of two kinds – involving reactions of phenolphthalein with sodium hydroxide and of ascorbic acid with diiodine – revealed that the SAR μ-reactor provided a smaller mixing length, reaction length and period than the SGM; the mixing performance of the SAR μ-reactor was much better than that of the SGM. We assessed the mixing behavior of fluorescent proteins (C-phycocyanin and R-phycoerythrin) in viscous fluids with a confocal microscope. Experimental results and simulations showed that the effect of fluid viscosity on the mixing efficiency of the SAR μ-reactor is less than for the SGM; the SAR mechanism effectively augmented the contact interface even though the intrinsic diffusivity of fluids was diminished.     

Structural insights into type I and type II of nsp4 porcine reproductive and respiratory syndrome virus (nsp4 PRRSV) by molecular dynamics simulations

Porcine reproductive and respiratory virus (PRRSV) causes major economic concerns for the swine industry worldwide. We have performed molecular dynamics simulations (MD) and principle component analysis (PCA) to investigate the role of the catalytic triad and conformational dynamics of type I and type II of nsp4 PRRSV. The results showed that the RMSF of residues 136–142 near the active site of all models was highly flexible. Moreover, we identified the effect of single structural mutations of the catalytic triad. The percentage of residue with a 0.1 nm RMSF value and PCA results revealed that the mutations affected domain I and II suggesting the wild types were more stable than the mutants. At the catalytic triad, the distances between H39 and S118 were very flexible while the distances between H39 and D64 were very stable. H39, D64 and S118 showed high occupancy percentage of the hydrogen bond interaction with many residues that are conserved in PRRSVAS, PRRSVES, LDVC, LDVP and EAV. Moreover, S118 of wild-type protein formed H-bonds with T134 and G135 but these interactions were lost in PRRSVAV (S118A) and PRRSVES (S117A) indicating that the substitution of important H-bond interaction in PRRSVAS (S118A) and PRRSVES (S117A) affected the flexibility around the catalytic triad, conformation and proteolytic activity. Overall, our study may provide the structural basic of the catalytic triad and be useful for testing the protein activity in future experiments.     

Temperature-induced unfolding pathway of a type III antifreeze protein: insight from molecular dynamics simulation

Molecular dynamics simulations of the temperature-induced unfolding reaction of a cold-adapted type III antifreeze protein (AFPIII) from the Antarctic eelpout Lycodichthys dearborni have been carried out for 10 ns each at five different temperatures. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. Plots of backbone root mean square deviation (RMSD) against radius of gyration (Rg) serve as phase space trajectories. These plots also indicate that the protein unfolds without many detectable intermediates suggestive of two-state unfolding kinetics. The transition state structures are identified from essential dynamics, which utilizes a principal component analysis (PCA) on the atomic fluctuations throughout the simulation. Overall, the transition state resembles an expanded native state with the loss of the three 3₁₀ helices and disrupted C-terminal region.Our study provides insight into the structure–stability relationship of AFPIII, which may help to engineer AFPs with increased thermal stability that is more desirable than natural AFPs for some industrial and biomedical purposes.     

First Vertical Hall Device in standard 0.35 μm CMOS technology

In order to lower the short-circuit effect due to the measurement contacts, Vertical Hall Devices (VHDs) are generally designed either in bulky N-type silicon or in the deep N-well of high-voltage CMOS technologies. In this last case, VHD can benefit from on chip circuitry for offset and 1/f noise reduction, but HVCMOS remains a costly technology. Using spinning-current, HVCMOS compatible VHDs with a resolution of 76 μT rms over a 1.6-kHz bandwidth have been demonstrated. The VHD presented here is designed in the shallow N-well of a low-cost 0.35 μm standard CMOS technology. Unlike conventional VHD, its measurement contacts are located outside the sensor active area. FEM simulations and experimental results show that the new geometry suppresses the short-circuit effect and strongly reduces the intrinsic offset and noise. Thus, without any noise and offset reduction method, this new small VHD (63 μm²) reaches a resolution of 79 μT rms over a (5 Hz–1.6 kHz) bandwidth, and opens the way to the integration of 3D Hall sensors in low-cost standard CMOS technologies.     

