Anchor chain length alters the apparent mechanism of chloride channel function in SCMTR derivatives

Two membrane-anchored heptapeptides have been prepared and their pore-formation behavior in phospholipid bilayer membranes has been found to differ profoundly as a result only of alkyl chain length.     

Simulation of proton transport in the gramicidin A channel

The free energy profiles for proton transfer along the oriented water file inside the gramicidin A channel were calculated. An original implementation of the rigid-body molecular dynamics method was used for describing the peptide groups of the channel and outer water molecules. The inner water wire was simulated using the PM6 force field parameters, which adequately describe the formation and cleavage of chemical and hydrogen bonds in water molecules. Different mechanisms of proton transfer through the gramicidin A channel were considered, namely, proton H⁺ translocation, transfer of the anion defect OH^?, and reorientation of the water file inside the channel. To facilitate parallel calculations of trajectories, the reaction coordinate was divided into segments, and the results were combined by the weighted histogram analysis method. The first two processes, H⁺ and OH^? transfers, were shown to be barrierless. Only the stage of reorientation of the water file inside the channel has an energy barrier.     

Cooperative transport in a potassium ion channel

Our current understanding of ion permeation through the selectivity filter of the KcsA potassium channel is based on the concept of a multi-ion transport mechanism. The details of this concerted movement, however, are not well understood. In the present paper we report on molecular dynamics simulations which provides new insights. It is shown that ion translocation is based on the collective hopping of ions and water molecules which is mediated by the flexible charged carbonyl groups lining the backbone of the pore. In particular, there is strong evidence for pairwise translocations where one ion and one water molecule form a bound state. We suggest a physical explanation of the observed phenomena employing a simple lattice model. It is argued that the water molecules can act as rectifiers during the hopping of ion-water pairs.     

A synthetic, chloride-selective channel that alters chloride transport in epithelial cells

An Ussing chamber was used to demonstrate that synthetic amphiphilic anion transporters function as chloride transporters in mammalian airway epithelial cells.     

Molecular dynamics simulations on the Escherichia coli ammonia channel protein AmtB: mechanism of ammonia/ammonium transport

Molecular dynamics (MD) simulations have been performed at the atomic level to study the ammonium/ammonia transport across the Escherichia coli AmtB membrane protein. Although ammonia primarily exists in the form of NH(4)(+) in aqueous solution, the recent X-ray structure determination of AmtB reveals that the ammonium/ammonia transporter proteins are ammonia-conducting channels rather than ammonium ion transporters [Khademi, S.; et al. Science 2004, 305, 1587; Zheng, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17090]. Our simulations showed that the entrance of NH(4)(+) into the periplasmic recruitment vestibule requires only 3.1 kcal/mol of energy. This is consistent with the X-ray crystal structure, where one NH(4)(+) is captured in the binding vestibule. In this vestibule, NH(4)(+) loses one water of hydration, but the loss is compensated by a hydrogen bond, first with the backbone carbonyl oxygen of Phe161 then with the hydroxyl group of Ser219, as well as the stabilizing pi-cation interactions with the aromatic rings of Trp148 and Phe107 in the AmtB protein. In the end of this recruitment vestibule, the phenyl ring of Phe107 dynamically switches to an open state. This is correlated with a slight rotation and shifting of the indole ring of Trp148, which eventually creates a slot for the initially buried carboxylate group of Asp160 to become exposed to the bulk solvent. A hydrogen bond wire between NH(4)(+) and the carboxylate group of Asp160 via two water molecules was observed. Thus, Asp160 is most likely the proton acceptor from NH(4)(+). This explains the high conservation of Asp160 in Amt proteins and why the D160A mutant would completely quench the activity of AmtB [Javelle, A.; et al. J. Biol. Chem. 2004, 279, 8530; Marini, A. M.; et al. Curr. Genet. 2006, 49, 364]. Once NH(4)(+) deprotonates, the phenyl ring of Phe215 rotates to open, and the subsequent passage of NH(3) through the channel is straightforward.     

