good photochemical 
 properties , chemical stability , and ease of 
 synthesis make coumarins an important class of fluorescent probes 
 for biological studies . in addition to being versatile fluorophores , coumarin 
 chromophores can be used as light - removable protecting groups , so - called 
  caging groups 
. caged molecules 
 have been extensively applied in the optical control of cellular processes . in particular , the 6-bromo-7-hydroxycoumarinmethyl caging group 
 undergoes fast two - photon photolysis at 740 nm and has been used to 
 optically control neurotransmitters , secondary messengers , and oligonucleotides .
two - photon irradiation enables optical activation of biological 
 processes with enhanced tissue penetration of up to 1 mm .
moreover , 
 two - photon caging groups can be released with greater precision in 
 three - dimensional space than simple one - photon caging groups . here
we report the site - specific incorporation of three coumarin 
 amino acids into proteins via genetic code expansion with unnatural 
 amino acids ( uaas ) to integrate the optical properties of coumarin probes 
 into cellular systems .
genetic code expansion requires the addition 
 of orthogonal translational machinery to achieve site - specific uaa 
 incorporation into proteins .
recent advances in engineering pyrrolysyl - trna 
 synthetase / trna pairs for the incorporation of sterically demanding 
 amino acids prompted us to synthesize coumarin lysines 13 ( figure 1a ) and to test their incorporation 
 into proteins .
the photochemical characteristics of these uaas complement 
 and enhance the properties of caged and fluorescent amino acids that 
 have been genetically encoded in bacterial and mammalian cells .
lysines 13 were assembled in three 
 steps from their corresponding coumarin alcohols ( supporting information , scheme s1 ) .
briefly , the coumarin 
 alcohols were activated with nitrophenyl chloroformate and coupled 
 to commercially available boc - lysine .
a global deprotection under 
 acidic conditions furnished the corresponding coumarin derivatives 13 in good yields .
all three coumarin 
 lysines 13 contain identical benzopyrone 
 cores as fluorescent probes . however , 
 subtle substitutions result in a set of coumarin derivatives with 
 unique photochemical properties .
introduction of a bromine at the 
 6-position enables decaging not only with uv ( single photon ) light 
 ( in case of 1 ) , but also near ir ( two - photon ) excitation 
 ( in case of 2 ) .
in contrast , 
 extension of the coumarin  carbamate linker by a single carbon 
 atom results in coumarin lysine 3 , which does not undergo 
 photolysis and thus represents a stable coumarin amino acid probe . 

thus , coumarin lysines 1 and 2 can be used 
 as both fluorescent and light - activated probes for optochemical control 
 of protein function using uv or near - ir light , while coumarin lysine 3 may serve as a stable fluorescent probe that does not decage 
 under uv excitation .
the methanosarcina barkeri pyrrolysyl trna synthetase / trnacua ( mbpylrs / trnacua ) is functional 
 and orthogonal in a wide range of organisms , such as escherichia 
 coli , yeast , mammalian cells , and animals such as caenorhabditis elegans and drosophila melanogaster .
furthermore , wild - type pylrs recognizes several unnatural amino 
 acids without accepting any of the 20 common amino acids as a substrate .
the active site of the pylrs can be further 
 engineered through directed evolution to enable the incorporation 
 of additional unnatural amino acids with new functions , including 
 post - translational modifications , bioconjugation handles , photo - cross - linkers , 
 photocaging groups , and others .
thus , 
 we generated and screened a panel of mbpylrs mutants , 
 guided by mutants that were previously reported , to direct the incorporation of 2 in response 
 to a tag amber codon in mammalian cells using a mcherry - tag - egfp reporter . 