Comparative structural studies of psychrophilic and mesophilic protein homologues by molecular dynamics simulation

Comparative molecular dynamics simulations of psychrophilic type III antifreeze protein from the North-Atlantic ocean-pout Macrozoarces americanus and its corresponding mesophilic counterpart, the antifreeze-like domain of human sialic acid synthase, have been performed for 10 ns each at five different temperatures. Analyses of trajectories in terms of secondary structure content, solvent accessibility, intramolecular hydrogen bonds and protein–solvent interactions indicate distinct differences in these two proteins. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their growth temperatures. However at higher temperatures psychrophilic protein shows increased overall flexibility than its mesophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are non-overlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar directions of motion of each protein especially at 298 K, 310 K and 373 K. Overall, the psychrophilic protein leads to increased conformational sampling of the phase space than its mesophilic counterpart.Our study may help in elucidating the molecular basis of thermostability of homologous proteins from two organisms living at different temperature conditions. Such an understanding is required for designing efficient proteins with characteristics for a particular application at desired working temperatures.     

Study of the water behavior into a ferrierite zeolite by molecular dynamics simulations

In this contribution, we present molecular dynamics (MD) simulations of water inside a ferrierite type framework. We stress the importance of introducing the long-range electrostatic contribution to the chosen adsorbate-adsorbent interaction potential, and present various thermodynamical, structural and dynamical results obtained from the analyses of the MD trajectories.     

Modeling of high-affinity binding of the novel atrial anti-arrhythmic agent, vernakalant, to Kv1.5 channels

Vernakalant (RSD1235) is an investigational drug that converts atrial fibrillation rapidly and safely in patients intravenously [Roy et al., J. Am. Coll. Cardiol. 44 (2004) 2355–2361; Roy et al., Circulation 117 (2008) 1518–1525] and maintains sinus rhythm when given orally [Savelieva et al., Europace 10 (2008) 647–665]. Here, modeling using AutoDock4 allowed exploration of potential binding modes of vernakalant to the open-state of the Kv1.5 channel structure. Point mutations were made in the channel model based on earlier patch-clamp studies [Eldstrom et al., Mol. Pharmacol. 72 (2007) 1522–1534] and the docking simulations re-run to evaluate the ability of the docking software to predict changes in drug–channel interactions. Each AutoDock run predicted a binding conformation with an associated value for free energy of binding (FEB) in kcal/mol and an estimated inhibitory concentration (K_i). The most favored conformation had a FEB of −7.12 kcal/mol and a predicted K_i of 6.08 μM (the IC50 for vernakalant is 13.8 μM; [Eldstrom et al., Mol. Pharmacol. 72 (2007) 1522–1534]). This conformation makes contact with all four T480 residues and appears to be clearly positioned to block the channel pore.     

Three hydrolases and a transferase: comparative analysis of active-site dynamics via the BioSimGrid database

Comparative molecular dynamics (MD) simulations enable us to explore the conformational dynamics of the active sites of distantly related enzymes. We have used the BioSimGrid (http://www.biosimgrid.org) database to facilitate such a comparison. Simulations of four enzymes were analyzed. These included three hydrolases and a transferase, namely acetylcholinesterase, outer-membrane phospholipase A, outer-membrane protease T, and PagP (an outer-membrane enzyme which transfers a palmitate chain from a phospholipid to lipid A). A set of 17 simulations were analyzed corresponding to a total of 0.1 μs simulation time. A simple metric for active-site integrity was used to demonstrate the existence of clusters of dynamic conformational behaviour of the active sites. Small (i.e. within a cluster) fluctuations appear to be related to the function of an enzymatically active site. Larger fluctuations (i.e. between clusters) correlate with transitions between catalytically active and inactive states. Overall, these results demonstrate the potential of a comparative MD approach to analysis of enzyme function. This approach could be extended to a wider range of enzymes using current high throughput MD simulation and database methods.     

