Molecular eyes: proteins that transform light into biological information

Abstract

Most biological photoreceptors are protein/cofactor complexes that induce a physiological reaction upon absorption of a photon. Therefore, these proteins represent signal converters that translate light into biological information. Researchers use this property to stimulate and study various biochemical processes conveniently and non-invasively by the application of light, an approach known as optogenetics. Here, we summarize the recent experimental progress on the family of blue light receptors using FAD (BLUF) receptors. Several BLUF photoreceptors modulate second messenger levels and thus represent highly interesting tools for optogenetic application. In order to activate a coupled effector protein, the flavin-binding pocket of the BLUF domain undergoes a subtle rearrangement of the hydrogen network upon blue light absorption. The hydrogen bond switch is facilitated by the ultrafast light-induced proton-coupled electron transfer (PCET) between a tyrosine and the flavin in less than a nanosecond and remains stable on a long enough timescale for biochemical reactions to take place. The cyclic nature of the photoinduced reaction makes BLUF domains powerful model systems to study protein/cofactor interaction, protein-modulated PCET and novel mechanisms of biological signalling. The ultrafast nature of the photoconversion as well as the subtle structural rearrangement requires sophisticated spectroscopic and molecular biological methods to study and understand this highly intriguing signalling process.

Keywords: flavin; photoreceptor; proton-coupled electron transfer; spectroscopy.

Figure 1.

Most biological photoreceptors can be described as light-activated switches, which thermally recover to the dark-adapted state. Upon transition from a dark-adapted state to a light-adapted state, the photosensory part of the protein modulates the activity of the cognate effector component.

Figure 2.

(a) BLUF domain of Slr1694 in dark- and light-adapted states illustrating the putative glutamine rotation mechanism. (b) The visible absorbance spectrum changes shifts by about 15 nm to the red upon illumination. (c) The light-minus-dark FT-IR difference spectrum shows the downshift of a carbonyl signature by approximately 20 cm^–1 predominantly assigned to the hydrogen bonding to the C₄=O of the flavin.

Figure 3.

Photocycle of BLUF domains as observed by ultrafast vis/IR and fluorescence spectroscopy on Slr1694. Lifetimes printed in parentheses correspond to H/D isotope affected reaction rates observed in D₂O.

Figure 4.

(a) The hydrogen bond network between tyrosine, glutamine and the flavin determines light-induced proton-coupled electron transfer. In contrast to the sequential ETPT reaction in the dark-adapted photocycle, the neutral semiquinone intermediate (b, green) in the light-adapted state is formed via a concerted ETPT (CEPT) reaction in about 1 ps. Lifetimes printed in parentheses correspond to the reaction rates observed in D₂O.

Figure 5.

Chemically modified BLUF domains. Roseoflavin (RoF) was reconstituted into the BLUF domain (RoSlr) in vivo using a genomically engineered E. coli expression strain, which is devoid of flavin biosynthesis and capable of taking up riboflavin from the medium. (a) Absorbance and emission properties of the isolated protein are shifted to the red accordingly. Additionally, the fluorescence of roseoflavin increases about 60-fold upon binding to the protein. (b) The protein may also be chemically modified by introduction of non-natural amino acids such as fluorotyrosine using a tyrosine auxotrophic expression strain. The time-resolved absorbance change at 701 nm is characteristic for excited state decay of flavins, which is mainly determined by electron transfer from a conserved tyrosine. Compared with WT, the ET reaction is significantly slowed down. (Online version in colour.)

Figure 6.

(a) Photocycle of BLUF domains as observed by ultrafast spectroscopy on redox-modulated Slr1694 proteins. Lifetimes printed in parentheses correspond to the reaction observed in D₂O. (b) Photoinduced electron transfer from flavin to tyrosine in BLUF domains is significantly influenced by changes in the redox potential of tyrosine (I) and flavin (II). The slowed down electron transfer allowed us to observe an excited state charge transfer state (FAD*CT (a)), which cannot be resolved in WT. Electron transfer is highly optimized and almost activation barrierless in Slr1694 WT (c, left) and is significantly slowed down by either an altered tyrosine redox potential which disfavours PET (c, middle) or a PET favouring redox potential of the flavin redox partner (c, right).