Increases in fluorescence intensity for different donor fluorescent proteins fused to Ultramarine after protease cleavage.

1<p>Increase in fluorescence emission was monitored until an endpoint was reached. Complete cleavage of the polypeptide linker was confirmed by subjecting endpoint samples to analysis SDS-PAGE.</p>2<p>R₀ values were calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041028#pone.0041028-Lakowicz1" target="_blank">[29]</a>.</p><p>For comparison data is included for the fusion protein ECFP^C3EYFP, a probe reported for measuring caspase 3 protease activation in cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041028#pone.0041028-Luo1" target="_blank">[30]</a>.</p

Kinetic mechanism and structure.

<p>(A) The DHDPS-catalyzed reaction follows a classic bi-bi substrate model, requiring the first substrate (PYR; pyruvate) bind the enzyme for the second substrate (ASA) to be recruited to the active site and ultimately liberate the reaction product (HTPA). The initial pyruvate-binding portion of the reaction scheme is highlighted in cyan. (B) Quaternary structure of the DHDPS dimer. (C) Licorice representation of key active site residues. Protein chains A and B are shown in yellow and green, respectively.</p

Umbrella sampling PMF.

<p>PMF curve calculated using 28 windows of umbrella sampling along an arbitrary <i>Z</i>-coordinate. Each curve, colored from red to blue, represents successive truncation of the data in 5% increments from the beginning of each simulation window until the final 50% of data (2.5 ns) remained. Several bound states identified are labelled according to their average <i>Z</i>-coordinate. State S13, which was bimodal with respect to the <i>Z</i>-coordinate, is highlighted as a gray box.</p

The major pyruvate-binding pathway is multi-step.

<p>(A) Pyruvate, indicated using green carbon atoms, must successively pass through several binding intermediates to reach the crystallographic bound pose. From bulk solvent, pyruvate forms a transient interaction with an arginine residue at the entryway to the active site (T1), moves into the active site cavity (T2), and in the penultimate step penetrates deeper into the active site to assume a ‘pre-bound’ pose (T3). Finally, from the ‘pre-bound’ pose pyruvate undergoes a twisting motion (T4) and achieves the crystallographic DHDPS-pyruvate complex. (B) Multiple sequence alignment of bacterial DHDPS enzymes. Several interacting residues from the binding pathway depicted in (A) are absolutely conserved across species. Sequence alignment was performed using CLUSTALO [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004811#pcbi.1004811.ref037" target="_blank">37</a>].</p

Binding data.

<p>(A) Ligand RMSD to crystal structure (PDB ID 3DI1) is shown as a function of time for 10 randomly selected trajectories, representing an eighth of the total simulation data set. Hydrogen atoms were excluded from RMSD calculations. (B) Ligand density plot. The <i>x</i> and <i>y</i> plane components of the geometric center of pyruvate were derived from each frame of the simulation data set and binned to form a 2-dimensional matrix. The color mapping reflects the number of counts within each of these bins (blue indicates low density, red indicates high density). For reference, the relative locations of several active site residues (Thr46, Tyr109, Tyr135, Arg140, and Lys163; <i>α</i>-carbons only) are indicated using black markers and labelled accordingly.</p

Grouping of states from binding trajectories into a coarse-grained metastable state model.

<p>(A) Unbound states. One hundred randomly selected conformations of pyruvate from unbound states S2 (yellow) and S6 (green) are presented against a cartoon representation of DHDPS. Collectively, poses within these states lack any conserved interactions with the protein surface. (B) The DHDPS-pyruvate bound complex (S13). Conformations of pyruvate and active site residues from this state (green) are contrasted with a crystallographic reference structure of pyruvate bound to DHDPS (PDB ID 3DI1; silver). Key active site residues Thr46, Tyr109, Tyr135, and Lys163 are indicated. Conformations of pyruvate within this state deviate from the reference structure by as little as 1.85 Å. Note that Tyr109 is shown from the opposing DHDPS subunit. (C) Individual metastable states, labelled accordingly, are shown as nodes within rounded boxes. Edges depict bidirectional interstate transitions, where edge shade reflects the transition probability (darker arrows indicate higher probabilities). States classified as unbound (S2, S6) are shaded in red, whereas the DHDPS-pyruvate bound state (S13) is shaded in green. For clarity, only highly-populated states (equilibrium populations greater than 4%) are shown.</p

Overlap of Ultramarine absorbance with fluorescence emission of some donor fluorescent proteins.

<p> The absorbance spectrum for Ultramarine (blue) is shown overlayed with the fluorescence emission spectra of selected donor FPs (Cerulean, cyan; cpT-sapphire and Sapphire, green; EYFP, yellow and mKO, orange). The overlap between Ultramarine absorbance and donor fluorescence emission is highlighted by light blue fill.</p

Some properties of Rtms5 variants.

<p>Properties were determined for purified protein in 20 mM Tris-HCl, pH 8.0 and 300 mM NaCl.</p>1<p>Determined by AUC.</p>2<p>Determined by SDS-PAGE for non-boiled samples (pseudo-native PAGE).</p>3<p>Determined by size-exclusion gel filtration chromatography.</p><p>ND: not determined.</p

Using FLIM to monitor caspase 3 activation in live cells expressing Ultramarine^hC3cpT-Sapphire.

<p>(A). Color maps of fluorescence lifetime data (Phase, τ_φ and modulation, τ_m) are shown for HeLa cells expressing Ultramarine^hC3cpT-Sapphire or cpT-Sapphire. For this experiements an Ultramarine expression cassette (Ultramarine^h) codon optimised for use in human cells was used. Cells were imaged in a time series before (t₀) and after addition of staurosporine (t₆₀–t₂₈₀) to initiate activation of Caspase 3. Fluorescence intensity and DIC images are shown. Lifetime images are shown for cells expressing cpT-Sapphire (donor alone). Colored arrows highlight individual cells for which lifetime distributions are plotted in (B). Lifetime images are shown for cells expressing cpT-Sapphire (donor alone). Time of incubation (mins) after addition of STS is shown at left. Scale bar  = 10 µm (B). The lifetime distributions for τ_φ are plotted for selected cells at different times of incubation. The line color corresponds to the cell highlighted by the same color arrow in A. The dashed line (centered on 1.6 ns) serves as a reference to highlight a progressive shift towards longer lifetimes. The lifetime distribution for the donor alone is plotted for all cells in the field of view.</p

The effect of pH on some optical properties of Ultramarine.

<p>(A). The absorbance spectrum was determined for Ultramarine diluted in buffers of different pH and constant ionic strength. The species absorbing at ∼450 nm and ∼590 nm correspond to the neutral and anionic form of the chromophore, respectively. (B). Absorbance at 586 nm was determined at different pH. The species absorbing at 586 nm corresponds to the anionic chromophore and has an estimated pK_a of 4.1. Excitation spectra were obtained for Ultramarine (λ_Em, 630 nm). Excitation scans are grouped into what are considered to be physiological (C), low (D) and high (E) pH ranges.</p