Direct electron transfer of myoglobin in mesoporous silica KIT-6 modified on screen-printed electrode

A disposable hydrogen peroxide biosensor was developed based on the direct electron transfer of myoglobin (Mb) on mesopores KIT-6 modified screen-printed electrode (SPE) which was manually performed to fabricate the planar carbon electrodes. KIT-6 is a new material which can absorb abundant of Mb molecules. A mixture of Mb and KIT-6 was immobilized with nafion on electrode. The cyclic voltammetry experiment indicated that a pair of stable and well-defined reduction peaks with a formal potentials of −0.35, and −0.28 V versus saturated calomel electrode (SCE) was obtained, using the present modified electrode in phosphate buffer saline (0.05 M, pH 7.0) at scan rate of 100 mV s⁻¹, characteristic of Mb heme Fe(III)/Fe(II) redox couple. The heterogeneous electron transfer rate constant k_s was estimated to be 16.93 s⁻¹. And the formal potential was pH-dependent, having two slopes of −54.7 and −49.3 mV/pH which illustrated one electron transfer. This modified electrode was applied to detect H₂O₂ with sensitivity of 55.68 mA M⁻¹ cm⁻². Infrared spectrum and UV–vis absorption spectra of immobilized Mb film were recorded. In conclusion, KIT-6 increases the electron transfer activity of Mb and this kind of H₂O₂ biosensor is low cost for using disposable.     

Position of helical kinks in membrane protein crystal structures and the accuracy of computational prediction

The structural features of helical transmembrane (TM) proteins, such as helical kinks, tilts, and rotational orientations are important in modulation of their function and these structural features give rise to functional diversity in membrane proteins with similar topology. In particular, the helical kinks caused by breaking of the backbone hydrogen bonds lead to hinge bending flexibility in these helices. Therefore it is important to understand the nature of the helical kinks and to be able to reproduce these kinks in structural models of membrane proteins. We have analyzed the position and extent of helical kinks in the transmembrane helices of all the crystal structures of membrane proteins taken from the MPtopo database, which are about 405 individual helices of length between 19 and 35 residues. 44% of the crystal structures of TM helices showed a significant helical kink, and 35% of these kinks are caused by prolines. Many of the non-proline helical kinks are caused by other residues like Ser and Gly that are located at the center of helical kinks. The side chain of Ser makes a hydrogen bond with the main chain carbonyl of the i − 4th or i + 4th residue thus making a kink. We have also studied how well molecular dynamics (MD) simulations on isolated helices can reproduce the position of the helical kinks in TM helices. Such a method is useful for structure prediction of membrane proteins. We performed MD simulations, starting from a canonical helix for the 405 TM helices. 1 ns of MD simulation results show that we can reproduce about 79% of the proline kinks, only 59% of the vestigial proline kinks and 18% of the non-proline helical kinks. We found that similar results can be obtained from choosing the lowest potential energy structure from the MD simulation. 4–14% more of the vestigial prolines were reproduced by replacing them with prolines before performing MD simulations, and changing the amino acid back to proline after the MD simulations. From these results we conclude that the position of the helical kinks is dependent on the TM sequence. However the extent of helical kinking may depend on the packing of the rest of the protein and the lipid bilayer.     

Control of the microparticle position in the channel based on dielectrophoresis

We report here the control of the microparticles position within fluid flow based on its size by using dielectrophoresis (DEP) with a microelectrode array consisted of rectangular features with the different size of width and gap. 3 μm- and 10 μm-diameter particles were introduced into the channel with 300 μm height at 30 μl/min. An AC electric field (20 V peak–peak and 2 MHz) was then applied to microelectrode arrays to form dielectrophoretic fluid cage, resulting in a formation of flow paths with low electric fields on the arrays. The microparticles separately flow in line streams along the paths formed between the rectangular features of the arrays, the 3 μm-diameter particles mainly flow through the narrow path and 10 μm-diameter particles through the wide path. These results indicated that positions of two types of microparticles in the fluidic channel were easily separated and controlled using the n-DEP.     

Three hydrolases and a transferase: Comparative analysis of active-site dynamics via the BioSimGrid database

Comparative molecular dynamics (MD) simulations enable us to explore the conformational dynamics of the active sites of distantly related enzymes. We have used the BioSimGrid (http://www.biosimgrid.org) database to facilitate such a comparison. Simulations of four enzymes were analyzed. These included three hydrolases and a transferase, namely acetylcholinesterase, outer-membrane phospholipase A, outer-membrane protease T, and PagP (an outer-membrane enzyme which transfers a palmitate chain from a phospholipid to lipid A). A set of 17 simulations were analyzed corresponding to a total of ∼0.1 μs simulation time. A simple metric for active-site integrity was used to demonstrate the existence of clusters of dynamic conformational behaviour of the active sites. Small (i.e. within a cluster) fluctuations appear to be related to the function of an enzymatically active site. Larger fluctuations (i.e. between clusters) correlate with transitions between catalytically active and inactive states. Overall, these results demonstrate the potential of a comparative MD approach to analysis of enzyme function. This approach could be extended to a wider range of enzymes using current high throughput MD simulation and database methods.     

