Figure 1.
Chromophore maturation mechanism.
The chromophore maturation mechanism proposed by Strack et al. [7] 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.
Figure 2.
Absorption and fluorescence spectra of various RFPs.
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.
Table 1.
Properties of the RFP mPlum and its mutants.
Figure 3.
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.
Figure 4.
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.
Table 2.
Maturation data.
Figure 5.
(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 [21]) adopts a slightly different conformation in the green versus red chromophore contexts. All proteins were aligned by the atoms of their imidazolinone ring.
Figure 6.
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.