Circular permutation sites scanned in mCherry and mKate.

<p>mCherry and mKate were numbered by primary amino acid sequences as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone-0020505-g001" target="_blank"><b>Fig. 1C</b></a>. Open circles indicate sites with fluorescence, closed circles sites without fluorescence. Three highly homologous regions of circular permutation tolerant sites in mCherry and mKate: Loop 7–8 region located in the loop between the 7^th and 8^th β-strands and flanking sites on the β-strands, Loop 8–9 region located in the loop between the 8^th and 9^th β-strands and flanking sites on the β-strands, and Loop 9–10 region flanked by the 9^th and 10^th β-strands.</p

Schematic plasmid and protein sequences of cp-Red Fluorescent Proteins.

<p><b>A.</b> Circular permutants were constructed from a tandem fusion template with forward (new N-terminal) and reverse (new C-terminal) primers, producing a full length amplicon beginning and ending at any desired site. Strategy for mKate circular permutation is shown. <b>B.</b> Diagram of cp-RFP final proteins with the N-terminal leader peptide from the expression vector pRSET-A; C (n+1 to 231) and N (1 to n) -terminal residues of mKate were joined by a GGTGGS linker and the LE linker between RSET and cp-mKate was encoded by the <i>Xho</i>I site. <b>C.</b> Amino acid sequences of DsRed from the <i>Discosoma sp.</i> coral, DsRed-derived mCherry, and mKate from the sea anemone <i>Entacmaea quadricolor</i>. Chromophore forming residues are underlined; beta-strands forming residues are shown in bold.</p

Absorption spectra of cp-mCherry and cp-mKate variants.

<p>Spectra were normalized to the 280 nm absorption for each protein. <b>A.</b> Absorption spectra of mCherry and cp-mCherry variants. <b>B.</b> Absorption spectra of mKate and cp-mKate variants.</p

Screening of circular-permutated mCherry variants in <i>E.coli</i>.

*<p>Circular-permutated mCherry variants were numbered by the primary amino acid sequence of mCherry as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone-0020505-g001" target="_blank"><b>Fig. 1C</b></a>. Variants are labeled with the new amino and carboxy termini (e.g. cp159-158 has the new N-terminus from the native carboxy amino acids 159 to 236 and the new C-terminus from the amino residues 1 to158).</p>a<p>Fluorescent variants from the initial screening in each region.</p>b<p>Aligns to cp-EGFP in GCaMP2.</p>c<p>Numbered by the amino acid sequence of DsRed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone-0020505-g001" target="_blank"><b>Fig. 1C</b></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone.0020505-Li1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone.0020505-Carlson1" target="_blank">[19]</a>. cp184-mCherry corresponds to cp191-190, with three duplicated residues at N-terminus (188-190). The cp193-mCherry and cp193g7 (with 6 mutated amino acids) correspond to cp197-196, with three duplicated residues (197-199) at the C-terminus.</p

Crystal structure of cp-mKate^154-153 and cp-mKate^168-167.

<p><b>A.</b> Cartoon presentation of mKate (pH 7.0), cp-mKate^154-153 and cp-mKate^168-167. The figures are drawn with PyMol (DeLano Scientific). <b>B.</b> 2mFo-Fc electron density map near chromophore region. The map is contoured at 1.0σ.</p

Screening of circular-permutated mKate variants in <i>E.coli</i>.

*<p>The circularly permuted variants are labeled with the new amino and carboxy termini.</p>a<p>Align to cp-EGFP in GCaMP2.</p>b<p>Aligns to cp-mCherry^159-158.</p>c<p>Aligns to cp-mCherry ^175-174.</p>d<p>Aligns to cp-mCherry^194-193.</p

Properties of circular-permutated mCherry variants.

a<p>All circular-permutated variants from native mCherry sequence shown in this table, except cp184 (not determined), have the same excitation (587 nm) and emission (610 nm) maxima as mCherry. The excitation and emission maxima of the cp193g7, with 6 amino acid mutations, are 580 nm and 602 nm, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone.0020505-Carlson1" target="_blank">[19]</a>.</p>b<p>Extinction coefficients were measured by alkali-denatured chromophore method.</p>c<p>Quantum yields were measured using mCherry as the reference standard.</p>d<p>Relative brightness of chromophore (extinction coefficient × quantum yield) was compared with mCherry (91,000×0.22).</p>e<p>Fluorescence of cp-mCherry relative to mCherry with fixed protein concentration (BCA assay).</p>f<p>Our data; the published data are 72,000 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone.0020505-Shaner1" target="_blank">[7]</a>, 78,000 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone.0020505-Shcherbo1" target="_blank">[9]</a>.</p>g<p>Published values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone.0020505-Li1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020505#pone.0020505-Carlson1" target="_blank">[19]</a>, which were based on the protein quantification (absorption at 280 nm).</p>i<p>Not determined.</p

X-ray Data Colletion and Refinement Statistics of cp-mKate^154-153 and cp-mKate^168-167.

<p>X-ray Data Colletion and Refinement Statistics of cp-mKate^154-153 and cp-mKate^168-167.</p

PI(3)k-Akt mediates stretch- induced Ca2+ release in smooth myocytes.

<p>Linescan images show the effect of LY492002 on Ca2+ spark at stretch length ΔL = 18%. LY492002 could not completely abrogated the immediately stretch-induced Ca2+ sparks (A), but completely abolished Ca2+ sparks occurred in stretch-maintaining stage (B). C, representatives of experiments in the absence and the presence of another PI3 kinase inhibitor, wortmannin. Like LY492002, wortmannin entirely abrogated Ca2+ sparks occurred in the stretch-maintaining stage. C, summary data of Ca2+ spark probability after sequential stretch. The numbers indicate the response experiments to stretch out of all experiments.</p

Stretch-induced NO production in single cell and intact smooth muscle tissues.

<p>A, ESR spectra of NO trapped by DETC-iron (II) complex in mouse bladder smooth muscle strips. Compared to control (slack tissue strips, gray line), NO was greatly increased in stretched tissue strips (black line). The inset shows the magnetic field range of NO in the presence of SNP. B, summary data of NO production. Note the significantly difference of NO production between the control group and stretch group; * P<0.01, n = 6. C, DAF-2 single cell experiments: by stretch the cell length to ΔL = 9% DAF-2 fluorescence transient occurred immediately. D, upper is x-y images taken from a tissue strip incubated with DAF-2; the lower is pseudo linescan images taken from a series of x-y image obtained from before (left) and after (right) stretch of the intact mouse bladder smooth muscle strip incubated with DAF-2, and at below of pseudo linescan images show NO transient profiles taken from slack and stretch. E, before ( left ) and after ( right ) stretch of tissue segment in the presence of L-name. Note that the stretch-induced NO production was completely inhibited by L-name.</p