cells containing a mbpylrs mutant with only two amino 
 acid mutations y271a and l274 m showed uaa - dependent expression of 
 full - length mcherry - egfp - ha .
the y271a mutation has previously been 
 reported to direct the incorporation of n - carbamate - linked lysines , while the 
 l274 m mutation was discovered to facilitate 
 higher amber suppression activities with 2 in vivo , because 
 it allows greater flexibility of the side chain and imposes less steric 
 bulk at the back of the hydrophobic pocket .
this synthetase , termed 
 bhckrs , enabled the site - specific incorporation of not only 2 but also 1 and 3 in response to 
 the amber codon tag within sfgfp - y151tag - his6 in e. coli ( figure 1 ) .
this is not surprising , 
 considering the very similar structures of 13 and previous observations of the high promiscuity of pylrs . to further rationalize the ability of bhckrs to incorporate 13 ,
molecular modeling was employed . 
 the wild - type pylrs structure ( pdb : 2q7h )
was used as a starting template for 
 which the y271a and l274 m mutations were introduced using modeller .
the mutant structure was energy minimized in 
 amber molecular dynamics before docking 13 into the active site pocket using 
 autodock4 . as expected , 13 adopt very similar poses , reflecting their 
 similarity in structure ( see supporting information , figure s1 ) .
the mutated synthetase model reveals that the y271a 
 and l274 m mutations greatly enlarge the binding pocket to accommodate 
 the bulky bicyclic caging group , while also orienting it in a favorable 
 -stacking interaction with w382 .
this orientation also benefits 
 from a favorable h - bond interaction between the coumarin hydroxyl 
 group and d373 .
similar to published crystal structures , the amino 
 group s positioning is maintained by interactions with a structural 
 water and y349 .
it has been previously 
 shown that interactions with n311 and r295 play an important role 
 in amino acid recognition by the pylrs system .
the docked structure maintains these key 
 interactions with the carbamate carbonyl forming a h - bond with n311 , 
 while the carboxylic acid forms a h - bond with r295 ( figure 1b , c ) .
sds - page analysis reveals coumarin fluorescence 
 of the expressed 
 proteins containing the coumarin lysines 13 .
no fluorescence is observed for wild - type sfgfp because 
 its excitation wavelength ( 488 nm ) does not match that of 13 ( 365 nm ) and because of the denaturing conditions 
 of the gel .
the dependence of protein expression on the presence of 13 demonstrates that the engineered bhckrs 
 synthetase has a high specificity for coumarin lysines and does not 
 significantly incorporate any of the common 20 amino acids .
similar 
 results were obtained for the incorporation of 13 into ubiquitin and myoglobin in e. coli .
electrospray ionization mass spectrometry ( esi - ms , supporting information , figures s2s4 ) 
 showed that recombinantly expressed sfgfp-1 and -3 have a mass of 28446.22 and 28460.60 da , in agreement with 
 the expected masses of 28446.03 and 28460.04 da , respectively .
esi - ms 
 analysis of sfgfp-2 showed a mass of 28445.97 da , indicating 
 a partial loss of bromine during e. coli expression , 
 possibly due to reductive dehalogenation .
overall , these results demonstrate that 13 can be incorporated into proteins in e. coli in good yields ( 8.0 mg / l , 1.6 mg / l , and 2.5 mg / l , respectively , 
 for sfgfp ) and with high specificity .
( a ) structures of the genetically encoded 
 coumarin amino acids 
 for fluorescence reporting and light activation of protein function . 

( b ) crystal structure of pylrs ( 2q7h ) with the pyrrolysine substrate ( yellow ) 
 in the active site . ( c )
( d ) sds - page analysis of sfgfp - y151tag containing 13 through incorporation in e. coli .
the gel 
 was stained with coomassie blue ( top ) , and coumarin fluorescence was 
 imaged via excitation at 365 nm ( bottom ) .
( e ) fluorescence micrographs 
 of hek 293 t cells expressing the bhckrs / trnacua pair and 
 mcherry - tag - egfp - ha in the presence or absence of 13 .
( f ) western blot analysis of cell lysates using an anti - ha 
 antibody and a gapdh antibody as a loading control .
full - length protein 
 expression is only observed in the presence of 13 , and incorporation efficiency with all three amino acids 
 is similar in mammalian cells . to demonstrate that the coumarin lysines 13 can also be genetically incorporated into proteins in mammalian 
 cells
, pbhckrs - mcherry - tag - egfp - ha and p4cmve - u6-pylt were cotransfected 
 into human embryonic kidney ( hek ) 293 t cells .
cells were incubated 
 for 24 h in the absence of any unnatural amino acid and in the presence 
 of 13 ( 0.25 mm ) .
fluorescence imaging 
 revealed egfp expression only in the presence of 13 , indicating specific incorporation of the coumarin lysines 
 in response to the tag codon , without measurable incorporation of 
 endogenous amino acids ( figure 1e ) .
this was 
 further confirmed by an anti - ha western blot on cell lysates from 
 the same experiment ( figure 1f ) .
furthermore , 
 full - length mcherry - egfp protein was immunoprecipitated from hek 293 t 
 cells using an immobilized antibody against the ha - tag and mass spectrometry 
 sequencing confirmed that 13 are 
 site - specifically incorporated into protein in mammalian cells ( supporting information , figure s5 ) .
importantly , 
 the presence of bromine was verified for protein containing 2 , confirming the genetic encoding of the bhc - caged lysine . because the coumarin groups on 1 and 2 are caging groups that can be removed via light exposure , loss of 
 their intrinsic fluorescence can be used as an indicator of protein 
 decaging through uv irradiation , as shown in figure 2a , b .
this was demonstrated through a uv exposure time - course 
 of purified sfgfp-1 , followed by sds - page analysis .
the 
 coumarin fluorescence intensity of sfgfp-1 gradually 
 decreases with extended uv exposure as more of the coumarin caging 
 group is removed from the protein , while the continued presence of 
 the coomassie - stained protein band indicates stability of the protein . 
 in a cellular context
, this may enable experiments that allow for 
 the determination of protein expression , protein localization , and 
 protein decaging using a single optochemical probe in a single experiment . 

in contrast , insertion of an extra methylene unit between the lysine 
 and the fluorophore fully abrogates photocleavage and thus establishes 3 as a stable amino acid for the site - specific fluorescent 
 labeling of proteins . no change in coumarin fluorescence is observed 
 after uv exposure of sfgfp-3 for 20 min ( figure 2c , d ) . due to the identical fluorophores in 1 and 3 and the stability of 3 to 
 the uv irradiation conditions ,
the loss of protein fluorescence in 
 sfgfp-1 is due to decaging and not due to photobleaching . 

