Controlled delivery of proteins into bilayer lipid membranes on chip

The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.     

Spin-state selective carbon-detected HNCO with TROSY optimization in all dimensions and double echo-antiecho sensitivity enhancement in both indirect dimensions

A carbon-detected TROSY-optimized experiment correlating 1HN, 15N, and 13C' resonances, referred to as c-TROSY-HNCO is presented, in which the 1HN and 15N TROSY effects are maintained in both indirect dimensions, while the directly detected 13C' is doubly TROSY-optimized with respect to 1HN and 15N. A new strategy for sensitivity enhancement, the so-called double echo-antiecho (dEA), is described and implemented in the c-TROSY-HNCO experiment. dEA offers sensitivity enhancement of square root of 2 in both indirect dimensions and is generally applicable to many multidimensional experiments. A carbon-detected HNCO experiment, c-HNCO, without TROSY optimization and sensitivity enhancement is also designed for comparison purposes. Relaxation simulations show that for a protein with a rotational correlation time of 10 ns or larger, the c-TROSY-HNCO experiment displays comparable or higher signal-to-noise (S/N) ratios than the c-HNCO experiment, although the former selects only 1/4 of the initial magnetization relative to the later. The high resolution afforded in the directly detected carbon dimension allows direct measurement of the doublet splitting to extract 1JCalphaC' scalar and 1DCalphaC' residual dipolar couplings. Simulations indicate that the c-TROSY-HNCO experiment offers higher precision (lower uncertainty) compared to the c-HNCO experiment for larger proteins. The experiments are applied to 15N/13C/2H/[Leu,Val]-methyl-protonated IIBMannose, a protein of molecular mass 18.6 kDa with a correlation time of approximately 10 ns at 30 degrees C. The experimental pairwise root-mean-square deviation for the measured 1JCalphaC' couplings obtained from duplicate experiments is 0.77 Hz. By directly measuring the doublet splitting, the experiments described here are expected to be much more tolerant to nonuniform values of 1JCalphaC' (or 1JCalphaC' + 1DCalphaC' for aligned samples) and pulse imperfections due to the smaller number of applied pulses in the "out-and-stay" coherence transfer in the c-HNCO-TROSY experiment relative to conventional 1H-detected "out-and-back" quantitative J correlation experiments. A carbon-detected TROSY-optimized experiment correlating 1HN, 15N, and 13C' resonances, referred to as c-TROSY-HNCO is presented, in which the 1HN and 15N TROSY effects are maintained in both indirect dimensions, while the directly detected 13C' is doubly TROSY-optimized with respect to 1HN and 15N. A new strategy for sensitivity enhancement, the so-called double echo-antiecho (dEA), is described and implemented in the c-TROSY-HNCO experiment. dEA offers sensitivity enhancement of in both indirect dimensions and is generally applicable to many multidimensional experiments.     

Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

Variations on a theme of Jost and Pais

We explore the extent to which a variant of a celebrated formula due to Jost and Pais, which reduces the Fredholm perturbation determinant associated with the Schrödinger operator on a half-line to a simple Wronski determinant of appropriate distributional solutions of the underlying Schrödinger equation, generalizes to higher dimensions. In this multi-dimensional extension the half-line is replaced by an open set , , n2, where Ω has a compact, nonempty boundary ∂Ω satisfying certain regularity conditions. Our variant involves ratios of perturbation determinants corresponding to Dirichlet and Neumann boundary conditions on ∂Ω and invokes the corresponding Dirichlet-to-Neumann map. As a result, we succeed in reducing a certain ratio of modified Fredholm perturbation determinants associated with operators in L²(Ω;dⁿx), , to modified Fredholm determinants associated with operators in L²(∂Ω;dⁿ⁻¹σ), n2. Applications involving the Birman–Schwinger principle and eigenvalue counting functions are discussed.     

Oligonuclear complexes as tectons in crystal engineering: structural diversity and magnetic properties

This article focuses on the employment of bi- and trinuclear complexes as building blocks in designing novel heterometallic systems. A large variety of polynuclear complexes, ranging from high-nuclearity clusters to high-dimensionality coordination polymers, can be constructed by taking advantage of the high flexibility of the multimetallic nodes. The following oligonuclear complexes are currently used as tectons in our laboratory: (a) bis(alkoxo)-bridged copper(ii) species; (b) homobinuclear species with metal ions held together by end-off, or macrocyclic compartmental ligands; (c) heterometallic complexes with dissymmetric compartmental ligands. The 3d-4f nodes are particularly interesting since the metal ions interact selectively with various spacers. The intra-node exchange interactions, as well as those between the resulting spins, generate interesting magnetic properties.     

Weighted Estimations from a Conjugate Operator

In this Letter we develop a general procedure leading from a Mourre-type estimation for a given self-adjoint operator H to a Hardy-type weighted inequality. We use this method in order to prove exponential decay for eigenvectors of a large class of perturbations of operators of convolution with bounded analytic functions.     

Complex Powers of the Neumann Laplacian in Lipschitz Domains

Let L^q_r(Ω) be the usual scale of Sobolev spaces and let Δ_N be the Neumann Laplacian in an arbitrary Lipschitz domain Ω. We present an interpolation based approach to the following question: for what range of indices does map isomorphically onto L^q_r(Ω)/ℝ?