Efficient electron transfer in a protein network lacking specific interactions

In many biochemical processes, proteins need to bind partners amidst a sea of other molecules. Generally, partner selection is achieved by formation of a single-orientation complex with well-defined, short-range interactions. We describe a protein network that functions effectively in a metabolic electron transfer process but lacks such specific interactions. The soil bacterium Paracoccus denitrificans oxidizes a variety of compounds by channeling electrons into the main respiratory pathway. Upon conversion of methylamine by methylamine dehydrogenase, electrons are transported to the terminal oxidase to reduce molecular oxygen. Steady-state kinetic measurements and NMR experiments demonstrate a remarkable number of possibilities for the electron transfer, involving the cupredoxin amicyanin as well as four c-type cytochromes. The observed interactions appear to be governed exclusively by the electrostatic nature of each of the proteins. It is concluded that Paracoccus provides a pool of cytochromes for efficient electron transfer via weak, ill-defined interactions, in contrast with the view that functional biochemical interactions require well-defined molecular interactions. It is proposed that the lack of requirement for specificity in these interactions might facilitate the integration of new metabolic pathways.     

Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins

In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.     

Interpretation of apparent activation energies for electron transport in dye-sensitized nanocrystalline solar cells

Electron transport in dye-sensitized nanocrystalline solar cells appears to be a slow diffusion-controlled process. Values of the apparent electron diffusion coefficient are many orders of magnitude smaller than those reported for bulk anatase. The slow transport of electrons has been attributed to multiple trapping (MT) at energy levels distributed exponentially in the band gap of the nanocrystalline oxide. In the MT model, release of immobile electrons from occupied traps to the conduction band is a thermally activated process, and it might therefore be expected that the apparent electron diffusion coefficient should depend strongly on temperature. In fact, rather small activation energies (0.1-0.25 eV) have been derived from time and frequency resolved measurements of the short circuit photocurrent. It is shown that the MT model can give rise to such anomalously low apparent activation energies as a consequence of the boundary conditions imposed by the short circuit condition and the quasi-static relationship between changes in the densities of free and trapped electrons. This conclusion has been confirmed by exact numerical solutions of the time-dependent generation/collection problem for periodic excitation that provide a good fit to experimental data.     

A study on the condition for differential electrophoretic transport at a channel entrance

Electrophoretic differential transport of ionic species in a solution moving from a large reservoir into a small channel is investigated numerically. The system setup is similar to the experiments of Polson, Savin, and Hayes (J. Microcol. Sep. 2000, 12, 98), where the bulk flow into a fused-silica capillary was driven by a pressure differential. A critical condition for achieving the defined differential transport near the channel entrance is found and this condition is solely determined by a dimensionless parameter when the geometry of the system is prescribed. This dimensionless parameter is the ratio between the electrophoretic migration velocity of the species based on the apparent electric intensity and the centerline fluid velocity of the fully developed channel flow. Species concentration distributions are also computed for various conditions. A separation technique can be derived from the experimental condition where a targeted division of species can be created at the channel entrance.     

A new productivity function and stability criterion in chemical vapor transport processes

The crystal growth rate in a chemical vapor transport process using a closed system is analyzed on the basis of a one-dimensional configuration. A simplified model of vapor transport enables one to obtain a set of equations yielding the rates of reaction without a complete evaluation of the partial pressure gradients. This linear set comprises as many equations as independent chemical reactions. The effect of finite interface kinetics is formally taken into account. The efficiency of a one-reaction process is given by a function involving the mole fractions of the gaseous species and the stoichiometric coefficients in the formula equation. The features of such a productivity function are discussed. Maximum growth rate is achieved if the gaseous components are present in stoichiometric quantities. The concept of the productivity function is illustrated by chemical vapor transport systems involving binary and ternary gaseous phases. Proceeding from a two-phase source material, stability criteria that define stable one-phase and stable two-phase crystal growth are given. The kind of deposit may be changed by altering the amount of transporting agent. It is shown that limited interface kinetics favors a two-phase deposit.     