this is further supported by mass spectroscopic analysis of the proteins 
 before and after uv exposure ( see supporting information , figures s2 and s4 ) . to demonstrate the ability of the genetically 
 encoded coumarin 
 lysines to act as reporters for protein localization in live cells , 
 we investigated their utility as a protein nuclear localization marker . 

a plasmid was constructed to express egfp - ha with an n - terminal nls 
 ( nuclear localization signal , pnls - linker - egfp - ha ) , which reliably localizes egfp to the nucleus ( supporting information , figure s6 ) .
a tag amber 
 codon was introduced in the linker between the nls and egfp , allowing 
 for site - specific unnatural amino acid incorporation without affecting 
 egfp formation or nuclear translocation .
cells cotransfected with 
 the pnls - ktag - egfp and bhckrs / pyltrnacua plasmid pair in 
 the presence of 1 ( 0.25 mm ) were analyzed for coumarin 
 fluorescence ( 405 nm excitation , 450480 nm emission ) and egfp 
 fluorescence ( 488 nm excitation , 490520 nm emission ) by confocal 
 microscopy .
the observation of complete colocalization of both fluorophores 
 in the nucleus ( merged micrographs ) demonstrates the ability to use 1 as a reporter of protein localization ( figure 2e and supporting information , movie 
 s1 and figure s7 ) .
sds - page fluorescence analysis shows photodecaging of 
 sfgfp-1 while sfgfp-3 is stable to uv exposure . 

( a ) 
 loss of coumarin fluorescence after extended sfgfp-1 in - gel 
 decaging for 050 min ( 365 nm , transilluminator ) .
( b ) coomassie 
 staining reveals identical sfgfp-1 protein amounts in 
 all lanes .
( c ) no loss of coumarin fluorescence is observed , since 
 sfgfp-3 does not decage .
( e ) nuclear 
 colocalization of coumarin and egfp fluorescence in cho k1 cells cotransfected 
 with pnls - tag - egfp - ha and the bhckrs / pylt pair ( pbhckrs-4pylt ) in 
 the presence of 1 ( 0.25 mm ) . a dic image and a merged 
 image of all three channels
to apply the coumarin lysines 13 in the optical control of protein function in live cells , 
 firefly 
 luciferase ( fluc ) was selected as an initial target because bioluminescence 
 measurements afford low background , high sensitivity , and easy quantification . 
 on the basis of the fluc crystal structure , a critical lysine residue , 
 k206 ,
was identified , which is positioned at the edge of the substrate - binding 
 pocket ( figure 3b ) .
it has been proposed that 
 this residue stabilizes and orients atp in the active site .
the -amino group on k206 provides a hydrogen - bond interaction 
 with the -phosphate of atp and promotes the adenylation reaction 
 with luciferin , thus being essential for catalytic activity as shown 
 by the dramatic decrease in enzymatic activity displayed by the k206r 
 mutant .
therefore , we hypothesized that 
 a sterically demanding coumarin caging group placed on k206 would 
 prevent the interaction with atp and limit the overall access of the 
 substrates to the active site ( figure 3a ) . 

photolysis of the coumarin lysine would remove the caging group and 
 produce a native lysine residue , restoring the catalytic activity 
 of the enzyme ( figure 3b ) .
a genetically encoded 
 photocaged lysine at k206 would enable the enhanced regulation of 
 the catalytic activity of firefly luciferase via light activation .
site - directed mutagenesis of the corresponding k206 residue to 
 the amber codon ( tag ) enabled incorporation of 13 into firefly luciferase in mammalian cells .
hek 293 t cells 
 were cotransfected with the mutated firefly luciferase plasmid ( pgl3-k206tag ) 
 and the mbbhckrs / pyltrnacua pair ( pbhckrs-4pylt ) 
 in the presence of 13 ( 0.25 mm ) . 

