Dynamic Inhomogeneity in the Photodynamics of Cyanobacterial Phytochrome Cph1

Abstract

Phytochromes are widespread red/far-red photosensory proteins well known as critical regulators of photomorphogenesis in plants. It is often assumed that natural selection would have optimized the light sensing efficiency of phytochromes to minimize nonproductive photochemical deexcitation pathways. Surprisingly, the quantum efficiency for the forward Pr-to-Pfr photoconversion of phytochromes seldom exceeds 15%, a value very much lower than that of animal rhodopsins. Exploiting ultrafast excitation wavelength- and temperature-dependent transient absorption spectroscopy, we resolve multiple pathways within the ultrafast photodynamics of the N-terminal PAS-GAF-PHY photosensory core module of cyanobacterial phytochrome Cph1 (termed Cph1Δ) that are primarily responsible for the overall low quantum efficiency. This inhomogeneity primarily reflects a long-lived fluorescent subpopulation that exists in equilibrium with a spectrally distinct, photoactive subpopulation. The fluorescent subpopulation is favored at elevated temperatures, resulting in anomalous excited-state dynamics (slower kinetics at higher temperatures). The spectral and kinetic behavior of the fluorescent subpopulation strongly resembles that of the photochemically compromised and highly fluorescent Y176H variant of Cph1Δ. We present an integrated, heterogeneous model for Cph1Δ that is based on the observed transient and static spectroscopic signals. Understanding the molecular basis for this dynamic inhomogeneity holds potential for rational design of efficient phytochrome-based fluorescent and photoswitchable probes.

Origins of Fluorescence in Evolved Bacteriophytochromes

“…The excited state lifetimes for fluorescent DrCBD derivatives are consistently longer and monoexponential compared with the value found for CBDs from Deinococcus radiodurans (32) and R. palustris (45). However, Cph1 lifetimes have been reported as long as 1.8 ns at room temperature (46,47). The longer decay lifetime and monoexponential decay behavior in the case of the DrCBD mon -Y263F variant indicates greater rigidity compared with the wild type and/or that the wild type lacks any type of fluorescent photoproduct.…”

The family of phytochrome-like photoreceptors: diverse, complex and multi-colored, but very useful

“…Given the difficulty to rationalize color-tuning in microbial and animal rhodopsins, which depend on the 'simpler' chromophore retinal, it is not surprising that theoretical approaches such as full-scale quantum chemical and QM/MM calculations on complex bilin chromophores in their protein environment are still in their infancy [49][50][51]. Here, progress is not only impeded by radiation effects on the chromophore during structural analyses [23 ,52], but also by intrinsic conformational heterogeneity of the P r state and [53][54][55][56] and to some extent also the P fr state [57]. The second issue is the chosen mode of downstream signaling, which apparently differs in the family of phytochrome-like photoreceptors (Figure 4c and d).…”

Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics

“…The phycobilisome terminal emitter L CM , which transfers energy to the chlorophylls in the photosynthetic membrane, binds PCB in a ZZZssa configuration as in phytochromes52 and has a fluorescence lifetime of 1.2 ns57. The BphP1-FP fluorescence lifetime is almost as long as that of the intensely fluorescent Y176F mutant of cyanobacterial phytochrome Cph1 (1.8 ns)1058, which binds PCB and is therefore not readily genetically encodable for in vivo fluorescence.…”