Paper Introduction 12 08 2015 Kazuya Matsuo J. Am. Chem. Soc. 2015, 137, 11218–11221. Kazuya Matsuo

Photocontrol of protein activity 1. Photoswitchable ligands Tamaoki, N. et al. ACS Nano 2014, 8, 4157-4165. Tamaoki, N. et al. Chem. Commun. 2013, 49, 9935–9937. Maruta, S. et al. J. Biochem. 2014, 155, 257–263. 2. Photoswitchable ligand tethered proteins D. Trauner et al. Angew. Chem. Int. Ed. 2011, 50, 12156-12182. 3. Labeling of photochromic molecules with two reactive groups A. Pingoud et al. PNAS 2010, 107, 1361-1366. 4. Genetically encodable small molecules with photoresponsivirity P. G. Schultz et al. J. Am. Chem. Soc. 2006, 128, 388-389.

Amber codon method for incorporating unnatural amino acids It is relatively easy to apply this technology into not only in vitro system but also in cell/in vivo system They already reported reactive amino acids incorporation to proteins L. Wang et al. Angew. Chem. Int. Ed. 2014, 53, 3932-3936. The drawback is to use UV light (365 nm)

They present the design and genetic incorporation of a new class of PhotoSwitchable Click amino acids (PSCaa), which generates a photoswitchable protein bridge To avoid infeasibility of tRNA system, they used pentafluoro PSCaa. Fluorine has similar size to proton Perfluorination can modulate photochromic performance Perfluorobenzene allows nucleophilic aromatic substitution (SNAr)

,although the photoreactivity in 540 nm irradiation was not so changed Photochromic properties and SNAr reactivity of Compound 1 Z E 540 nm 77% Z E 540 nm 78% Z 365 nm Similar to 540nm 405 nm 73% E Z SNAr reaction was completely done after about 6h at 25oC Pentafluoro compound 1 resulted in distinct separation in wavelength of both transition bands (π-π*, n-π*) between E and Z isomers, Especially, the separation of n-π* band enabled the use of green light to get Z isomer Compound 3 showed less sharper changes in 405 nm irradiation than pentafluoro compound 1 ,although the photoreactivity in 540 nm irradiation was not so changed

They expressed (Cys83)CaM76 in the presence/absence of PSCaa 1 using their original tRNA system based on TAG amber codon (a) SDS-PAGE & (b) MS (after Ni-NTA purification) → Target protein expression was observed ◯ PSCaa 1 was genetically encodable amino acids ◯ No peak corresponding to non-bridged CaM (= intramolecular SNAr reaction go well with high efficiency) CaM = Calmodulin : A small, highly conserved protein composed of 148 amino acids, which can bind Ca2+ ion.

(a) Bridged CaM was controllable by blue/green light irradiation π-π* Before irradiation 470 nm irradiation (Z→E : pss of E state) n-π* 540 nm irradiation (E→Z : pss of Z state) (a) Bridged CaM was controllable by blue/green light irradiation (b) Reversible transformation between the E and Z states (c) CD (circular dichroism) spectra was typical for a-helical CaM (222/208 nm) Upon E to Z state, band at 208nm decreased = wild type of CaM’s change before/after Ca2+ binding → clear conformational change of CaM

Green light induced Z state In the absence of substrate peptide Test the binding of CaM Dye : dansyl No Fluorescence Fluorescence NOS-I peptide Ca2+ Ca2+ Dansyl-labeled CaM Green light induced Z state (22% E) E state In the absence of substrate peptide 215% increment 50% increment Z state CaM was inhibited on the binding of NOS-I peptide But, no experiments about photoreversibility

Conclusion In this paper, they achieved… 1. Genetically encodable unnatural amino acids with pentafuluoroazobenzene 2. Perfect and quantitative site-specific incorporation 3. Proximity-accelerated SNAr reaction on proteins 4. Protein structure/activity control by not UV but biocompatible visible light irradiation 5. Applicability in vitro and in cells