Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore

Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore

Importance of Secondary Interactions in Twisted Doubly Hydrogen Bonded Complexes

Three model hydrogen bond arrays that form complexes with large twist angles between their heterocyclic rings were synthesized differing only in the sequence of their hydrogen bond donors and acceptors. The complementary and self-complementary association of the arrays to form complexes was studied computationally and in solution. The analysis reveals the significant impact secondary interactions have on complex stability in such an arrangement despite the very different topology in comparison to typical planar arrays

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

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

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 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

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

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