Additional file 1: of Aggregation Control by Multi-stimuli-Responsive Poly(N-vinylamide) Derivatives in Aqueous System

Supporting information. (DOCX 343 kb

Time-Resolved Observation of Chiral-Index-Selective Wrapping on Single-Walled Carbon Nanotube with Non-Aromatic Polysilane

In the present paper, we ascertain two novel findings on chiral-index-selective binding/separating of single-walled carbon nanotubes (SWNTs) with a nonaromatic polymer, poly­(dialkylsilane) (PSi). PSi is a typical σ-conjugated polymer, composed of alkyl side chains attached to the silicon (Si)-catenated main chain. First, PSi’s with linear alkyl side chains showed significant diameter-selective wrapping for SWNTs with ca. 0.9 nm in diameter, resulting in the selective separation of (7,6) and (9,4) SWNTs. Its driving force was demonstrated to be cooperative CH−π interactions among the alkyl side chains of PSi’s and the curved graphene of SWNTs. Second, the dynamic wrapping behavior of PSi’s onto SWNTs was elucidated with time-resolved UV spectroscopy. Highly anisotropic UV absorption of PSi along the Si main chain was utilized as a “chromophoric indicator” to monitor the global/local conformations, which enabled us to track kinetic structural changes of PSi’s on SWNTs. Consequently, we concluded that upon wrapping, flexible/helical PSi with an average dihedral angle (φ) of 145° and Kuhn’s segment length (λ^–1) of 2.6 nm interconverted to the more stiffer/planar conformation with 170° and λ^–1 of 7.4 nm. Furthermore, through kinetic analyses of the time-course UV spectra, we discovered the fact that PSi’s involve three distinct structural changes during wrapping. That is, (i) the very fast adsorption of several segments within dead time of mixing (<30 ms), following (ii) the gradual adsorption of loosely wrapped segments with the half-maximum values (τ₁) of 31.4 ms, and (iii) the slow rearrangement of the entire chains with τ₂ of 123.1 ms, coupling with elongation of the segment lengths. The present results may be useful for rational design of polymers toward chiral-index-selective binding/separating of desired (<i>n</i>,<i>m</i>) SWNTs

Fe–His16 and Fe–Met61 distances in monomeric and dimeric WT PA cyt <i>c</i>₅₅₁.

<p>^a PDB ID: 351C.</p><p>^b There are two independent WT PA cyt <i>c</i>₅₅₁ molecules in the asymmetric unit of dimeric WT PA cyt <i>c</i>₅₅₁ crystal.</p><p>Fe–His16 and Fe–Met61 distances in monomeric and dimeric WT PA cyt <i>c</i>₅₅₁.</p

Active site structures of monomeric and dimeric WT PA cyt <i>c</i>₅₅₁.

<p>(A) Structure of monomeric WT PA cyt <i>c</i>₅₅₁ (PDB ID: 351C). (B) Structure of dimeric WT PA cyt <i>c</i>₅₅₁ (PDB ID: 3X39). The heme and side-chains of amino acid residues near the heme (Phe7, Cys12, Ala14, Cys15, His16, Val23, Pro25, Val30, Leu44, Arg47, Ile48, Ser52, Trp56, Pro60, Met61, Pro62, Pro63, Asn64, Leu74, and Val78) are shown as stick models. The sulfur atoms of the heme axial Met ligand and heme-linked Cys are shown in yellow, and the nitrogen atoms of the heme axial His ligand are shown in blue. The cyan strand in the dimeric structure is a region from another molecule. The hemes and Thr20–Met22 residues (hinge loop) are depicted in dark and pale colors, respectively.</p

CD spectra and small angle X-ray scattering curves of WT and M61A PA cyt <i>c</i>₅₅₁.

<p>(A) CD spectra of oxidized monomeric WT (red) and M61A (green) PA cyt <i>c</i>₅₅₁. Measurement conditions: Sample concentration, 10 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature. (B) Small angle X-ray scattering curves of oxidized monomeric WT (red) and M61A (green) PA cyt <i>c</i>₅₅₁ shown by Kratky plots. The intensities are normalized at their maximum intensities. Measurement conditions: sample concentration, 500 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, 20°C.</p

<i>In vitro</i> import of t75-EGFP variants.