after 24 h incubation , the cells were either irradiated for 4 min 
 ( 365 nm , 25 w ) or kept in the dark .
the incorporation of 13 into fluc caused complete inhibition of luciferase 
 activity before uv irradiation , as determined by a bright - glo luciferase 
 assay , comparable to the negative control ( no unnatural amino acid ) . 
 after uv irradiation , 1 and 2 were decaged 
 to produce native lysine , resulting in the activation of firefly luciferase 
 by 34-fold and 31-fold , respectively ( figure 3c ) .
as expected , 3 did not show any activation of luciferase 
 enzymatic activity upon illumination , as it does not undergo decaging . 
 therefore , the activity of firefly luciferase can be tightly optochemically 
 regulated by incorporation of a coumarin lysine residue into the active 
 site of the luciferase protein .
interestingly , attempts to apply 1 and 2 at position k529 , another site that can 
 be used for optical control of luciferase function , led to greatly diminished luciferase activity , while introduction 
 of our previously reported o - nitrobenzyl - caged lysine worked at both positions k206 
 and k529 .
western blots confirmed that both fluc - k206  1 and fluc - k529  1 were expressed at 
 similar levels in mammalian cells ( supporting 
 information , figures s8 and s9 ) .
engineering of an optochemically 
 controlled photinus pyralis firefly luciferase through 
 unnatural amino acid mutagenesis .
( a ) 
 caging groups at position k206 are blocking access to the binding 
 pocket by luciferin and atp and are disrupting a required hydrogen 
 bonding network .
( b ) after decaging , wild - type fluc is generated and 
 the substrates can now enter the active site .
( c ) bright - glo 
 luciferase assay of cells that were either kept in the dark or irradiated 
 ( 365 nm , 4 min ) .
no enzymatic activity was observed for the caged proteins , 
 and significant increases in luminescence were observed after photolysis 
 of luciferase containing 1 or 2 , while the 
 k206  3 mutant was permanently deactivated , as 
 expected .
error bars represent standard deviations from three independent 
 experiments . to observe the optical 
 triggering of protein function via decaging 
 of 1 and 2 in real time ,
enhanced green 
 fluorescent protein ( egfp ) was selected as a second target protein 
 for caging .
egfp consists of an 11-stranded -barrel and a central 
 -helix with the thr65-tyr66-gly67 chromophore .
correctly folded egfp 
 is a prerequisite for mature chromophore formation , with a number 
 of lysine residues being essential to its successful folding .
most notable is that only 1 lysine ( k85 ) out 
 of 20 is buried within the protein .
k85 
 forms a salt bridge with d82 and h - bonding interactions with the backbone 
 of c70 and s72 , all of which are in close 
 proximity to the chromophore ( figure 4a ) .
it 
 has been shown that c70 , s72 , and d82 are key residues for control 
 of chromophore formation and oxidation .
we hypothesized 
 that introduction of coumarin - caged lysines 1 and 2 at k85 would affect d82 , c70 , and s72 , interrupting the 
 -helix bending and thus indirectly inhibiting chromophore maturation . 
 to this end , we envisioned that uv activation would yield native egfp 
 that rapidly undergoes maturation .
an egfp mutant with an amber codon 
 at position k85 ( pegfp - k85tag ) was generated as a fusion construct 
 with mcherry , to provide a second reporter for successful plasmid 
 transfection and incorporation of 1 and 2 .
hek 293 t cells were cotransfected with pegfp - k85tag - mcherry and 
 the bhckrs / pyltrna pair in the presence of 1 and 2 ( 0.25 mm ) .
after 24 h , the cells were washed and incubated 
 in fresh media for 1 h. cells expressing mcherry were observed by 
 fluorescence imaging to confirm that egfp-1/2-mcherry 
 is generated in the presence of 1 or 2 . 

cells were irradiated for 30 s at 365 nm , and fluorescence was imaged 
 by time - lapse microscopy .
after photolysis of egfp-1 , 
 green fluorescence started to appear around 10 min , and over time 
 the fluorescence intensity gradually increased , reaching a plateau 
 at 120 min ( figure 4d and supporting information , movie s2 ) .
a half - life of 49 min was 
 observed , matching reports of egfp chromophore maturation as the rate - limiting 
 step .
previous measurements of egfp folding 
 and maturation have been exclusively performed in test tubes .
no cellular studies have been conducted , as 
 a precise starting point for kinetic analysis could not be provided .
( a ) location 
 of k85 ( yellow ) and interactions with d82 , c70 , and 
 s72 in egfp .
( b ) schematic of 
 the pegfp - k85tag - mcherry construct and its application in light activation 
 studies .
( c ) fluorescence imaging of hek 293 t cells expressing egfp - k85tag - mcherry , 
 90 min after irradiation at 365 nm ( 30 s , dapi filter , 358365 
 nm ) in the presence of 1 ( nikon a1r confocal microscope , 
 20 objective , 2-fold zoom ) .
( d ) normalized egfp fluorescence 
 as a function of time after 365 nm light activation ( error bars represent 
 standard deviations from the measurement of three independent cells , t1/2 = 49 min ) .
given that the coumarin lysines have relatively broad absorption 
 bands in the 300420 nm range that enable decaging at longer 
 wavelengths , we speculated that irradiation at 405 nm may efficiently activate 1 and 2 .
thus , 
 activation through blue light irradiation using a standard laser - scanning 
 confocal microscope was tested .
as expected , exposure at 405 nm induced 
 fluorophore formation of egfp-1 and -2 ( figure 5a , b ) . because attempts to decage a previously incorporated 
 nitrobenzyloxycarbonyl lysine and nitrobenzyl tyrosine at 405 nm were not successful on comparable time scales and at comparable 
 illumination power ( data not shown ) , the caged lysines 1 and 2 may enable multiwavelength activation of proteins 
 caged with the two different optical probes .
taking advantage 
 of the two - photon decaging feature of 2 , photocontrol of egfp folding by two - photon 
 activation of egfp-2 was performed .
hek 293 t cells were 
 cotransfected with pegpf - k85tag - mcherry and pbhckrs-4pylt in the absence 
 or presence of 1 and 2 ( 0.25 mm ) .
after 
 a 24 h incubation , the cells were washed and incubated in fresh media 
 for 1 h and irradiated with a multiphoton laser ( 760 nm , 130 mw , 2 
 m / s dwell time , 30 cycles , olympus fluoview fv1000 mpe multiphoton 
 laser scanning microscope fv10-asw , maitai dsbb - ol ir pulsed laser ) . 

