One-step formation of three-dimensional interconnected T-shaped microstructures inside composites by orthogonal bidirectional self-assembly method

The fillers inside a polymer matrix should typically be self-assembled in both the horizontal and vertical directions to obtain 3-dimentional (3D) percolation pathways, whereby the fields of application can be expanded and the properties of organic-inorganic composite films improved. Conventional dielectrophoresis techniques can typically only drive fillers to self-assemble in only one direction. We have devised a one-step dielectrophoresis-driven approach that effectively induces fillers self-assembly along two orthogonal axes, which results in the formation of 3D interconnected T-shaped iron microstructures (3D-T CIP) inside a polymer matrix. This approach to carbonyl iron powder (CIP) embedded in a polymer matrix results in a linear structure along the thickness direction and a network structure on the top surface of the film. The fillers in the polymer were controlled to achieve orthogonal bidirectional self-assembly using an external alternating current (AC) electric field and a non-contact technique that did not lead to electrical breakdown. The process of 3D-T CIP formation was observed in real time using in situ observation methods with optical microscopy, and the quantity and quality of self-assembly were characterized using statistical and fractal analysis. The process of fillers self-assembly along the direction perpendicular to the electric field was explained by finite element analogue simulations, and the results indicated that the insulating polyethylene terephthalate (PET) film between the electrode and the CIP/prepolymer suspension was the key to the formation of the 3D-T CIP. In contrast to the traditional two-step method of fabricating sandwich-structured film, the fabricated 3D-T CIP film with 3D electrically conductive pathways can be applied as magnetic field sensor.</p

Fluorescence Analysis of the Lipid Binding-Induced Conformational Change of Apolipoprotein E4

Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix–helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipid-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL₃ suggests that not only apoE–lipid interactions but also protein–protein interactions are important for apoE4 binding to HDL₃

Kinetic and Thermodynamic Analyses of Spontaneous Exchange between High-Density Lipoprotein-Bound and Lipid-Free Apolipoprotein A‑I

It is thought that apolipoprotein A-I (apoA-I) spontaneously exchanges between high-density lipoprotein (HDL)-bound and lipid-free states, which is relevant to the occurrence of preβ-HDL particles in plasma. To improve our understanding of the mechanistic basis for this phenomenon, we performed kinetic and thermodynamic analyses for apoA-I exchange between discoidal HDL-bound and lipid-free forms using fluorescence-labeled apoA-I variants. Gel filtration experiments demonstrated that addition of excess lipid-free apoA-I to discoidal HDL particles promotes exchange of apoA-I between HDL-associated and lipid-free pools without alteration of the steady-state HDL particle size. Kinetic analysis of time-dependent changes in NBD fluorescence upon the transition of NBD-labeled apoA-I from HDL-bound to lipid-free state indicates that the exchange kinetics are independent of the collision frequency between HDL-bound and lipid-free apoA-I, in which the lipid binding ability of apoA-I affects the rate of association of lipid-free apoA-I with the HDL particles and not the rate of dissociation of HDL-bound apoA-I. Thus, C-terminal truncations or mutations that reduce the lipid binding affinity of apoA-I strongly impair the transition of lipid-free apoA-I to the HDL-bound state. Thermodynamic analysis of the exchange kinetics demonstrated that the apoA-I exchange process is enthalpically unfavorable but entropically favorable. These results explain the thermodynamic basis of the spontaneous exchange reaction of apoA-I associated with HDL particles. The altered exchangeability of dysfunctional apoA-I would affect HDL particle rearrangement, leading to perturbed HDL metabolism

Molecular Complex Composed of β‑Cyclodextrin-Grafted Chitosan and pH-Sensitive Amphipathic Peptide for Enhancing Cellular Cholesterol Efflux under Acidic pH

Excess of cholesterol in peripheral cells is known to lead to atherosclerosis. In this study, a molecular complex composed of β-cyclodextrin-grafted chitosan (BCC) and cellular cholesterol efflux enhancing peptide (CEEP), synthesized by modifying pH sensitive amphipathic GALA peptide, is introduced with the eventual aim of treating atherosclerosis. BCC has a markedly enhanced ability to induce cholesterol efflux from cell membranes compared to β-cyclodextrin, and the BCC-CEEP complex exhibited a 2-fold increase in cellular cholesterol efflux compared to BCC alone under weakly acidic conditions. Isothermal titration calorimetry and fluorescence spectroscopy measurements demonstrated that the random coil structure of CEEP at neutral pH converted to the α-helical structure at acidic pH, resulting in a three-order larger binding constant to BCC (<i>K</i> = 3.7 × 10⁷ at pH 5.5) compared to that at pH 7.4 (<i>K</i> = 7.9 × 10⁴). Such high-affinity binding of CEEP to BCC at acidic pH leads to the formation of 100-nm-sized aggregate with positive surface charge, which would efficiently interact with cell membranes and induce cholesterol efflux. Since the cholesterol efflux ability of HDL is thought to be impaired under acidic environments in advanced atherosclerotic lesions, the BCC-CEEP complex might serve as a novel nanomaterial for treating atherosclerosis

Effects of the Iowa and Milano Mutations on Apolipoprotein A‑I Structure and Dynamics Determined by Hydrogen Exchange and Mass Spectrometry

The Iowa point mutation in apolipoprotein A-I (G26R) leads to a systemic amyloidosis condition, and the Milano mutation (R173C) is associated with hypoalphalipoproteinemia, a reduced plasma level of high-density lipoprotein. To probe the structural effects that lead to these outcomes, we used amide hydrogen–deuterium exchange coupled with a fragment separation/mass spectrometry analysis (HX MS). The Iowa mutation inserts an arginine residue into the nonpolar face of an α-helix that spans residues 7–44 and causes changes in structure and structural dynamics. This helix unfolds, and other helices in the N-terminal helix bundle domain are destabilized. The segment encompassing residues 116–158, largely unstructured in wild-type apolipoprotein A-I, becomes helical. The helix spanning residues 81–115 is destabilized by 2 kcal/mol, increasing the small fraction of time it is transiently unfolded to ≥1%, which allows proteolysis at residue 83 in vivo over time, releasing an amyloid-forming peptide. The Milano mutation situated on the polar face of the helix spanning residues 147–178 destabilizes the helix bundle domain only moderately, but enough to allow cysteine-mediated dimerization that leads to the altered functionality of this variant. These results show how the HX MS approach can provide a powerful means of monitoring, in a nonperturbing way and at close to amino acid resolution, the structural, dynamic, and energetic consequences of biologically interesting point mutations