Ab initio molecular orbital study of the flavin-catalyzed dehydrogenation reaction of glycine - protein transport channel driving hydride-transfer mechanism

The reaction mechanism of flavin-catalyzed dehydrogenation of glycine has been studied by ab initio molecular orbital calculations using the 6-31G* basis set. 10-Methyl isoalloxazine (10-MIA) has been used as the flavin model compound. The results showed that when we assume a proton transport channel in amino acid oxidase, which is switched on by the substrate anion, the O12-protonated 10-MIA [10-MIAH⁺(O12)] is generated. The main structure of 10-MIAH⁺(O12) is one in which the central ring is expressed by an NAD⁺-like structure, which is favorable for driving the hydride-transfer reaction, i.e., the abstraction of the α-hydrogen of glycine by the hydride-transfer mechanism. We have found that this protonation results in a dramatic lowering of the activation energy of the reaction. The proposed mechanism is summarized as follows: the hydride transfer proceeds via two-electron transfer and synchronous intramolecular proton transfer → intermolecular proton transfer. Received: 10 August 1998 / Accepted: 17 September 1998 / Published online: 8 February 1999     

A new protein graph model for function prediction

As several structural proteomic projects are producing an increasing number of protein structures with unknown function, methods that can reliably predict protein functions from protein structures are in urgent need. In this paper, we present a method to explore the clustering patterns of amino acids on the 3-dimensional space for protein function prediction. First, amino acid residues on a protein structure are clustered into spatial groups using hierarchical agglomerative clustering, based on the distance between them. Second, the protein structure is represented using a graph, where each node denotes a cluster of amino acids. The nodes are labeled with an evolutionary profile derived from the multiple alignment of homologous sequences. Then, a shortest-path graph kernel is used to calculate similarities between the graphs. Finally, a support vector machine using this graph kernel is used to train classifiers for protein function prediction. We applied the proposed method to two separate problems, namely, prediction of enzymes and prediction of DNA-binding proteins. In both cases, the results showed that the proposed method outperformed other state-of-the-art methods.     

Hindered transmembrane protein transport in hollow-fibre devices

Protein transport in the extracapillary and intracapillary spaces (ECS and ICS) as well as across the membranes of hollow-fibre devices was investigated. Regenerated-cellulose ultrafiltration membrane cartridges were loaded with solutions of myoglobin and operated in the closed-shell mode for a period of several days. The transmembrane leakage and spatial distribution of myoglobin were dependent on the protein loading, the ICS flow rate and the presence of bovine serum albumin in the ECS. A mathematical model was developed to characterise the time-dependent protein redistribution, taking into account the effects of osmotic pressure, fibre expansion under wet conditions and the fibre-free manifold regions of the ECS. According to the model, the transmembrane transport of protein can be described using one semi-empirical parameter, the membrane constant, defined as the ratio of the square of pore tortuosity to surface porosity, from which the osmotic reflection coefficient, the partition coefficient, and the diffusive and convective hindrance factors can be calculated. The experimental data were used to test the model and to find the values of the membrane constant for the system under consideration.     

Top-down proteomics reveals novel protein forms expressed in methanosarcina acetivorans

Combining our previous work with current platforms presented here that identified 99 new proteins (Supplemental Table 1), a total of 200 protein identities in M. acetivorans have been confirmed. Of these, five proteins were unannotated, 15 were mispredicted, two exhibited variable removal of the start methionine, and one was a proteolysis fragment from the identical C-terminus of two possible proteins. The incorporation of LC-MS/MS and offline automation speeds up the processing of samples through automation of the top-down process. For five data files that were automatically processed and iteratively searched, 2 h were required to complete analysis. In these, a total of 835 identifications were detected by the search algorithm in this time period. The expanded use of top-down will use both online and offline strategies, with increasingly sophisticated data acquisition strategies tailored for the challenges of top-down fragmentation [[16], [18], [19], [32] and [33]].     

