Development of a compact capillary electrophoresis–chemiluminescence system with ultra‐fast peroxyoxalate reaction to monitor the hydrolysis of rhodamine 6G

A simple and effective capillary electrophoresis–chemiluminescence (CE–CL) detection system was developed based on an ultra‐fast bis(2,4,6‐trichlorophenyl)oxalate (TCPO) chemiluminescence (CL) reaction (0.6 s duration) that avoided overlapping peaks and peak tailing. Through a series of static injection experiments, this unusually rapid CL reaction was ascribed to the catalytic effect of imidazole in the tetrahydrofuran solvent, which has been rarely utilized in such investigations. A possible mechanism is given to explain the results. Under the optimized conditions, rhodamine 6 G (R6G) and its hydrolysis product (R6G‐COOH) could be efficiently separated through electrophoresis in 7 min, with sensitive CL detection in the proposed CE–CL system. In this way, the alkaline hydrolysis of R6G was monitored, followed by estimation of relative rate constants and activation energy. This finding and application should be helpful in further study for the TCPO CL reaction, and revealed an attractive opportunity for simplifying the CE–CL system, such as in a microchip device. Copyright © 2011 John Wiley & Sons, Ltd.     

Oscillations of the energy gap for the initial electron-transfer step in bacterial reaction centers

Oscillations in the electrostatic energy gap [V_elec(t)] for electron transfer from the primary electron donor (P) to the adjacent bacteriochlorophyll (B) in photosynthetic bacterial reaction centers are examined by molecular-dynamics simulations. Autocorrelation functions of V_elec in the reactant state (PB) include prominent oscillations with an energy of 17 cm^–1. This feature is much weaker if the trajectory is propagated in the product state P⁺B^–. The autocorrelation functions also include oscillations in the regions of 5, 80 and 390 cm^–1 in both states, and near 25 and 48 cm^–1 in P⁺B^–. The strong 17-cm^–1 oscillation could involve motions that modulate the distance between P and B, because a similar oscillation occurs in the direct electrostatic interactions between the electron carriers.     

Distinct encounter complexes of PAI‐1 with plasminogen activators and vitronectin revealed by changes in the conformation and dynamics of the reactive center loop

Plasminogen activator inhibitor‐1 (PAI‐1) is a biologically important serine protease inhibitor (serpin) that, when overexpressed, is associated with a high risk for cardiovascular disease and cancer metastasis. Several of its ligands, including vitronectin, tissue‐type and urokinase‐type plasminogen activator (tPA, uPA), affect the fate of PAI‐1. Here, we measured changes in the solvent accessibility and dynamics of an important unresolved functional region, the reactive center loop (RCL), upon binding of these ligands. Binding of the catalytically inactive S195A variant of tPA to the RCL causes an increase in fluorescence, indicating greater solvent protection, at its C‐terminus, while mobility along the loop remains relatively unchanged. In contrast, a fluorescence increase and large decrease in mobility at the N‐terminal RCL is observed upon binding of S195A‐uPA to PAI‐1. At a site distant from the RCL, binding of vitronectin results in a modest decrease in fluorescence at its proximal end without restricting overall loop dynamics. These results provide the new evidence for ligand effects on RCL conformation and dynamics and differences in the Michaelis complex with plasminogen activators that can be used for the development of more specific inhibitors to PAI‐1. This study is also the first to use electron paramagnetic resonance (EPR) spectroscopy to investigate PAI‐1 dynamics. Significance : Balanced blood homeostasis and controlled cell migration requires coordination between serine proteases, serpins, and cofactors. These ligands form noncovalent complexes, which influence the outcome of protease inhibition and associated physiological processes. This study reveals differences in binding via changes in solvent accessibility and dynamics within these complexes that can be exploited to develop more specific drugs in the treatment of diseases associated with unbalanced serpin activity.     

