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

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

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

Maturation kinetics plots.

<p>All spectral data is normalized to the maximum peak intensity observed over the course of maturation for each wavelength depicted. Suppression of spectral interference involving the 410 nm absorbance peak is illustrated for mPlum when maturation is tracked at higher pH (A and B). A shift to faster red chromophore maturation half-time and faster arrival at the 410 nm peak maximum occurs when tracking maturation at pH 9.5 in mPlum-E16P (C). This shift to shorter half-times can be seen in mPlum as well for pH 7.5 (A) versus pH 9.5 (B). In mPlumAYC (D), green chromophore maturation half-time is equivalent when tracking both the neutral green chromophore (396 nm) and the anionic green chromophore (508 nm). This result indicates that green chromophore ionization and maturation occur on much different timescales.</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

Chromophore maturation mechanism.

<p>The chromophore maturation mechanism proposed by Strack et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052463#pone.0052463-Strack1" target="_blank">[7]</a> is a branched pathway ending with either red or green chromophore formation. Intermediates and final products on this pathway that are observable by absorption spectroscopy are color-coded and labeled with the approximate wavelength of their peak absorption. The branch point is indicated by a box.</p

Properties of the RFP mPlum and its mutants.

a<p>Neutral green chromophore.</p>b<p>Anionic green chromophore.</p>c<p>Quantum yields for all proteins were measured at pH 7.5. mPlum exhibits measureable green-yellow fluorescence emission at pH >7.5, but at pH 7.5, it was not possible to measure a <b>Φ_F</b> (green) value.</p><p>N.D. = not determined.</p><p>“–” indicates an absence of corresponding species and associated properties.</p

Maturation data.

a<p>Maturation half-times are for the anionic green chromophore that absorbs at ∼510 nm.</p>b<p>Crystallographic NZ-O2 distances are presented for the major conformer of Lys70 in each structure.</p>c<p>Does not mature to the 410 nm absorbing blue intermediate or to the anionic red chromophore.</p

Bioanalysis for Biocatalysis: Multiplexed Capillary Electrophoresis–Mass Spectrometry Assay for Aminotransferase Substrate Discovery and Specificity Profiling

In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections–incubation–separation–enzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion. We used the MINISEP-MS assay to profile the substrate specificity of three aminotransferases (<i>E. coli</i> aspartate aminotransferase, <i>E. coli</i> branched-chain amino acid aminotransferase, and <i>Bacillus sp.</i> YM-1 d-amino acid aminotransferase) for 33 potential amino acid substrates and to measure steady-state kinetics. Using MINISEP-MS, we were able to recapitulate the known substrate specificities and to discover new amino acid substrates for these industrially relevant enzymes. Additionally, we were able to measure the apparent <i>K</i>_M and <i>k</i>_cat parameters for amino acid donor substrates of these aminotransferases. Because of its many advantages, the MINISEP-MS assay has the potential of becoming a useful tool for researchers aiming to identify or create novel enzymes for specific biocatalytic applications

Brighter Red Fluorescent Proteins by Rational Design of Triple-Decker Motif

Red fluorescent proteins (RFPs) are used extensively in chemical biology research as fluorophores for live cell imaging, as partners in FRET pairs, and as signal transducers in biosensors. For all of these applications, brighter RFP variants are desired. Here, we used rational design to increase the quantum yield of monomeric RFPs in order to improve their brightness. We postulated that we could increase quantum yield by restricting the conformational degrees of freedom of the RFP chromophore. To test our hypothesis, we introduced aromatic residues above the chromophore of mRojoA, a dim RFP containing a π-stacked Tyr residue directly beneath the chromophore, in order to reduce chromophore conformational flexibility via improved packing and steric complementarity. The best mutant identified displayed an absolute quantum yield increase of 0.07, representing an over 3-fold improvement relative to mRojoA. Remarkably, this variant was isolated following the screening of only 48 mutants, a library size that is several orders of magnitude smaller than those previously used to achieve equivalent gains in quantum yield in other RFPs. The crystal structure of the highest quantum yield mutant showed that the chromophore is sandwiched between two Tyr residues in a triple-decker motif of aromatic rings. Presence of this motif increases chromophore rigidity, as evidenced by the significantly reduced temperature factors compared to dim RFPs. Overall, the approach presented here paves the way for the rapid development of fluorescent proteins with higher quantum yield and overall brightness