Hierarchical Structure and Crystal Orientation in Poly(ethylene oxide)/Clay Nanocomposite Films

Water-cast nanocomposite films formed by poly­(ethylene oxide) (PEO) and Laponite clay were found to display three characteristic levels of structure with large-scale orientation. The first level with the length scale of ca. 30–50 nm was the clay lamellar bundles, which tended to stack perpendicularly to the film surface. The second level with the characteristic length of 1.8 nm was associated with the alternating stacking of the silicate layers and the PEO chains sandwiched between them. The preferred orientations of these two levels of structure were independent of clay content, solvent removal rate for the film preparation, and the crystallization temperature of the PEO chains situating outside the clay bundles. The third level of structure was characterized by the preferred orientation of the PEO crystalline stems with respect to the surface of the silicate layers. Perpendicular orientation always dominated in the nanocomposite films prepared by slow solvent removal irrespective of crystallization temperature. In the films prepared by fast solvent removal, however, parallel crystal orientation set in as the clay concentration exceeded ca. 33 wt %. The preferred crystal orientation was ascribed to the confinement effect imposed by the clay bundles to the crystallization of the PEO chains situating in the interbundle region. In the films cast by slow solvent removal, the weaker confinement associated with the larger interbundle distance led to perpendicular crystal orientation. When the interbundle distance was reduced to ca. 30 nm in the films prepared by rapid solvent evaporation, the strong confinement directed the crystals to form parallel orientation

Two-hybrid analysis of interactions between Pxa2_NBD and Pxa2_CT.

<p>Pxa2_NBD was fused to Gal4 AD and Pxa2_CT was fused to Lex BD in yeast two-hybrid plasmids. Two-hybrid analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104892#s4" target="_blank">Materials and Methods</a>.</p

The CT of Pxa2_NBD-CT (Pxa2_CT) promotes its interaction with Pxa1_NBD in an indirect and Pxa2_NBD-CT₁-dependent manner.

<p>(A) Two-hybrid analysis of the interaction between the CT of Pxa2_NBD-CT (Pxa2_CT) and Pxa1_NBD. Two-hybrid plasmids containing Pxa2_CT and Pxa1_NBD were co-transformed into EGY48 (pSH18-34). The yeast transformants were streaked on an X-gal plate and incubated for 16 h at 30°C. Blue indicates the existence of a protein-protein interaction. (B) The effect of the NBD-CT₁ substitution in Pxa2_NBD-CT on its interaction with Pxa1_NBD. The yeast two-hybrid plasmid containing Pxa1_NBD-CT or Pxa2_NBD-CT with the substitution by NBD-CT₁ was co-transformed with the plasmid containing Pxa1_NBD into EGY48 (pSH18-34). The yeast transformants were streaked on an X-gal plate and incubated for 16 h at 30°C. Blue indicates the existence of a protein-protein interaction.</p

General schematic of the functional ABC transporter consisting of two half ABC transporters.

<p>In general, each half ABC transporter contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD) with or without the carboxyl-terminal region (CT).</p

Analysis of co-purification of the CT-deleted mutant proteins of Pxa2_NBD-CT with Pxa1_NBD.

<p>The various CT-deleted proteins of Pxa2_NBD-CT-His₆ were co-expressed with Pxa1_NBD-HA in yeast strain BJ2168, and yeast cells were disrupted by sonication. The total yeast extracts were subjected to a pull-down assay using Ni²⁺-NTA chromatography. The presence of Pxa2-His₆ or Pxa1-HA in the total extract (T), flow-through (F), wash-one (W1), wash-final (WF), elute-one (E1), elute-two (E2), and elute-three (E3) fractions was revealed by Western blot probed with anti-HA antibody for Pxa1-HA (right panel) and anti-His antibody for Pxa2-His6 (left panel). Protein intensity was analyzed by using GeneTools software (Syngene). Protein intensity of the E2 fraction was normalized to that of the T fraction for each experiment.</p

Limited proteolysis analysis of Pxa2_NBD-CT_Y726L and Pxa2_NBD-CT_F731A by proteinase K.

<p>(A) Equal aliquots of total yeast extracts containing Pxa2_NBD-CT_Y726L were placed into each tube. Proteinase K was added to each tube with a serial dilution of 0, 4, 8, and 12 µg/ml. After treatment, proteins were analyzed by SDS-PAGE and Western blot probed with anti-His-tag antibodies. The vehicle represented the vector-only control. The different peptide bands between wild type and mutant were marked with an asterisk and labeled band 1, band 2, and band 3. (B) The experimental procedure for limited proteolysis of Pxa2_NBD-CT_F731A was the same as in A. The different peptide bands between wild type and mutant were labeled band 1 and band 2.</p

Effect of the C-terminal deletion of Pxa2_NBD-CT on the interaction between Pxa2_NBD-CT and Pxa1_NBD.

<p>(A) Multiple sequence alignment of the C-terminal amino acid sequences of the peroxisomal ABC transporter protein Pxa2p (678–853) and Pxa1p (775–870) by the ClustalW2 program. The C-terminal sequences of Pxa1p (775–870) and Pxa2p (678–853) were analyzed with the ClustalW2 program on the EMBL-EBI website (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). Pxa2p has an extreme C-terminal region (774–853) that does not exist in Pxa1p. We designated this as the CT₃ region. The region before CT₃ was divided into two regions, designated CT₁ (678–723) and CT₂ (724–773). * indicates identical residues; indicates conserved residues; indicates semi-conserved residues. (B) Two-hybrid analysis of the interactions between Pxa2_NBD-CT (or its deletion mutants) and Pxa1_NBD. Two-hybrid plasmids were co-transformed into EGY48 (pSH18-34). The yeast transformants were streaked on an X-gal plate and incubated for 16 h at 30°C. Blue indicates the existence of the heterodimeric interaction. (C) Expression levels of the yeast two-hybrid proteins in yeast transformants. The total yeast extracts were prepared by the glass bead method and assayed by Western blot probed with anti-LexA BD and anti-Gal4 AD antibodies. Lane 1: yeast co-expressing LexA BD-Pxa2_NBD-CT and Gal4 AD-Pxa1_NBD; lane 2: yeast co-expressing LexA BD-Pxa2_NBD-CT₁₊₂ and Gal4 AD-Pxa1_NBD; lane 3: yeast co-expressing LexA BD-Pxa2_NBD-CT₁ and Gal4 AD-Pxa1_NBD.</p

Two-hybrid analysis of interactions between Pxa2_NBD-CT (or Pxa2_NBD) and Pxa1_NBD-CT (or Pxa1_NBD).

<p>The EGY48 (pSH18-34) reporter strains that express the indicated hybrid proteins (with the Pxa2 fragment fused to LexA BD and the Pxa1 fragment fused to Gal4 AD) were analyzed for β-galactosidase activity on an X-gal plate. Plates were incubated for 16 h at 30°C. Blue indicates the existence of a protein-protein interaction between hybrid proteins.</p