Computational characterization of the chemical step in the GTP hydrolysis by Ras‐GAP for the wild‐type and G13V mutated Ras

The free energy profiles for the chemical reaction of the guanosine triphosphate hydrolysis GTP + H₂O → GDP + Pi by Ras‐GAP for the wild‐type and G13V mutated Ras were computed by using molecular dynamics protocols with the QM(ab initio)/MM potentials. The results are consistent with the recent measurements of reaction kinetics in Ras‐GAP showing about two‐order reduction of the rate constant upon G13V mutation in Ras: the computed activation barrier on the free energy profile is increased by 3 kcal/mol upon the G13V replacement. The major reason for a higher energy barrier is a shift of the “arginine finger” (R789 from GAP) from the favorable position in the active site. The results of simulations provide support for the mechanism of the reference reaction according to which the Q61 side chain directly participates in chemical transformations at the proton transfer stage. Proteins 2015; 83:1046–1053. © 2015 Wiley Periodicals, Inc.     

Effects of slight structural distortion on the luminescence performance in (Ca1‐xEux)WO4 luminescent materials

(Ca_1‐xEu_x)WO₄ (x = 0–21 mol%) phosphors were prepared using the classical solid‐state reaction method. The influence of Eu³⁺ ion doping on lattice structure was observed using powder X‐ray diffraction and Fourier transform infrared spectroscopy. Furthermore, under this influence, the luminescence properties of all samples were analyzed. The results clearly illustrated that the element europium was successfully incorporated into the CaWO₄ lattice with a scheelite structure in the form of a Eu³⁺ ion, which introduced a slight lattice distortion into the CaWO₄ matrix. These lattice distortions had no effect on phase purity, but had regular effects on the intrinsic luminescence of the matrix and the f–f excitation transitions of Eu³⁺ activators. When the Eu³⁺ concentration was increased to 21 mol%, a local luminescence centre of [WO₄]^2? groups was detected in the matrix and manifested as the decay curves of [WO₄]^2? groups and luminescence changed from single exponential to double exponential fitting. Furthermore, the excitation transitions of Eu³⁺ between different energy levels (such as ⁷F₀→⁵L₆, ⁷F₀→⁵D₂) also produced interesting changes. Based on analysis of photoluminescence spectra and the chromaticity coordinates in this study, it could be verified that the nonreversing energy transfer of [WO₄]^2?→Eu³⁺ was efficient and incomplete.     

Chromatography process development in the quality by design paradigm I: Establishing a high‐throughput process development platform as a tool for estimating “characterization space” for an ion exchange chromatography step

This article describes the development of a high‐throughput process development (HTPD) platform for developing chromatography steps. An assessment of the platform as a tool for establishing the “characterization space” for an ion exchange chromatography step has been performed by using design of experiments. Case studies involving use of a biotech therapeutic, granulocyte colony‐stimulating factor have been used to demonstrate the performance of the platform. We discuss the various challenges that arise when working at such small volumes along with the solutions that we propose to alleviate these challenges to make the HTPD data suitable for empirical modeling. Further, we have also validated the scalability of this platform by comparing the results from the HTPD platform (2 and 6 μL resin volumes) against those obtained at the traditional laboratory scale (resin volume, 0.5 mL). We find that after integration of the proposed correction factors, the HTPD platform is capable of performing the process optimization studies at 170‐fold higher productivity. The platform is capable of providing semi‐quantitative assessment of the effects of the various input parameters under consideration. We think that platform such as the one presented is an excellent tool for examining the “characterization space” and reducing the extensive experimentation at the traditional lab scale that is otherwise required for establishing the “design space.” Thus, this platform will specifically aid in successful implementation of quality by design in biotech process development. This is especially significant in view of the constraints with respect to time and resources that the biopharma industry faces today. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 403–414, 2013     

Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus‐infected cell

Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A^pro). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET‐based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP²) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real‐time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A^pro catalytic activity, irrespective of other viral‐encoded protease, the activated caspases or general inhibition of protein synthesis in the EV‐infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease–substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus‐induced host translation inhibition. Biotechnol. Bioeng. 2009; 104: 1142–1152. © 2009 Wiley Periodicals, Inc.     