A fluorescence aptasensor based on DNA charge transport for sensitive protein detection in serum

A novel fluorescence aptasensor based on DNA charge transport for sensitive protein detection has been developed. A 15nt DNA aptamer against thrombin was used as a model system. The aptamer was integrated into a double strand DNA (dsDNA) that was labeled with a hole injector, naphthalimide (NI), and a fluorophore, Alexa532, at its two ends. After irradiation by UV light, the fluorescence of Alexa532 was bleached due to the oxidization of Alexa532 by the positive charge transported from naphthalimide through the dsDNA. In the presence of thrombin, the binding of thrombin to the aptamer resulted in the unwinding of the dsDNA into ssDNA, which led to the blocking of charge transfer and the strong fluorescence emission of Alexa532. By monitoring the fluorescence signal change, we were able to detect thrombin in homogeneous solutions with high selectivity and high sensitivity down to 1.2 pM. Moreover, as DNA charge transfer is resistant to interferences from biological contexts, the aptasensor can be used directly in undiluted serum with similar sensitivity as that in buffer. This new sensing strategy is expected to promote the exploitation of aptamer-based biosensors for protein assays in complex biological matrixes.     

A parallel finite element simulator for ion transport through three‐dimensional ion channel systems

A parallel finite element simulator, ichannel, is developed for ion transport through three‐dimensional ion channel systems that consist of protein and membrane. The coordinates of heavy atoms of the protein are taken from the Protein Data Bank and the membrane is represented as a slab. The simulator contains two components: a parallel adaptive finite element solver for a set of Poisson–Nernst–Planck (PNP) equations that describe the electrodiffusion process of ion transport, and a mesh generation tool chain for ion channel systems, which is an essential component for the finite element computations. The finite element method has advantages in modeling irregular geometries and complex boundary conditions. We have built a tool chain to get the surface and volume mesh for ion channel systems, which consists of a set of mesh generation tools. The adaptive finite element solver in our simulator is implemented using the parallel adaptive finite element package Parallel Hierarchical Grid (PHG) developed by one of the authors, which provides the capability of doing large scale parallel computations with high parallel efficiency and the flexibility of choosing high order elements to achieve high order accuracy. The simulator is applied to a real transmembrane protein, the gramicidin A (gA) channel protein, to calculate the electrostatic potential, ion concentrations and I – V curve, with which both primitive and transformed PNP equations are studied and their numerical performances are compared. To further validate the method, we also apply the simulator to two other ion channel systems, the voltage dependent anion channel (VDAC) and α‐Hemolysin (α‐HL). The simulation results agree well with Brownian dynamics (BD) simulation results and experimental results. Moreover, because ionic finite size effects can be included in PNP model now, we also perform simulations using a size‐modified PNP (SMPNP) model on VDAC and α‐HL. It is shown that the size effects in SMPNP can effectively lead to reduced current in the channel, and the results are closer to BD simulation results. © 2013 Wiley Periodicals, Inc.     

Development and validation of HTS assay for screening the calcium-activated chloride channel modulators in TMEM16A stably expressed CHO cells

Calcium-activated chloride channels (CaCCs), for example TMEM16A, are widely expressed in a variety of tissues and are involved in many important physiological functions. We developed and validated an atomic absorption spectroscopy (AAS)-based detection system for high-throughput screening (HTS) of CaCC modulators. With this assay, Cl^? flux from CHO cells stably transfected with TMEM16A is assayed indirectly, by measuring excess silver ions (Ag⁺) in the supernatant of AgCl precipitates. The screening process involved four steps: (1) TMEM16A CHO cells were incubated in high-K⁺ and high-Cl^? buffer with test compounds, and with ionomycin as Ca²⁺ ionophore, for 12 min; (2) cells were washed with a low-K⁺, Cl^?-free and Ca²⁺-free buffer; (3) CaCC/TMEM16A were activated in high-K⁺, Cl^?-free buffer with ionomycin (10 μmol L^?1) for 12 min; and (4) excess Ag⁺ concentration was measured using an ion channel reader (ICR, an AAS system). The assay can be used to screen CaCC activators and inhibitors at the same time. With this assay, positive control drugs, including NPPB, CaCCinh-A01, flufenamic acid (Flu) and E_act, all had good concentration-dependent effects on CaCC/TMEM16A. NPPB and CaCCinh-A01 inhibited the CaCC/TMEM16A currents completely at 300 μmol L^?1, with IC₅₀ values of 39.35?±?4.72 μmol L^?1 and 6.35?±?0.27 μmol L^?1, respectively; and E_act, activated CaCC/TMEM16A, with an EC₅₀ value of 3.92?±?0.87 μmol L^?1.