An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway

Abstract

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm−1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.

Fluorescence Modulation by Ultrafast Chromophore Twisting Events: Developing a Powerful Toolset for Fluorescent-Protein-Based Imaging

“…Therefore, it remains necessary to study the chromophore twisting events directly inside the protein matrix for various applications across bioimaging and bioengineering fields, which commonly pose various challenges for high-level computations and high-fidelity simulations. Moreover, it is notable that such investigations are not limited to the autocatalytically formed three-residue chromophores inside FPs, as demonstrated by our recent elucidation of the highly efficient reversible photoswitching (twisting) events of one of the conjugated tetrapyrrole rings as a cofactor for a wild-type cyanobacteriochrome photoreceptor AnPixJg2 (with phycocyanobilin cofactor) 114 and its newly engineered version AnPixJg2_BV4 (with only four mutations to incorporate a mammalian cofactor biliverdin with redder absorption and emission) 115 mainly on the fs to ps time scales.…”

Red Light Optogenetics in Neuroscience

“…Still, several advances have been made to ease the bilin availability for red light-sensing optogenetic actuators. For example, the genetically engineered production of bilins has been further improved ( Kyriakakis et al, 2018 ; Uda et al, 2020 ), and biliverdin-utilizing CBCRs have been either discovered or created by mutations ( Fushimi et al, 2019 ; Moreno et al, 2020 ; Tachibana et al, 2021 ).…”

The Physical Chemistry and Chemical Physics (PCCP) Section of the International Journal of Molecular Sciences in Its Publications: The First 300 Thematic Articles in the First 3 Years

“…In this Thematic Area, 56 articles have been published [ 48 , 64 , 104 , 108 , 110 , 124 , 195 , 252 , 253 , 254 , 255 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 264 , 265 , 266 , 267 , 268 , 269 , 270 , 271 , 272 , 273 , 274 , 275 , 276 , 277 , 278 , 279 , 280 , 281 , 282 , 283 , 284 , 285 , 286 , 287 , 288 , 289 , 290 , 291 , 292 , 293 , 294 , 295 , 296 , 297 , 298 , 299 , 300 ], an...…”

“…A more detailed examination shows that, in this direction, as well as in the previous ones, articles in which the objects of research are organic materials clearly prevail over articles in which the objects of research are inorganic materials (36 and 20, respectively). Wherein, the overwhelming majority of articles of the “organic” profile are, in one way or another, connected with the participation of the objects with a biological origin; these include [ 64 , 124 , 254 , 255 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 274 , 277 , 278 , 282 , 284 , 289 , 291 , 292 , 293 , 294 , 295 , 296 , 298 , 299 , 300 ] and the processes in abiogenic organic materials were considered only in [ 48 , 195 , 266 , 271 , 272 , 273 , 283 , 288 , 290 ]. The thematic focus of these works, as well as the chemical compounds studied in them, is also very diverse, and even a simple listing of them here is clearly beyond the scope of this article, too.…”