Interactions of acetylcholine binding site residues contributing to nicotinic acetylcholine receptor gating: Role of residues Y93, Y190, K145 and D200

The nicotinic acetylcholine receptor exhibits multiple conformational states, resting (channel closed), active (channel open) and desensitized (channel closed). The resting state may be distinguished from the active and desensitized states by the orientation of loop C in the extracellular ligand binding domain (LBD). Homology modeling was used to generate structures of the Torpedo californica α₂βδγ nAChR that initially represent the resting state (loop C open) and the desensitized state (loop C closed). Molecular dynamics (MD) simulations were performed on the extracellular LBD on each nAChR conformational state, with and without the agonist anabaseine present in each binding site (the αγ and the αδ sites). Three MD simulations of 10 ns each were performed for each of the four conditions. Comparison of dynamics revealed that in the presence of agonist, loop C was drawn inward and attains a more stable conformation. Examination of side-chain interactions revealed that residue αY190 exhibited hydrogen-bonding interactions either with residue αY93 in the ligand binding site or with residue αK145 proximal to the binding site. αK145 also exhibited side chain (salt bridge) interactions with αD200 and main chain interactions with αY93. Residues αW149, αY198, γY116/δT119, γL118/δL121 and γL108/δL111 appear to play the role of stabilizing ligand in the binding site. In MD simulations for the desensitized state, the effect of ligand upon the interactions among αK145, αY190, and αY93 as well as ligand-hydrogen-bonding to αW149 were more pronounced at the αγ interface than at the αδ interface. Differences in affinity for the desensitized state were determined experimentally to be 10-fold. The changes in side chain interactions observed for the two conformations and induced by ligand support a model wherein hydrogen bond interactions between αD200 and αY93 are broken and rearrange to form a salt-bridge between αK145 and αD200 and hydrogen bond interactions between αY93 and αY190 and between αK145 and αY190.     

Study of symmetric microstructures for CMOS multilayer residual stress

This study presents a fabrication-based approach to improve the curl-up effect in complementary metal oxide semiconductor (CMOS) multilayer large-area planar structures. Control of the residual stress of CMOS multilayer microstructures is necessary for development of microelectromechanical systems (MEMS) sensors such as accelerometers and micromirrors. In this work, 3D symmetric geometry can be used to overcome effectively the residual stresses in CMOS multilayer microstructures. To demonstrate this concept, a symmetric multilayer flat-plane is fabricated and release-etched using an isotropic plasma etching process. The isotropic etch characteristics and lateral undercut can be controlled using a chamber pressure of 0.47 ± 0.2 Torr. A flat-plane structure with an area of 500 μm × 500 μm is fabricated using multilayer materials, including four metal and three silicon dioxide layers. Based on this approach, the measured results show the residual stress effect can be minimized in CMOS multilayer microstructures, and furthermore the curl-up effect of flat-plane is less than 2 μm across the 500 μm × 500 μm area.     

Improved field emission characteristics of screen-printed CNT-FED cathode by interfusing Fe/Ni nano-grains

Carbon nanotube (CNT) cathode with and without interfusing nano-metal particles was prepared using screen-printing technology. For the good electric conductivity of metal, the turn-on electric field of the Fe/Ni and CNT composite film (Fe/CNT film) decreases to 1.42 V/μm comparing with the usual CNT film of 2.45 V/μm, and the emission current increases from 60 μA to 440 μA at an applied electric field of 2.3 V/μm. Furthermore, the field enhancement factor β increases from 1721 to 3242. By characterizing the prepared samples via X-ray diffraction (XRD) and field emission scanning electron microscopy (FESEM), it is found that carbide Fe₃C phase is formed in Fe/CNT film, and the metal particles are filled in the interspaces of CNTs. It is evaluated that benefiting from good electrical conductivity and chemical inertness of metal carbide, Fe/CNT film achieves high emission characteristics and emission uniformity.     

