Nanoporous Crystalline Templates from Double-Crystalline Block Copolymers by Control of Interactive Confinement

Single, double, and coincident crystallizations under hard or soft confinement are all carried out using a single type of syndiotactic poly­(<i>p</i>-methyl­styrene)-<i>block</i>-poly­(l-lactide) (<i>s</i>PPMS–PLLA) block copolymers. The single crystallization of <i>s</i>PPMS matrix can lead to the disordered arrangement of hexagonally packed PLLA cylinders under soft confinement. In contrast, the lamellar nanostructure remained unchanged regardless of the PLLA crystallization under hard or soft confinement. Crystallization-induced morphological transitions from the confined monosized lamella to the metastable dual-sized lamella and finally to the breakout morphology are evident by transmission electron microscopy and small-angle X-ray scattering. The dual-sized lamella is attributed to the thermodynamically and kinetically controlled nanocrystallite growth templating along the ordered microphase separation. Despite crystalline sequences, the double-crystallized morphologies are determined by the first-crystallized event even though the subsequent crystallization temperature is performed under soft confinement. By the control of interactive confinement, ordered crystalline nanosheets and cylindrical monoliths are obtained, providing a novel means for the fabrication of nanoporous crystalline templates

Average lengths and widths of the plasmid and nucleoprotein complexes.

<p>EM images were measured using ImageJ software.</p

FRET efficiency and distance of the DNA duplex alone or bound to Sso7c4 in bulk FRET experiments.

<p>FRET efficiency and distance of the DNA duplex alone or bound to Sso7c4 in bulk FRET experiments.</p

Crystal structure of the wild-type Sso7c4 protein.

<p>(A) Ribbon diagram of wild-type Sso7c4 with the two arginine pairs (R11/R22′ and R11′/R22) and three sulfates (X201, X202 and X203) depicted as balls and sticks with (2<i>F</i>o-<i>F</i>c) Fourier electron density maps (contoured at 1σ level). (B) Top view of the Sso7c4 dimer. The ribbon diagrams of the Sso7c4 dimer are colored green and pink to represent each monomer. All the lysine residues, the two arginine pairs, the three sulfates and the two PEG 400 molecules are depicted as balls and sticks. The water molecules surrounding the lysine side chains are drawn as purple spheres.</p

Model of the interaction of Sso7c4 with DNA.

<p>(A)The DNA-binding surface of the Sso7c4 dimer was manually docked into the major groove such that the sulfates were located as close as possible to the phosphates in the DNA backbone. A symmetrically bent DNA conformation was constructed using a known curved DNA (from the CAP-DNA complex; 1O3T). A brief minimization was performed to remove steric hindrance between the protein side chains and DNA. (B) The models of three DNA binding modes induced by Sso7c4 variants. The wild-type Sso7c4 binds to DNA in two different modes, bending and bridging. In contrast, the C-terminally truncated Sso7c4 binds side-by-side along the DNA molecule leading to DNA stiffening.</p

The Arginine Pairs and C-Termini of the Sso7c4 from <i>Sulfolobus solfataricus</i> Participate in Binding and Bending DNA

<div><p>The Sso7c4 from <i>Sulfolobus solfataricus</i> forms a dimer, which is believed to function as a chromosomal protein involved in genomic DNA compaction and gene regulation. Here, we present the crystal structure of wild-type Sso7c4 at a high resolution of 1.63 Å, showing that the two basic C-termini are disordered. Based on the fluorescence polarization (FP) binding assay, two arginine pairs, R11/R22′ and R11′/R22, on the top surface participate in binding DNA. As shown in electron microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and bending interactions, whereas the binding of C-terminally truncated proteins rigidifies and opens DNA molecules, and no compaction of the DNA occurs. Moreover, the FP, EM and fluorescence resonance energy transfer (FRET) data indicated that the two basic and flexible C-terminal arms of the Sso7c4 dimer play a crucial role in binding and bending DNA. Sso7c4 has been classified as a repressor-like protein because of its similarity to <i>Escherichia coli</i> Ecrep 6.8 and Ecrep 7.3 as well as <i>Agrobacterium tumefaciens</i> ACCR in amino acid sequence. Based on these data, we proposed a model of the Sso7c4-DNA complex using a curved DNA molecule in the catabolite activator protein-DNA complex. The DNA end-to-end distance measured with FRET upon wild-type Sso7c4 binding is almost equal to the distance measured in the model, which supports the fidelity of the proposed model. The FRET data also confirm the EM observation showing that the binding of wild-type Sso7c4 reduces the DNA length while the C-terminal truncation does not. A functional role for Sso7c4 in the organization of chromosomal DNA and/or the regulation of gene expression through bridging and bending interactions is suggested.</p></div

Binding affinities for the arginine mutant/wild-type/C-terminally truncated/proteins in complex with three dsDNA fragments of different lengths.

<p>Binding affinities for the arginine mutant/wild-type/C-terminally truncated/proteins in complex with three dsDNA fragments of different lengths.</p

Electron micrographs of the phiX174 plasmid with or without the wild-type/C-terminally truncated Sso7c4 proteins.

<p>Representative images of Sso7c4-DNA complexes visualized by EM. Nicked phiX174 plasmids were incubated with different stoichiometries (5 bp/dimer or 0.5 bp/dimer) of either the wild-type (wt) or C-terminally truncated (ctt) proteins. (A) High magnification images of the relaxed, circular phiX174 plasmid alone. (B) and (C) High magnification images of the wild-type Sso7c4-plasmid complex. (D) and (E) High magnification images of the C-terminally truncated Sso7c4-plasmid complex. The scale bar represents 100 nm.</p