images were acquired before and after two - photon irradiation using 
 both egfp ( 488 nm ) and mcherry ( 561 nm ) excitation .
gratifyingly , 
 an egfp fluorescent signal was observed after photolysis of 2 at 760 nm ( figure 5c ) .
the cells 
 expressing egfp containing 1 , as a control , were also 
 exposed to two - photon excitation ( 760 nm ) and imaged in the same fashion 
 ( figure 5d ) ; no egfp activation was observed . 

in addition to the increased three - dimensional resolution that is 
 provided through two - photon excitation , effectively shifting the activation 
 wavelength to the near - ir will enable multiwavelength activation in 
 conjunction with other optically triggered biological processes , while 
 also preventing any overlap with established fluorescent reporter 
 proteins .
fluorescence confocal imaging of cos-7 cells expressing egfp - ktag - mcherry , 
 before and after irradiation at 405 nm ( 30 mw diode laser , 20% laser 
 power , 12.6 s dwell time , 8 cycles ) in the presence of 2 ( a ) or 1 ( b ) ( zeiss confocal lsm710 microscope , 
 40 water objective ) .
similar light - activation experiments before 
 and after irradiation of hek 293 t cells at 760 nm ( 130 mw , 2 m / s 
 dwell time , 30 cycles , olympus fluoview fv1000 mpe , maitai dsbb - ol 
 ir pulsed laser ) , in the presence of 2 ( c ) or 1 ( d ) , imaged with a olympus fluoview1000 , 40 oil objective .
the site - specific genetic 
 incorporation of three new coumarin lysine 
 analogues 13 into proteins was achieved 
 in bacterial and mammalian cells using an engineered bhckrs synthetase 
 system .
the genetically encoded coumarin lysines were successfully 
 applied as fluorescent cellular probes for protein localization , and 
 the small size of these coumarin lysines is expected to minimally 
 perturb protein structure and function , unless they are placed at 
 critical sites .
in addition to their small size , the spectral properties 
 of 13 do not interfere with common 
 fluorescent proteins ( e.g. , egfp ) .
while the amino acid 3 showed stability under irradiation conditions , the coumarins 1 and 2 were readily decaged , generating wild - type 
 lysine residues . as a proof - of - principle , photoregulation of firefly 

luciferase was achieved in live cells by caging a key lysine residue , 
 and excellent off to on light - switching ratios were observed for 1 and 2 . as expected
furthermore , 
 two - photon and single - photon optochemical control of egfp maturation 
 was demonstrated , enabling the use of different , potentially orthogonal , 
 excitation wavelengths ( 365 , 405 , and 760 nm ) for the sequential activation 
 of protein function in live cells . while the caged lysine 2 could be activated using two - photon irradiation at 760 nm , the lysine 1 was stable under these conditions .
however , decaging of 1 was readily achieved with blue light of 405 nm , while a 
 previously encoded o - nitrobenzyl - caged lysine requires 
 uv activation .
these results demonstrate that coumarin lysines are a new and valuable 
 class of optical probes that can potentially be used for the investigation 
 and regulation of protein structure , dynamics , function , and localization 
 in live cells .
the small size of coumarin , the application as both 
 a light - activated caging group and a fluorescent probe , and the broad 
 range of excitation wavelengths are advantageous over other genetically 
 encoded photocontrol systems and provide a unique and multifunctional 
 tool for cellular biology .
the ability to incorporate all three coumarin 
 lysines with the same pylrs / trnacua pair further facilitates 
 their application .
( 1 ) construction of pnls - tag - egfp - ha : the ptag - egfp - ha 
 fragment was amplified from pmcherry - tag - egfp - ha using the pcr primers 
 g1/g2 , digested with hindiii and bglii , and ligated into pegfp - n1 ( clontech ) , generating the ptag - egfp - ha 
 plamid .
the pnls pcr fragment was obtained by using primers n1/n2 
 and then ligated into the hindiii and xbai sites of ptag - egfp - ha to generate the pnls - tag - egfp - ha plasmid . 

( 2 ) construction of pnls - wt - egfp - ha : plasmids were obtained by converting 
 the tag codon of pnls - tag - egfp - ha into an aag ( lys ) codon using primers 
 qc1/qc2 and a quikchange site - directed mutagenesis kit ( agilent ) . 