Simultaneous measurement of Pb, Cd and Zn using differential pulse anodic stripping voltammetry at a bismuth/poly(p-aminobenzene sulfonic acid) film electrode

A novel sensor was developed for simultaneous detection of Pb, Cd and Zn, based on the differential pulse anodic stripping response at a bismuth/poly(p-aminobenzene sulfonic acid) (Bi/poly(p-ABSA)) film electrode. This electrode was generated in situ by depositing simultaneously bismuth and the metals by reduction at −1.40 V on the poly(p-ABSA) modified electrode. Compared with the bismuth film electrode, the Bi/poly(p-ABSA) film electrode can yield a larger stripping signal for Pb, Cd and Zn. Under the optimum conditions, a linear response was observed for Cd and Zn in the range from 1.00 to 110.00 μg L⁻¹ and for Pb in the range from 1.00 to 130.00 μg L⁻¹. The detection limits of Pb(II), Cd(II) and Zn(II) were 0.80, 0.63 and 0.62 μg L⁻¹, respectively. Finally this sensor had been applied to the simultaneous determination of Pb(II), Cd(II) and Zn(II) in river water samples and the results were quite corresponding to the value obtained by atomic absorption spectrometry.     

Tensile and high cycle fatigue test of Al–3% Ti thin films

This paper presents the results of tensile and high cycle fatigue tests with stress ratio R = 0.1 for an Al–3% Ti thin film of 1 μm thickness in atmospheric air at room temperature. Specimens with three different widths (50, 100, and 150 μm) were fabricated to study the width effects of each sample. Test results show that tensile and fatigue properties for the Al–3% Ti thin film with different widths are very close, and, thus, width effects were found to be minimal. The elastic moduli ranged from 80 to 82 GPa, and the tensile strengths ranged from 369 to 379 MPa. Fatigue strength coefficients of the specimens with 50, 100, and 150 μm width were 193, 181, and 164 MPa, respectively. In addition, fatigue strength exponents of the specimens with 50, 100, and 150 μm width were −0.023, −0.020, and −0.013, respectively. When present test results are compared with typical properties of bulk aluminium, the Al–3% Ti thin film is found to have longer life at the same stress, but it is more sensitive to the stress level.     

Simulation, fabrication and characterization of a 3D piezoresistive force sensor

In this contribution we report on a miniaturized bulk micro-machined three-axes piezoresistive force sensor. The force sensor consists of a full membrane with 16 conventional two terminal p-type diffused piezoresistors on the surface of the membrane. The die size of the chip is 6.5 mm × 6.5 mm. Piezoresistors with four different designs were placed on the membrane. Sensitivities were found to be in the range of 0.37–0.79 mV/(V mN) and 1.68–2.92 mV/(V mN) in Z-direction and X- or Y-direction, respectively. The stiffness of the measured microprobes in the range of 5–8 mN/μm and 0.27–0.48 mN/μm were obtained in vertical and lateral direction, respectively. Various single and twin membranes designs were simulated to calculate stiffness of the microprobe. The measurement results show a cross-axis sensitivity of <2.5% at full scale of 25 mN.     

Comparative structural studies of psychrophilic and mesophilic protein homologues by molecular dynamics simulation

Comparative molecular dynamics simulations of psychrophilic type III antifreeze protein from the North-Atlantic ocean-pout Macrozoarces americanus and its corresponding mesophilic counterpart, the antifreeze-like domain of human sialic acid synthase, have been performed for 10 ns each at five different temperatures. Analyses of trajectories in terms of secondary structure content, solvent accessibility, intramolecular hydrogen bonds and protein–solvent interactions indicate distinct differences in these two proteins. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their growth temperatures. However at higher temperatures psychrophilic protein shows increased overall flexibility than its mesophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are non-overlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar directions of motion of each protein especially at 298 K, 310 K and 373 K. Overall, the psychrophilic protein leads to increased conformational sampling of the phase space than its mesophilic counterpart.Our study may help in elucidating the molecular basis of thermostability of homologous proteins from two organisms living at different temperature conditions. Such an understanding is required for designing efficient proteins with characteristics for a particular application at desired working temperatures.