Avena sativa LOV2

In the C-terminal light-oxygen-voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix)

In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix)

In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513.

Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains.

The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in a loss of the interaction between the side chain of N414 and the backbone C═O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains.

In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513.

The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct.

The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct.

Finally, to investigate the requirements of an active-site cysteine for photocycling, we moved the nearby cysteine to alternative locations and found that some variants can still photocycle.

In addition, we demonstrate that dehydration leads to drastically slower photocycle times.

In Avena sativa LOV2 (AsLOV2), the photocycle is accompanied by an allosteric conformational change that activates the attached phototropin kinase in the full-length protein.

Mutations to the N414 and Q513 residues identify a potential water gate and H₂O coordination sites. These residues affect the electronic nature of the chromophore and photocycle time by helping catalyze the N5 reduction leading to the completion of the photocycle.

Both the conformational change and formation of the cysteinyl-flavin adduct are stabilized by the reduction of the N5 atom in the flavin's isoalloxazine ring.

However, electronegative side chains in the vicinity of the chromophore accelerate the N5 deprotonation and the return to the dark state.

We mutated all the residues that interact with the chromophore isoalloxazine ring to inert functional groups but none could fully inhibit the photocycle except those to the active-site cysteine.