<p>(A) Import of radiolabeled t75-EGFP variants into isolated chloroplasts, post-import fractionation, and analysis of the results were done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.g002" target="_blank">Fig 2A</a>. The experiments were done concurrently with those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.g002" target="_blank">Fig 2A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.s002" target="_blank">S1 Fig</a>. The precursor proteins containing the entire t75 variants, the intermediates containing the c75 variants but not n75, and the mature form that lacks the entire t75 variants are indicated as t75*-EGFP, c75*-EGFP, and m, respectively. For the CBB panel, large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and light-harvesting chlorophyll a/b-binding protein are indicated as LS and LHCP, respectively. (B) Import of radiolabeled t75-EGFP variants into isolated chloroplasts, post-import protease treatment, and analysis of the results were done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.g002" target="_blank">Fig 2B</a>. The experiments were done concurrently with those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.g002" target="_blank">Fig 2B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.s003" target="_blank">S2 Fig</a>. The 27-kD protease-protected EGFP is indicated as 27. For other labels, see the legend to panel (A). (C) Import of radiolabeled t75-EGFP variants into isolated chloroplasts were performed for 5 or 30 min followed by post-import treatment with thermolysin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.g002" target="_blank">Fig 2B</a>. The precursor proteins containing the entire t75 variants, the intermediates containing the c75 variants but not n75, and the mature form that lacks the entire t75 variants are indicated as t75-EGFP/t75_GGA-EGFP, c75-EGFP/c75_GGA-EGFP, and m, respectively. The 27-kD protease-protected EGFP is indicated as 27.</p

Affinity of the EspB fragments to α-catenin635–906 analyzed by fluorescence anisotropy.

<p>Each EspB peptide was modified with a fluorescein moiety at the N-terminus. (A), Titration curves for the peptides bound to α-catenin. EspB (○), EspB41–60 (l>), EspB51–70 (◊), EspB61–80 (•), EspB41–70 (◂). (B) Titration results obtained for the peptides unable to bind to α-catenin635–906. EspB1–20(□), EspB11–30 (Δ), EspB21–40 (∇), EspB31–50 (</p

<i>In vitro</i> import of t75-mSS variants.

<p>(A) Radiolabeled t75-mSS variants indicated above were incubated with isolated chloroplasts under the import condition. After 30-min of import, intact chloroplasts were reisolated and separated into two aliquots. The first aliquot was kept as total chloroplasts (T). The second aliquot was hypotonically lysed and fractionated by centrifugation to a supernatant (S1) and the pellet. The pellet was then resuspended with 0.1M Na₂CO₃ and fractionated by centrifugation to the second supernatant (S2) and the final pellet fraction (P). Samples equivalent to 3 μg chlorophyll were separated by SDS-PAGE, and radiolabeled proteins and total proteins in each sample were visualized by phosphorimaging (PI) and Coomassie Brilliant Blue staining (CBB), respectively. The experiments were done concurrently with those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.g003" target="_blank">Fig 3A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.s002" target="_blank">S1 Fig</a>. tl contained the translation product corresponding to the one used for the import assay with 3 μg chlorophyll-equivalent chloroplasts. The precursors containing the entire t75 variants, the intermediates that carrying the c75 variants, and the mature forms lacking the entire t75 variant, respectively, are indicated at right; mSS is indicated with the letter m. For the CBB panel, large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and light-harvesting chlorophyll a/b-binding protein are indicated as LS and LHCP, respectively. (B) After the import reaction as described in the legend to panel (A), intact chloroplasts were reisolated and separated into six aliquots. Three of them were resuspended in import buffer containing 1 mM CaCl₂ with or without 1 μg thermolysin (tlysin) per μg chlorophyll equivalent chloroplasts and 1% Triton X-100 (TX) as indicated, incubated for 30 min on ice in the dark. Other three aliquots were resuspended in import buffer with or without 0.5 μg trypsin (tryp) per μg chlorophyll equivalent chloroplasts and 1% Triton X-100 (TX) as indicated, incubated for 60 min at room temperature in the dark. The activities of thermolysin and trypsin were quenched by 10 mM EDTA and 10 μg trypsin inhibitor per μg trypsin, respectively. Samples equivalent to 3 μg chlorophyll were separated by SDS-PAGE and radiolabeled proteins visualized by phosphorimaging. The experiments were done concurrently with those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.g003" target="_blank">Fig 3B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167802#pone.0167802.s003" target="_blank">S2 Fig</a>. For the labels at right, see the legend to panel (A).</p

Far-UV CD spectra of EspB fragments.

<p>(A) Schematic representation of the fragments prepared in this study. (B) Raw ellipticity data (<i>θ</i>_obs) obtained by full length EspB (circles), EspB1–176 (squares) and EspB177–312 (triangles). The protein concentration for each protein was 15 µM. Broken line indicates the sum of the spectra of EspB1–176 and EspB177–312. (C) Another representation of spectra shown in panel B expressed as mean residue ellipticity, [<i>θ</i>].</p

Thermodynamic parameters of unfolding of EspB and EspB41–70 in the presence of GdnHCl at pH 7.0.

a<p>Estimated from linear extrapolation of Δ<i>G</i>_20°C obtained by the analysis of the data in the presence of GdnHCl as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071618#pone-0071618-g001" target="_blank">Fig. 1</a>.</p>b<p>Not available.</p>c<p>Same as above.</p