Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface

Abstract

Protein-chromophore interactions in photoreceptors often shift the chromophore absorbance maximum to a biologically relevant spectral region. A fundamental question regarding such spectral tuning effects is how the electronic ground state S(0) and excited state S(1) are modified by the protein. It is widely assumed that changes in energy gap between S(0) and S(1) are the main factor in biological spectral tuning. We report a generally applicable approach to determine if a specific residue modulates the energy gap, or if it alters the equilibrium nuclear geometry or width of the energy surfaces. This approach uses the effects that changes in these three parameters have on the absorbance and fluorescence emission spectra of mutants. We apply this strategy to a set of mutants of photoactive yellow protein (PYP) containing all 20 side chains at active site residue 46. While the mutants exhibit significant variation in both the position and width of their absorbance spectra, the fluorescence emission spectra are largely unchanged. This provides strong evidence against a major role for changes in energy gap in the spectral tuning of these mutants and reveals a change in the width of the S(1) energy surface. We determined the excited state lifetime of selected mutants and the observed correlation between the fluorescence quantum yield and lifetime shows that the fluorescence spectra are representative of the energy surfaces of the mutants. These results reveal that residue 46 tunes the absorbance spectrum of PYP largely by modulating the width of the S(1) energy surface.

Fig. 1.

Spectral tuning in PYP. (A) Structure of the pCA chromophore in the native state of PYP with active site residues Tyr42 and Glu46. (B) Amplitude-normalized absorbance spectra for free pCA, pCA attached to fully unfolded PYP in its neutral (pU) and ionized (pU-) state, and PYP in its native pG state. All four samples are in aqueous solution. Note that besides the absorbance maximum also the shape and width of the absorbance band is affected.

Fig. 2.

Strategy for probing excited state surfaces in spectral tuning. Three archetypal changes in energy surfaces by mutations and the resulting spectral tuning effects for absorbance and fluorescence emission spectra are shown. The energy surfaces, electronic transitions, and spectra for both the wild-type protein (black) and mutant protein (red) are indicated schematically for each of the three cases. Changes in both peak position and shape of the absorbance spectra (solid lines) and fluorescence emission spectra (dotted lines) are depicted. Shifts in the positions of the absorbance and fluorescence emission maxima are indicated by horizontal blue arrows. In case 1, which represents the classical explanation of spectral tuning, the energy gap ΔE between the electronic ground state S₀ and the first electronically excited state S₁ is altered. In case 2 the difference in nuclear equilibrium geometry ΔR_e is altered. In case 3 the width W of the S₁ energy surface is altered.

Fig. 5.

Analysis of spectral tuning by residue 46 in PYP based on the correlation between absorbance maxima and fluorescence emission maxima. The predicted correlations for spectral tuning by a change in ΔE (red), ΔR_e (green), and S₁ W (blue) are shown together with the experimentally observed correlation (black).

Fig. 4.

Analysis of the variation in the absorbance and fluorescence emission spectra of the E46X mutants of PYP. Histograms are shown for the peak positions (A), widths at 3/4 height (B), and band asymmetries (C) of the absorbance spectra (broad gray bars) and fluorescence emission spectra (narrow black bars) of the mutants.

Fig. 3.

Absorbance and fluorescence emission spectra of the 20 E46X mutants of PYP. (A) The amplitude-normalized absorbance and fluorescence emission spectra of the E46X mutants are shown. (B) Analysis of positions and bandwidths of the E46X mutants. Absorbance (closed circles) and fluorescence emission (open circles) maxima are indicated. The vertical bars depict the width of the spectra at 3/4 height. The horizontal axis represents the residue substituted at position 46. The residues were sorted from most red-shifted to most blue-shifted absorbance maximum.

Fig. 6.

Correlation of excited state lifetime and fluorescence quantum yield Φ_fl in wt PYP and the E46H, E46K, and E46Y mutants. (A) Determination of excited state lifetime by sub-ps transient absorbance pump-probe spectroscopy of wt PYP (black line) and its E46H (blue line), E46K (green line), and E46Y (red line) mutants. (B) Correlation between excited state lifetime and Φ_fl in wt PYP and the three mutants. The error bars depict the standard deviations.