Triplet Excitons in Highly Efficient Solar Cells Based on the Soluble Small Molecule p‐DTS(FBTTh2)2

Triplet exciton formation in neat 7,7‐(4,4‐bis(2‐ethylhexyl)‐4H‐silolo[3,2‐b:4,5‐b′] dithiophene‐2,6‐diyl)bis(6‐fluoro‐4‐(5′‐hexyl‐[2,2′‐bithiophen]‐5‐yl)benzo[c][1,2,5]thiadiazole) (p‐DTS(FBTTh₂)₂) and blends with [6,6]‐Phenyl C₇₀ butyric acid methyl ester (PC₇₀BM), with and without the selective solvent additive 1,8‐diiodooctane, is investigated by means of spin sensitive photoluminescence measurements. For all three material systems, a significant amount of long living triplet excitons is detected, situated on the p‐DTS(FBTTh₂)₂ molecules. The characteristic zero‐field splitting parameters for this state are identified to be D = 42 mT (1177 MHz) and E = 5 mT (140 MHz). However, no triplet excitons located on PC₇₀BM are detectable. Using electrically detected spin resonance, the presence of these triplet excitons is confirmed even at room temperature, highlighting that triplet excitons form during solar cell operation and influence the photocurrent and photovoltage. Surprisingly, the superior performing blend is found to have the largest triplet population. It is concluded, that the formation of triplet excitons from charge transfer states via electron back transfer has no crucial impact on device performance in p‐DTS(FBTTh₂)₂:PC₇₀BM based solar cells.     

Insight into the redox partner interaction mechanism in cytochrome P450BM‐3 using molecular dynamics simulations

Flavocytochrome P450BM‐3 is a soluble bacterial reductase composed of two flavin (FAD/FMN) and one HEME domains. In this article, we have performed molecular dynamics simulations on both the isolated FMN and HEME domains and their crystallographic complex, with the aim to study their binding modes and to garner insight into the interdomain electron transfer (ET) mechanism. The results evidenced an interdomain conformational rearrangement that reduces the average distance between the FMN and HEME cofactors from 1.81 nm, in the crystal structure, to an average value of 1.41 ± 0.09 nm along the simulation. This modification is in agreement with previously proposed hypotheses suggesting that the crystallographic FMN/HEME complex is not in the optimal arrangement for favorable ET rate under physiological conditions. The calculation of the transfer rate along the simulation, using the Pathways Path method, demonstrated the occurrence of seven ET pathways between the two redox centers, with three of them providing ET rates (K_ET) comparable with the experimental one. The sampled ET pathways comprise the amino acids N319, L322, F390, K391, P392, F393, A399, C400, and Q403 of the HEME domain and M490 of the FMN domain. The values of K_ET closer to the experiment were found along the pathways FMN(C7) → F390 → K391 → P392 → HEME(Fe) and FMN(C8) → M490 → F393 → HEME(Fe). Finally, the analysis of the collective modes of the protein complex evidences a clear correlation of the first two essential modes with the activation of the most effective ET pathways along the trajectory. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 197–209, 2014.     

Essential function of the N‐termini tails of the proteasome for the gating mechanism revealed by molecular dynamics simulations

Proteasome is involved in the degradation of proteins. Proteasome activators bind to the proteasome core particle (CP) and facilitate opening a gate of the CP, where Tyr8 and Asp9 in the N‐termini tails of the CP form the ordered open gate. In a double mutant (Tyr8Gly/Asp9Gly), the N‐termini tails are disordered and the stabilized open‐gate conformation cannot be formed. To understand the gating mechanism of the CP for the translocation of the substrate, four different molecular dynamics simulations were carried out: ordered‐ and Tyr8Gly/Asp9Gly disordered‐gate models of the CP complexed with an ATP‐independent PA26 and ordered‐ and disordered‐gate models of the CP complexed with an ATP‐dependent PAN‐like activator. The free‐energies of the translocation of a polypeptide substrate moving through the gate were estimated. In the ordered‐gate models, the substrate in the activator was more stable than that in the CP. The conformational entropy of the N‐termini tails of the CP was larger when the substrate was in the activator than in the CP. In the disordered‐gate models, the substrate in the activator was more destabilized than in the ordered‐gate models. The mutated N‐termini tails became randomized and their increased conformational entropy could no longer increase further even when the substrate was in the activator, meaning the randomized N‐termini tails had lost the ability to stabilize the substrate in the activator. Thus, it was concluded that the dynamics of the N‐termini tails entropically play a key role in the translocation of the substrate. Proteins 2014; 82:1985–1999. © 2014 Wiley Periodicals, Inc.     