( 3 ) construction of pbhckrs-4pylt : the plasmid was obtained by ligating 
 the p4cmve - u6-pylt fragment from pmbpylt between 
 the restriction sites nhei and mfei sites of pmbbhckrs
. the plasmid , pbad - sfgfp - y151tag - pylt 
 was cotransformed 
 with pbk - bhckrs into e. coli top10 cells .
a single colony was grown in lb media overnight , and 
 500 l of the overnight culture was added to 25 ml of lb media , 
 supplemented with 1 mm of the designated unnatural amino acid and 
 25 g / ml of tetracycline and 50 g / ml of kanamycin .
cells 
 were grown at 37 c , 250 rpm , and protein expression was induced 
 with 0.1% arabinose when the od600 reached 0.6 . 

after overnight expression at 37 c , cells were harvested and 
 washed by pbs .
the cell pellets were resuspended in 6 ml of phosphate 
 lysis buffer ( 50 mm , ph 8.0 ) and triton x-100 ( 60 l , 10% ) , 
 gently mixed , and incubated for 1 h at 4 c .
the cell mixtures 
 were sonicated , and the cell lysates were centrifuged at 4 c , 
 13  000 g , for 10 min .
the supernatant was transferred to a 
 15 ml conical tube , and 100 l of ni - nta resin ( qiagen ) was 
 added .
the resin was then collected by centrifugation ( 1000 g , 10 min ) , washed twice with 400 l of lysis buffer , and followed 
 by two washes with 400 l of wash buffer containing 20 mm imidazole . 

the protein was eluted with 400 l of elution buffer containing 
 250 mm imidazole .
the purified proteins were analyzed by 10% sds - page 
 and stained with coomassie blue .
two different instruments 
 were used : ( a ) protein samples were analyzed using capillary lc esi - tof 
 ms .
the protein samples were loaded onto a prlp - s column ( thermo fisher 
 5 m , 1000 a , 300 m i.d .
 100 mm ) on an lc system 
 ( ultimate 3000 , dionex , sunnyvale , ca ) .
the lc system was directly 
 coupled to an electrospray ionization time - of - flight mass spectrometer 
 ( microtof , brukerdaltonics , billerica , ma ) .
chromatographic separation 
 was performed at a constant flow rate of 3.5 l / min using a 
 binary solvent system ( solvent a : 2.5% acetonitrile and 0.1% formic 
 acid ; solvent b : 80% acetonitrile and 0.1% formic acid ) and a linear 
 gradient program ( 05 min , 5% b ; 510 min , 530% 
 b ; 1030 min , 3075% b ; 3035 min , 75100% 
 b ; 3545 min , 1005% b ; 4560 min , 5% b ) .
mass 
 spectra were acquired in positive ion mode over the mass range m / z 50 to 3000 .
esi spectra were deconvoluted 
 with the maxent algorithm ( data analysis 3.3 , bruker daltonics , billerica , 
 ma ) , obtaining molecular ion masses with a mass accuracy of 12 
 da .
( b ) high - resolution exact mass measurement were conducted on an 
 agilent technologies ( santa clara , ca ) 6210 lc - tof mass spectrometer . 

samples were analyzed via a 1 l flow injection at 300 l / min 
 in a water : methanol mixture ( 25:75 v / v ) with 0.1% formic acid .
the 
 mass spectrometer was operated in positive ion mode with a capillary 
 voltage of 4 kv , nebulizer pressure of 35 psi , and a drying gas flow 
 rate of 12 l / min at 350 c .
reference ions of purine at m / z 121.0509 and hp-0921 at m / z 922.0098 were simultaneously introduced via a 
 second orthogonal sprayer and used for internal calibration .
human 
 embryonic kidney ( hek ) 293 t cells were grown in dmem ( dulbecco s 
 modified eagle medium , gibco ) supplemented with 10% fbs ( gibco ) , 1% 
 pen - strep ( corning cellgro ) , and 2 mm l - glutamine ( alfa aesar ) 
 in 96-well plates ( costar ) in a humidified atmosphere with 5% co2 at 37 c .
hek 293 t cells were transiently transfected 
 with the pmbbhckrs - mcherry - tag - egfp - ha and p4cmve - u6-pylt at 75% confluency in the presence or 
 absence of 1 , 2 , and 3 ( 0.25 
 mm ) in 96-well plates .
after an overnight incubation at 37 c , 
 the cells were washed by pbs and imaged with a zeiss axio observer.z1microscope 
 ( 10 objective ) . to confirm the expression of the fusion protein 
 and also differentiate between expression levels ,
hek 293 t cells were cotransfected with pmbbhckrs - mcherry - tag - egfp - ha and p4cmve - u6-pylt in the presence or 
 absence of 1 , 2 , and 3 ( 0.25 
 mm ) in six - well plates .
after 24 h of incubation , the cells were washed 
 by chilled pbs , lysed in mammalian protein extraction buffer ( ge healthcare ) 
 with complete protease inhibitor cocktail ( sigma ) on ice , and the 
 cell lysates were cleared at 13  200 rpm centrifugation ( 4 c , 
 20 min ) .
the protein lysate was boiled with loading buffer and then 
 analyzed by 10% sds - page . after gel electrophoresis and transfer to 
 a pvdf membrane ( ge healthcare )
, the membrane was blocked in tbs with 
 0.1% tween 20 ( fisher scientific ) and 5% milk for 1 h. the blots were 
 probed and incubated with the primary antibody , -ha - probe ( y-11 ) 
 rabbit polyclonal lgg ( sc-805 , santa cruz biotech ) , overnight at 4 
 c , followed by a fluorescent secondary antibody , goat--rabbit 
 lgg cy3 ( ge healthcare ) , for 1 h at room temperature .
hek 293 t cells were 
 transfected with pbhckrs - mcherry - tag - egfp - ha and p4cmve - u6-pylt in 
 a 10 cm petri dish and incubated with dmem containing 1 , 2 , or 3 ( 0.25 mm ) for 24 h. cells were 
 lysed with extraction buffer ( ge healthcare ) and the mcherry-1/2/3-egfp - ha protein was immunoprecipitated 
 using the pierce ha tag ip / co - ip kit ( pierce ) according to manufacturer s 
 protocol .
the proteins were separated on sds - page gels and stained 
 with silver stain .
regions corresponding to the expected molecular 
 weight of mcherry - egfp - ha were excised , washed with hplc water , and 
 destained with 50% acetonitrile/25 mm ammonium bicarbonate until no 
 visible staining .
gel pieces were dehydrated with 100% acetonitrile 
 and reduced with 10 mm dithiothreitol at 56 c for 1 h , followed 
 by alkylation with 55 mm iodoacetamide at room temperature for 45 
 min in the dark .
gel pieces were then again dehydrated with 100% acetonitrile 
 to remove excess alkylating and reducing agents and rehydrated with 
 20 ng/l trypsin/25 mm ammonium bicarbonate and digested overnight 
 at 37 c .
the resultant tryptic peptides were extracted with 
 70% acetonitrile/5% formic acid , speed - vac dried , and reconstituted 
 in 18 l of 0.1% formic acid .
tryptic digests were analyzed 
 by reverse - phased lc - ms / ms using a nanoflow lc ( waters nanoacquity 
 uplc system , waters corp . ,
milford , ma ) coupled online to an ltq / orbitrap 
 velos hybrid mass spectrometer ( thermo - fisher , san jose , ca ) .
separations 
 were performed using a c18 column ( picochip column packed with 10.5 
 cm reprosil c18 3 m 120  chromatography media with a 
 75 m i d column and a 15 m tip , new objective , inc . , 

