Comprehensive Analysis of the Green-to-Blue Photoconversion of Full-Length Cyanobacteriochrome Tlr0924

Samantha J.O Hardman,Anna F.E Hauck,Ian P Clark,Derren J Heyes,Nigel S Scrutton

doi:10.1016/j.bpj.2014.09.020

Abstract

Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 μs. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms.

Highlights

Cyanobacteriochromes (CBCRs), a photochemically versatile class of proteins, mediate a variety of molecular outputs via the reversible E/Z isomerization of a sensory bilin chromophore in their cyanobacterial host organism [1]
Data collected with a depolarized pump beam yielded kinetics and spectra similar to those shown in Fig. 2; we assume that any polarization effects would not affect the model derived from these data
The comparatively small population of the PCB chromophore combined with the selective wavelength constant illumination allows almost complete isolation of the PVB photodynamics

Summary

Introduction

Cyanobacteriochromes (CBCRs), a photochemically versatile class of proteins, mediate a variety of molecular outputs via the reversible E/Z isomerization of a sensory bilin chromophore in their cyanobacterial host organism [1]. The general protein structure is divided into domains, which always include the photosensory chromophore binding GAF (cGMP-specific phosphodiesterase/adenylyl cyclase/FhlA protein) domain in the N-terminus and an output domain (e.g., histidine kinase or GGDEF domain) in the C-terminus [1,2]. The different subgroups of this protein class show great functional versatility. CBCRs have attracted a great deal of attention as novel photoreceptors. Variations in the domain architecture and chromophore structure permit the creation of proteins with a specific function for applications such as fluorescence biomarking [5], optogenetics [6], and the production of biofuels [7]. At the heart of these developments is a thorough understanding of the protein as a photoreceptor

Methods

Results

Conclusion