Evaluation of glucose sensitive affinity binding assay entrapped in fluorescent dissolved‐core alginate microspheres

The feasibility of dissolved‐core alginate‐templated fluorescent microspheres as “smart tattoo” glucose biosensors was investigated in simulated interstitial fluid (SIF). The sensor works on the principle of competitive binding and fluorescence resonance energy transfer. The sensor consists of multilayer thin film coated alginate microspheres incorporating dye‐labeled glucose receptor and competing ligand within the partially dissolved alginate core. In this study, different approaches for the sensing and detection chemistry were studied, and the response of encapsulated reagents was compared with the solution‐phase counterparts. The glucose sensitivity of the encapsulated TRITC‐Con A/FITC‐dextran (500 kDa) assay in DI water was estimated to be 0.26%/mM glucose while that in SIF was observed to be 0.3%/mM glucose. The glucose sensitivity of TRITC‐apo‐GOx/FITC‐dextran (500 kDa) assay was estimated to be 0.33%/mM glucose in DI water and 0.5%/mM glucose in SIF and both demonstrated a response in the range of 0–50 mM glucose. Therefore, it is hypothesized that the calcium ion concentration outside the microsphere (in the SIF) does not interfere with the response sensitivity. The sensor response was observed to exhibit a maximum response time of 120 s. The system further exhibited a sensitivity of 0.94%/mM glucose with a response in range of 0–50 mM glucose, using near‐infrared dyes (Alexa Fluor‐647‐labeled dextran as donor and QSY‐21‐conjugated apo‐GOx as acceptor), thereby making the sensor more amenable to in vivo use, when implanted in scattering tissue. Biotechnol. Bioeng. 2009; 104: 1075–1085. © 2009 Wiley Periodicals, Inc.     

Folding propensity and biological activity of peptides: the effect of a single stereochemical isomerization on the conformational properties of bombinins in aqueous solution

Folding propensities of bombinins H2 and H4, two members of amphibian bombinins H, a family of 17-20 residue alpha-helical peptides, have been investigated by means of circular dichroism (CD) measurements and molecular dynamics (MD) simulations. The two peptides, with primary structure IIGPVLGLVGSALGGLLKKI-NH2 and differing only for the configuration of the second aminoacid (an L-isoleucine in H2 and a D-alloisoleucine in H4) behave rather differently in solution. In particular both CD measurements and MD simulations indicate that bombinin H2 shows a markedly higher tendency to fold. From a careful inspection of MD trajectories it emerges that the stereochemical isomerization mutation of residue 2 to D-alloisoleucine in H4 peptide, drastically decreases its ability to form intrapeptide contacts. MD simulations also indicate that the conformational sampling in both systems derives from a subtle combination of energetic and entropic effects both involving the peptide itself and the solvent. The present results have been finally paralleled with preliminary information on bombinins H2 and H4 biological activity, i.e. interaction with membrane, supporting the hypothesis of an "already folded" conformation in water rather than interfacial folding tenet.     

Theoretical investigation on the glycan‐binding specificity of Agrocybe cylindracea galectin using molecular modeling and molecular dynamics simulation studies

Galectins are β‐galactoside binding proteins which have the ability to serve as potent antitumor, cancer biomarker, and induce tumor cell apoptosis. Agrocybe cylindracea galectin (ACG) is a fungal galectin which specifically recognizes α(2,3)‐linked sialyllactose at the cell surface that plays extensive roles in the biological recognition processes. To investigate the change in glycan‐binding specificity upon mutations, single point and double point site‐directed in silico mutations are performed at the binding pocket of ACG. Molecular dynamics (MD) simulation studies are carried out for the wild‐type (ACG) and single point (ACG1) and double point (ACG2) mutated ACGs to investigate the dynamics of substituted mutants and their interactions with the receptor sialyllactose. Plausible binding modes are proposed for galectin–sialylglycan complexes based on the analysis of hydrogen bonding interactions, total pair‐wise interaction energy between the interacting binding site residues and sialyllactose and binding free energy of the complexes using molecular mechanics–Poisson–Boltzmann surface area. Our result shows that high contribution to the binding in different modes is due to the direct and water‐mediated hydrogen bonds. The binding specificity of double point mutant Y59R/N140Q of ACG2 is found to be high, and it has 26 direct and water‐mediated hydrogen bonds with a relatively low‐binding free energy of −47.52 ± 5.2 kcal/mol. We also observe that the substituted mutant Arg59 is crucial for glycan‐binding and for the preference of α(2,3)‐linked sialyllactose at the binding pocket of ACG2 galectin. When compared with the wild‐type and single point mutant, the double point mutant exhibits enhanced affinity towards α(2,3)‐linked sialyllactose, which can be effectively used as a model for biological cell marker in cancer therapeutics. Copyright © 2015 John Wiley & Sons, Ltd.     

