mCherry Core Reversion Variants.

<p>Peak emission wavelength, brightness, and thermostability resulting from reversion mutations to mCherry’s core.</p><p>mCherry Core Reversion Variants.</p

Structure of DsRed (PDB ID: 1ZGO).

<p>(A) Positions mutated in the core of the protein during the directed evolution of mCherry. DsRed is shown in light gray ribbon, mutated residues are shown in sticks, and those within 5 Å of the chromophore (CRO) are highlighted in orange. The aligned residues from mCherry (PDB ID: 2H5Q) are overlaid in pink. (B) DsRed chains B, C, and D are shown as light gray, dark gray, and gray surfaces respectively, while chain A is shown as an orange ribbon. The C-terminal tail of chain A, shown as spheres, stabilizes the AC interface between chain A and chain C. Below chain A in the image is chain B, with which chain A forms the AB dimeric interface.</p

Properties of DsRed, mCherry, DsRmCh, and Selected Variants.

<p>Spectroscopic properties of RFP variants. “mLib Avg” is the mean of mLib members;, “mLib Top” is the largest value of each attribute seen in mLib. “mLib Top” does not refer to a single, specific variant.</p><p>^† “Δ” indicates number of residues deleted from the C-terminal tail; “+” indicates number of residues added to the C-terminal tail.</p><p>^‡ Fluorescence in culture was measured with the following wavelengths: 570 nm excitation and 610 nm emission.</p><p>Properties of DsRed, mCherry, DsRmCh, and Selected Variants.</p

Low Throughput Oligomerization State Analysis of FPs by AUC and SEC.

<p>(<i>A</i>) c(m) distribution from AUC sedimentation velocity experiments. (<i>B</i>) Oligomerization state of FP variants analyzed by SEC (Superdex 75). In both A and B, the experiments are normalized to 1 for clarity of presentation.</p

Characterization of Designed Libraries.

<p>Graphical representation of quantum yield (<i>Top</i>), extinction coefficient (<i>Middle</i>), and brightness (<i>Bottom</i>) of mLib, rLib, and controls. For mLib and rLib, light gray bars indicate individual variants, the larger blue bar represents the library mean, and blue horizontal bars represent one standard deviation from the mean. For standards, a legend is shown in the top chart, and error bars represent the standard deviation from three independent measurements.</p

High Throughput Oligomerization State Analysis of mLib by Homo-FRET.

<p>Grey, 96 members of mLib; red, mCherry monomer control; yellow, DsRed tetramer control; blue, DsRmCh; black, Rose Bengal. Position along the x-axis serves only to separate individual variants for better visibility.</p

Absorption and fluorescence spectra of various RFPs.

<p>Absorption spectra (full lines) are normalized to the largest intensity absorbance peak present in each spectrum. Fluorescence emission spectra (dotted lines) are normalized to the absorbance peak in each spectrum corresponding to the excitation wavelength used to induce fluorescence. All spectra were measured at pH 7.0.</p

Maturation experiments.

<p>All spectra are normalized to the 280 nm absorbance peak. Heavy black and blue traces represent the beginning (t = 0 h) and end (t = 20 h) of the maturation experiment, respectively. The distance in time between each gray or black trace is 1.0 h. Arrows indicate the primary direction of peak movement during maturation. Each heavy red trace indicates the point in time when the 410 nm absorbance peak reached its maximum during the course of maturation. Black traces occur before the 410 nm peak reaches its maximum level; gray traces occur after the maximum.</p

Crystal structures.

<p>(A and B) Introduction of the AYC motif results in π-stacking interactions between the chromophore and Tyr197 in both mPlumAYC (A) and mPlumAYC-E16A (B). H-bonding interactions with Lys70 are illustrated with dashed lines. These interactions combine to sequester the terminal amino group of Lys70 away from the chromophore. (C and D) Comparisons of Lys70-to-chromophore distance are illustrated between mPlum (purple), mPlumAYC (yellow), and mPlumAYC-E16A (blue) (C), as well as between mCherry (pink), mPlum (purple), and mPlum-E16P (red) (D). A dotted line connects the NZ atom of Lys70 in mPlum to the O2 atom of the mPlum green (C) or red (D) chromophore. Note that Lys70 in mPlum (PDB code 2QLG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052463#pone.0052463-Shu2" target="_blank">[21]</a>) adopts a slightly different conformation in the green versus red chromophore contexts. All proteins were aligned by the atoms of their imidazolinone ring.</p

Electron density maps.

<p>Omit maps contoured at 3σ were constructed for the chromophore and surrounding main chain atoms in mPlumAYC, mPlumAYC-E16A, and mPlum-E16P. Arrows indicate the sp² or sp³-hybridized alpha carbon atom of Met66 observable in each chromophore.</p