Effect of the PHY Domain on the Photoisomerization Step of the Forward P_r→P_fr Conversion of a Knotless Phytochrome

Abstract

Phytochrome photoreceptors operate via photoisomerization of a bound bilin chromophore. Their typical architecture consists of GAF, PAS and PHY domains. Knotless phytochromes lack the PAS domain, while retaining photoconversion abilities, with some being able to photoconvert with just the GAF domain. Therefore, we investigated the ultrafast photoisomerization of the Pr state of a knotless phytochrome to reveal the effect of the PHY domain and its “tongue” region on the transduction of the light signal. We show that the PHY domain does not affect the initial conformational dynamics of the chromophore. However, it significantly accelerates the consecutively induced reorganizational dynamics of the protein, necessary for the progression of the photoisomerization. Consequently, the PHY domain keeps the bilin and its binding pocket in a more reactive conformation, which decreases the extent of protein reorganization required for the chromophore isomerization. Thereby, less energy is lost along nonproductive reaction pathways, resulting in increased efficiency.

Ultrafast Photoconversion Dynamics of the Knotless Phytochrome SynCph2

“…The dominant feature of the lifetime density maps (LDM) is located at ~200 ps with a positive-amplitude distribution in the range of the ESA signal (400–575 nm) and a negative-amplitude distribution in the GSB-SE range (600–740 nm) indicating the decay of the excited state, the recovery of the ground state and the formation of the primary photoproduct Lumi-R. The broadness of the lifetime distributions reflects the distributed type of the excited state decay kinetics as shown by the detailed analysis in our previous studies [ 50 , 53 ]. The final pair of lifetime distribution amplitudes at 3 ns represents the remaining GSB and Lumi-R absorption at 680 nm.…”

“…LDMs can be viewed as a kinetic footprint of a given system. In this regard, we noticed that the LDMs describing the P r kinetics in the single GAF domain All2699g1 of a knotless phytochrome [ 53 ], the complete PSMs of two knotless phytochromes (All2699g1g2 [ 50 ], Syn Cph2 ( Figure 2 )) and the red-green CBCR Slr1393g3 [ 41 ] show a striking level of similarity despite the structural and evolutionary differences of these phytochromes ( Figure 4 ). The LDMs, as well as the transient data, of all these longer-lived phytochromes exhibit a common pattern of a sub-200 fs departure from the FC-region followed by two pairs of lifetime distributions at ~0.6 ps and 2–8 ps and a distributed excited state decay in the hundreds of ps time range.…”

“…As we previously concluded based on theoretical calculations and kinetic modelling, the origin of the distributed kinetics can be linked to the protein environment being the controlling factor for excited state relaxation [ 50 , 53 ]. As it constrains the bilin chromophore, and in particular the D-ring, the reaction cannot proceed unless the protein binding pocket undergoes rearrangements that facilitate chromophore isomerization.…”

“…However, we do not observe distinctly heterogeneous dynamics in terms of evolution of separable substates, instead some ground state heterogeneity might add to the distributed character of the dynamics. Therefore, we believe that for the longer-lived phytochromes (e.g., knotless phytochromes [ 50 , 53 ], some bacteriophytochromes [ 35 , 36 , 56 ] and some CBCRs [ 40 , 41 , 42 , 43 ]) the reorganization of the binding pocket after photoexcitation plays a dominant role in determining the isomerization kinetics.…”

“…In contrast, little is known about the ultrafast forward and reverse dynamics of Group II phytochromes. To date, only the forward photoisomerization direction of the knotless phytochrome All2699g1g2 has been examined [ 50 ], but no details on the reverse reaction are available. To shed light on the ultrafast primary photoreaction of knotless phytochromes, here we present both the ultrafast forward (P r → P fr ) and reverse (P fr → P r ) dynamics of the most prominent group member, Syn Cph2.…”