Detection of Pseudo-nitzschia (Bacillariophyceae) species and amnesic shellfish toxins in Scottish coastal waters using oligonucleotide probes and the Jellet Rapid Test™

Species specific LSU rRNA targeted fluorescent oligonucleotide probes, designed by researchers at the Monterey Bay Aquarium Research Institute (USA) for a limited range of Pseudo-nitzschia species, were applied to unialgal cultures and Scottish field samples, to investigate possible applications in Scottish phytoplankton monitoring programmes to detect potential amnesic shellfish poisoning (ASP) toxin producing species. The existing available probe for Pseudo-nitzschia australis gave good results, positively labelling cells from cultures and field samples. However, application of the P. pungens, P. delicatissima and P. fraudulenta probes gave poor results, with little or no fluorescence label observed in field samples, while transmission electron microscopy (TEM) showed these species to be present. Comparison of the same region of the LSU sequence from cultures of P. delicatissima, isolated from Scottish waters, with the probe designed for detection of P. delicatissima isolated from Monterey Bay revealed the presence of a single base difference between the two sequences, which may have prevented the probe from hybridising to Scottish isolates and cells from field samples. In an attempt to assess the potential ASP toxin production by field populations of Pseudo-nitzschia a rapid immunodiagnostic test (the Jellet Rapid Test, JRT) for ASP toxins was examined. Results indicate that additional development of molecular probes for the detection of a range of Pseudo-nitzschia species detected in Scottish coastal waters and the use of JRT for toxin detection could conceivably provide an effective tool for broad-scale mapping of toxin events and management of coastal zone activities.     

Helix-coil transition of alanine peptides in water: force field dependence on the folded and unfolded structures

The force fields used in classical modeling studies are semiempirical in nature and rely on their validation by comparison of simulations with experimental data. The all-atom replica-exchange molecular dynamics (REMD) methodology allows us to calculate the thermodynamics of folding/unfolding of peptides and small proteins, and provides a way of evaluating the reliability of force fields. We apply the REMD to obtain equilibrium folding/unfolding thermodynamics of a 21-residue peptide containing only alanine residues in explicit aqueous solution. The thermodynamics of this peptide is modeled with both the OPLS/AA/L and the A94/MOD force fields. We find that the helical content and the values for the helix propagation and nucleation parameters for this alanine peptide are consistent with measurements on similar peptides and with calculations using the modified AMBER force field (A94/MOD). The nature of conformations, both folded and unfolded, that contributes to the helix-coil transition profile, however, is quite different between these two force fields.     

Let's think again about using mammalian temperature‐sensitive mutants to investigate functional molecules—The perspectives from the studies on three mutants showing chromosome instability

This review evaluates the use of temperature‐sensitive (ts) mutants to investigate functional molecules in mammalian cells. A series of studies were performed in which mammalian cells expressing functional molecules were isolated from ts mutants using complementation by the introduction and expression of the responsible protein tagged with the green fluorescent protein. The results showed that chromosome instability and cell‐cycle arrest were caused by ts defects in the following three molecules: the largest subunit of RNA polymerase II, a protein involved in splicing, and ubiquitin‐activating enzyme. The cells expressing functional protein were then isolated by introducing the responsible gene tagged with the green fluorescent protein to complement the ts phenotype. These cells proved to be useful in analyzing the dynamics of RNA polymerase II in living cells. Analyses of the functional interaction between proteins involved in splicing were also useful in the investigation of ts mutants and their derivatives. In addition, these cells demonstrated the functional localization of ubiquitin‐activating enzyme in the nucleus. Mammalian ts mutants continue to show great potential to aid in understanding the functions of the essential molecules in cells. Therefore, it is highly important that studies on the identification and characterization of the genes responsible for the phenotype of a mutant are carried out.