The Orange Carotenoid Protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 milliseconds. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid β1-ring in picoseconds occurs at a low yield of <1 %, whereby the β1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 μs, which allow the carotenoid to translocate into the N-terminal domain in 10 μs. We identified infrared signatures of helical elements that dock on the C-terminal domain β-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer timescales, lagging carotenoid translocation by at least 2 decades of time.

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λ_max = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λ_max = 690 nm) highly fluorescent state (Q_F = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors.

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a β-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D₂O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 μs. We observe a two-step unfolding of the Jα helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 μs, along with hydrogen-bond rupture of the FMN C4═O with Gln-513, motion of the β-sheet, and an additional helical element. The second phase occurs in approximately 240 μs. The final spectrum at 500 μs is essentially identical to the steady-state light-minus-dark Fourier transform infrared spectrum, indicating that Jα helix unfolding is complete on that time scale.

Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 3₁₀-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting family that may control thylakoid inter-protein interactions driving photoregulatory responses.

Bacteriophytochromes (Bphs) are red-light photoreceptor proteins with a photosensory core that consists of three distinct domains, PAS, GAF and PHY, and covalently binds biliverdin (BV) to a conserved cysteine in the PAS domain. In a recent development, PAS–GAF variants were engineered for use as a near-infrared fluorescent marker in mammalian tissues (Tsien and co-workers, Science, 2009, 324, 804–807). Here, we report the fluorescence quantum yield and photochemistry of two highly-related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We applied ultrafast spectroscopy to wild type P3 and P2 PAS–GAF proteins and their P3 D216A, Y272F and P2 D202A PAS–GAF–PHY mutant proteins. In these mutants hydrogen-bond interactions between a conserved aspartate (Asp) which connects the BV chromophore with the PHY domains are disrupted. The excited-state lifetime of the truncated P3 and P2 PAS–GAF proteins was significantly longer than in their PAS–GAF–PHY counterparts that constitute the full photosensory core. Mutation of the conserved Asp to Ala in the PAS–GAF–PHY protein had a similar but larger effect. The fluorescence quantum yields of the P3 D216A and Y272F mutants were 0.066, higher than that of wild type P3 (0.043) and similar to the engineered Bph of Tsien and co-workers. We conclude that elimination of a key hydrogen-bond interaction between Asp and a conserved Arg in the PHY domain is responsible for the excited-state lifetime increase in all Bph variants studied here. H/D exchange resulted in a 1.4–1.7 fold increase of excited-state lifetime. The results support a reaction model in which deactivation of the BV chromophore proceeds via excited-state proton transfer from the BV pyrrole nitrogens to the backbone of the conserved Asp or to a bound water. This work may aid in rational structure- and mechanism-based conversion of constructs based on P3 and other BPhs into efficient near-IR, deep tissue, fluorescent markers.