The interplay between chromophore and protein determines the extended excited state dynamics in a single-domain phytochrome

Abstract

Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their photochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes undergo reversible photoconversion triggered by theZ⇄Ephotoisomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photoreceptor All2699g1 (Nostocsp. PCC 7120) that exhibits photoconversion between the red light-absorbing (Pr) and far red-absorbing (Pfr) states just like canonical phytochromes. We present the crystal structure and examine the photoisomerization mechanism of the Prform as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations. We show that the unusually long excited state lifetime (broad lifetime distribution centered at ∼300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environment, thereby providing a structural framework for engineering bilin-based optical agents for imaging and optogenetics applications.

Effect of the PHY Domain on the Photoisomerization Step of the Forward Pr→Pfr Conversion of a Knotless Phytochrome

“…The signs of these signals indicate the orientation of the peripheral rings Aa nd Dw ith respect to the co-plane of rings B and C. [8,36] This overall orientation appearsu naffected by the presence of the "tongue", whichi si nl ine with the slight changes in the dihedrala ngle of rings Aa nd Do bserved by NMR. [32] In comparison, other phytochromes like Cph1 [37] and Cph2 [35] exhibit as imilar sign change of the Q-band, while most CBCRs and bacteriophytochromes show no sign change upon switching.…”

“…The role of the protein environment on the ultrafast photoisomerization dynamics of the PCB chromophorew as investigated by femtosecond transient absorption (TA) measurements on g1g2 as comparedw ith the single-domain g1. [8] The TA data of g1g2 show three main features ( Figure 3A): i) ab road positive signal below 575 nm which can be assigned to excited state absorption (ESA), ii)a negative signal above 575 nm due to ground state bleach (GSB) and stimulated emission (SE), and iii)a positive photoproduct absorption (PA) appearinga tl ater times at 670 nm associated with the formation of the primary Figure 1. Schematic representation of the structural homology model of the P r state of the All2699g1g2 construct.…”

“…[1,2] Ac omplete PAS-GAF-PHY array is required for photoconversion in canonical phytochromes( e.g.,P hyA, Cph1, Agp1), [3][4][5] while knotless phytochromes (e.g.,Cph2) [6,7] lack the PASd omain. [1,2] Interestingly,s ingle GAF domains of cyanobacteriochromes (CBCRs) [9] and some knotless phytochromes [8,10] preserve their photoconversion capability,w hichm akes them attractive for biotechnological applications. [11,12] The parental and the photoproduct states of canonical and knotless phytochromes are red-light (P r )a nd far-red-light (P fr )a bsorbing, respectively.…”

“…Recently,w ec ould show that the excited state decay kinetics in as ingleG AF domain (g1) derived from ak notless phytochrome (All2699g1g2 from Nostoc sp.P CC7120) is strongly distributed, and we assigned this behavior to conformationalc hanges in the bilin-binding pocket that control the photoisomerization of the chromophore. [8] Here, we report on the forward (P r !P fr )p hotoisomerization dynamics of the complete knotless phytochromeA ll2699g1g2( structurally similar to Cph2). [31,32] The homology model of All2699g1g2 [32] (Figure 1) shows that the "tongue" region of the g2 domain interacts with the chromophore bound to g1, just like the PHYd omains of canonical phytochromes.…”

Photocycle of Cyanobacteriochrome TePixJ

“…The excited state lifetimes of other bilin-containing photoreceptors are often reported as multiexponential, which can be explained either as ground state heterogeneity 26 or as the initial excited state affecting the surrounding protein environment. 29 …”