mobile phase a was 0.1% formic acid in water , and mobile 
 phase b was 0.1% formic acid in acetonitrile .
samples were injected 
 onto a trap column ( nanoacquity uplc trap column , waters corp . ,
milford , 
 ma ) and washed with 1% mobile phase b at a flow rate of 5 l / min 
 for 3 min .
peptides were eluted from the column using a 90 min gradient 
 running at 300 nl / min ( 5% b for 3 min , 536% b in 62 min , 3695% 
 b in 2 min , 95% b for 8 min , 95%5% b in 1 min , 5% b for 16 
 min ) .
the ltq / orbitrap instrument was operated in a data - dependent 
 ms / ms mode in which each high resolution broad - band full ms spectra 
 ( r = 60  000 at mass to charge ( m / z ) 400 , precursor ion selection range of m / z 300 to 2000 ) was followed by 13 ms / ms 
 scans in the linear ion trap where the 13 most abundant peptide molecular 
 ions dynamically determined from the ms scan were selected for tandem 
 ms using a relative collision - induced dissociation ( cid ) energy of 
 35% .
dynamic exclusion was enabled to minimize redundant selection 
 of peptides previously selected for cid .
ms / ms spectra were searched 
 with the mascot search engine ( version 2.4.0 , matrix science ltd . ) 
 against a uniprot jellyfish proteome database ( june 2014 release ) 
 from the european bioinformatics institute ( http://www.ebi.ac.uk/integr8 ) combined with endogenous mcherry - egfp fasta sequences .
the following 
 modifications were used : static modification of cysteine ( carboxyamidomethylation , 
 + 57.0214 da ) and variable modification of methionine ( oxidation , + 15.9949 
 da ) for all searches , variable modifications of lysine for mcherry - egfp - ha 
 ( 1 , + 218.17 da ; 2 , + 295.93 da ; 3 , + 231.03 da ) .
the mass tolerance was set at 20 ppm for the precursor 
 ions and 0.8 da for the fragment ions .
peptide identifications were 
 filtered using peptideprophet and proteinprophet algorithms with a 
 protein threshold cutoff of 99% and peptide threshold cutoff of 95% 
 implemented in scaffold ( proteome software , portland , or ) . hek 293 t 
 cells were cultured in dmem ( dulbecco s modified 
 eagle medium , gibco ) supplemented with 10% fbs ( gibco ) , 1% pen - strep 
 ( gibco ) , and 2 mm l - glutamine ( alfa aesar ) in 96-well plates 
 ( bd falcon ) in a humidified atmosphere with 5% co2 at 37 
 c . at 8090% confluency
, cells seeded on plates were 
 transfected and the medium was changed to fresh dmem supplemented 
 without or with 1 , 2 , or 3 ( 0.25 
 mm ) .
the plasmid pmbbhckrs-4pylt was constructed 
 containing both cmv - mbbhckrs and 4cmve - u6-pylt .
a 
 tag amber stop codon was introduced at the k206 site using primers 
 gl1/gl2 and a quikchange mutagenesis kit ( agilent technologies ) . a 
 pgl3-control plasmid containing the gene encoding p. pyralis firefly luciferase with the tag amber mutation at residue k206 ( pgl3-k206tag ) 
 was cotransfected into cells with the plasmid pbhckrs-4pylt using 
 linear pei according to the manufacturer s protocol ( millipore ) . 

