DNA binding of Ms1.

<p>A: EMSA experiment of ABD2 and FOXO3a as a positive control with a random 18mer library (2 μM). B: consensus motif of the AT-hook shown as web logo [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144614#pone.0144614.ref040" target="_blank">40</a>] (based on PROSITE entry PDOC00306) and in short form on top of a sequence alignment of the AT-hook motif in selected Ms1 sequences. Colours are: purple—hydrophilic; green—hydrophobic; orange—proline and glycine; blue—positive charge. Phosphorylation sites identified experimentally are indicated by a star [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144614#pone.0144614.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144614#pone.0144614.ref041" target="_blank">41</a>]. C: EMSA of N-terminally extended ABD2 constructs with the same 18mer random library used in A. The overall Ms1 topology is shown together with the detail of the N-terminus of the various constructs. Residues at the N-terminus of ABD2 are shown in blue and those of the AT-hook in red. D: NMR analysis of the interaction of Ms1 270–375 with AT-rich DNA ds oligonucleotides. Spectra of Ms1 270–375 without (black) and with (1:0.5 red; 1:1 blue; 1:2 green) of the oligonucleotide are shown superimposed together with a detailed view for two selected residues from the N-terminus. The chemical shift perturbations are summarised in a plot against the amino acid sequence that is annotated with the functional and structural properties of the 270–375 construct.</p

Structure determination of rat ABD2.

<p>A: Stereo view of the family of the best 20 structures superimposed on the lowest energy structure. B: Secondary structure of the lowest energy structure in the same orientation as in A. Key structural features are labeled, helices are coloured red and strands are coloured yellow. C: Structural superposition of Ms1 ABD2 (red, PDB ID 2KRH) and COSTARS (blue, PDB ID 2L2O). Shown in the same orientation as in B as well as rotated about 90° around the horizontal axis to reveal the structural differences in the segment connecting α1 and β1 (arrow). D: Structural superposition of Ms1 ABD2 (red) on the three top hits of the DALI search in the PDB (1HR3 (green), 1SAX (blue), 1SD4 (yellow); for a list of the top 50 DALI hits see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144614#pone.0144614.s003" target="_blank">S3 Fig</a>).</p

Subcellular localisation of Ms1.

<p>A: Immunofluorescent detection of endogenous Ms1 in NRCs. Staining is (left-to-right) DAPI (nuclei, blue), α-actinin (green) and Ms1 (red). B: as A but in ARCs. C: western blot detection of Ms1 in subcellular fractions isolated from ARC and NRC cultures. C = cytoplasm, M = membrane, SN = soluble nuclear, CH = chromatin bound, CS = cytoskeleton. D: Sequence alignment of Ms1 in the region of the nuclear localisation signal (NLS). Phosphorylation sites identified experimentally are indicated by a star [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144614#pone.0144614.ref041" target="_blank">41</a>]. Colours and the subset of sequences are identical to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144614#pone.0144614.g002" target="_blank">Fig 2B</a>. Note that Ms1 from lower organisms lack this region of the protein.</p

Regulation of the subcellular localisation of Ms1.

<p>A: Overexpression of wt myc-tagged Ms1 in culture medium with phenylephrine; detection with anti-myc antibody. B: Overexpression of wt Ms1 in NRCs in culture medium without phenylephrine; detection with anti-myc antibody. C: Detection of endogenous Ms1 with aABD2chn in NRCs grown in culture medium without phenylephrine. D: Detection of endogenous Ms1 with aABD2chn in NRCs grown in culture medium with phenylephrine. E: Overexpression of Ms1 with RR->AA mutation of NLS in NRCs grown in presence of phenylephrine; detection with anti-myc antibody.</p

The specificity of DNA binding.

<p>A: Gel shift assays with DNA pools from stages 1 (first) and 8 (last) of the SELEX procedure with Ms1 270–375. Positions of free and bound DNA are indicated. B: Sequencing results from the final SELEX pool after stage 8. Only random region shown, constant region is omitted. C: Web logo [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144614#pone.0144614.ref040" target="_blank">40</a>] plot of the consensus DNA recognition motif of ABD2.</p

Halide and Proton Binding Kinetics of Yellow Fluorescent Protein Variants

A T203Y substitution in green fluorescent protein causes a red shift in emission to yield a class of mutants known as yellow fluorescent protein (YFP). Many of these YFP mutants bind halides with affinities in the millimolar range, which often results in the chromophore p<i>K</i> values being shifted into the physiological range. While such sensitivities may be exploited for halide and pH sensors, it is desirable to reduce such environmental sensitivities in other studies, such as in Förster resonance energy transfer probes to measure conformational changes within fusion proteins. Venus and Citrine are two such variants that have been developed with much reduced halide sensitivities. Here we compare the kinetics of halide binding, and the coupled protonation reaction, for several YFP variants and detect slow kinetics (dissociation rate constants in the range of 0.1–1 s^–1), indicative of binding to an internal site, in all cases. The effective halide affinity for Venus and Citrine is much reduced compared with that of the original YFP 10C construct, primarily through a reduced association rate constant. Nuclear magnetic resonance studies of YFP 10C confirm halide binding occurs on a slow time scale (<4 s^–1) and that perturbations in the chemical shift occur throughout the sequence and structure

Schematic representation of the evolutionary conservation of SR units.

<p>(A) Nesprin-1. (C) Nesprin-2. Each SR unit is coloured according the percentage of conserved residues based on the alignments of vertebrate nesprin-1 and of nesprin-2, separately. The yellow star represents the invariant motif in the unstructured region. Arrows indicate N-termini of short isoforms. (B and D) Number of conservation pbs in each SR unit. Contiguous SR units with >5 pbs are coloured red for nesprin-1 (B) and for nesprin-2 (D).</p

RMSF of the single and double SR units.

<p>(A) RMSF of single units (black and blue lines) and replica simulations performed on the double SR unit (red and green lines) of ^NES1SR70-71. (B) RMSF of single units (black and blue lines) and the replica simulations performed on the double SR unit ^NES2SR52-53. (C) Comparison of the RMSF of the following double SR units: 1S35 (black line), ^NES1SR70-71 replicas (red and green lines) and ^NES2SR52-53 replicas (blue and yellow lines).</p

Ribbon representation of ^NES1SR70-71 (A-B) and ^NES2SR52-53 (C-D).

<p>Close up of the interaction between Phe8113 (magenta spheres) and Gln8169 (cyan spheres) in ^NES1SR70-71 (B) and between Phe6244 Phe8113 (magenta spheres) and Gln8169 (cyan spheres) in ^NES2SR52-53 (D).</p

Template structures containing spectrin repeats (SR).

<p>Template structures containing spectrin repeats (SR).</p