after double transfection and 24 h incubation , the medium was changed 
 to dmem without phenol red , and the cells were irradiated with uv 
 light ( 365 nm ) for 4 min using a 365 nm uv lamp ( high performance 
 uv transilluminator , uvp , 25 w ) or kept in the dark .
cells were lysed 
 by addition of 100 l of substrate solution ( promega ) in a 96-well 
 plate ( bd falcon ) , and luminescence was measured on a synergy 4 multimode 
 microplate reader with an integration time of 2 s and a sensitivity 
 of 150 or on a tecan m1000 microplate reader with an integration time 
 of 1 s. cho k1 cells were plated into a polylysine - coated 
 four - well chamber slide ( lab - tek ) and , after incubation to 75% confluency , 
 were transfected with 1 g of pnls - ktag - egfp and pbhckrs-4pylt 
 each .
after 16 h incubation at 37 c/5% co2 in dmem 
 with 10% fbs in the presence of 1 ( 0.25 mm ) , cells were 
 washed with dmem without phenol red and then incubated for 2 h. the 
 cells were washed with pbs , fixed with 4% formaldehyde , and stained 
 with rhodamine  phalloidin ( life technologies ) .
the chamber 
 slide was dried in the dark overnight and cells were imaged on a zeiss 
 710 confocal microscope ( 40 water objective ) . hek 293 t cells 

were plated into a poly - d - lysine - coated eight - well chamber 
 slide ( lab - tek ) .
after incubation to 70% confluency , cells were transfected 
 with pegfp - k85tag - mcherry and pbhckrs-4pylt ( 200 ng each ) . after a 
 20 h incubation at 37 c/5% co2 in dmem with 10% fbs 
 in the presence of 1 ( 0.25 mm ) ,
cells were washed with 
 dmem without phenol red and then incubated for 1 h. before light activation , 
 mcherry - expressing cells were identified using the txred channel , 
 and imaged with a nikon a1rsi confocal microscope ( 20 objective , 
 2-fold zoom , egfp ( ex .
subsequently , cells were illuminated for 15 s at 365 nm light ( dapi 
 filter , 358365 nm ) , and egfp and mcherry fluorescence was 
 acquired by time - lapse imaging ( every 1 min for the first 15 min , 
 every 5 min for the following 150 min , scan resolution 512  
 512 , scan zoom 2 , dwell time 1.9 ms ) .
hek 293 t cells 
 were plated into a polylysine - coated -dish ( ibidi ) , and after 
 incubation to 50% confluency , the cells were transfected with 1 g 
 each of pegfp - ktag - mcherry and pbhckrs-4pylt .
after a 20 h incubation 
 at 37 c/5% co2 in dmem with 10% fbs in the presence 
 of 1 or 2 ( 0.25 mm , 0.5% dmso ) , cells were 
 washed with dmem without phenol red and then incubated for 1 h. cells 
 were imaged with an olympus fluoview confocal microscope before two - photon 
 irradiation ( 40 oil objective , egfp ( ex .
560 nm ) channels ) , imaging positions for mcherry - expressing cells 
 were recorded , and the cell -dish was transferred to an olympus 
 multiphoton microscope for irradiation ( olympus fluoview fv1000 mpe ) . 

cells were localized at the previously recorded positions , focused 
 using the mcherry channel , and then irradiated using a 760 nm laser 
 ( 130 mw , 5% of laser power , 30 cycles of scanning , 2 m / s dwell 
 time , maitai dsbb - ol ir pulsed laser ) .
after irradiation , the cell 
 -dish was transferred back to the original microscope for imaging .
the initial template structure of pdb 2q7h was chosen as a starting point for all 
 modeling .
the missing loops were remodeled using modeler and the two point mutations ( y271a and l274 m ) were constructed using 
 the mutate_model.py script provided by modeler .
superposition of pdb 2q7 g on top of 2q7h provided the coordinates for the incorporation of 
 atp and magnesium ions into the newly mutated structure .
the atp and 
 magnesium ions were parametrized in antechamber using 
 previously developed parameters .
the mutated 
 structure was imported into amber12 software using 
 the amber ff99sbildn force field .
the 
 protein was placed into a cubic box with a 12.0- border , solvated 
 with 17  316 water molecules , and charge neutralized with the 
 addition of six sodium ions .
this system was energy minimized first 
 with 5000 steps steepest descent method , followed by 15  000 
 steps conjugate gradient method with 5 kcal / mol restraints on all 
 atoms .
this was followed by another 5000 steps steepest descent method , 
 followed by 15  000 steps conjugate gradient method with 2 kcal / mol 
 restraints on all atoms except y271a and l274 m .
the resulting energy - minimized 
 structure was used as the starting structure for all our docking experiments . 

all amber12 computational experiments were completed 
 on the center for simulation and modeling ( sam ) frank supercomputer 
 at the university of pittsburgh .
the energy - minimized 
 mutant structure was prepared for docking with autodock4 by removing all sodium ions , and all water molecules except for 
 a single water molecule which exists in the active site pocket of 
 the protein .
this structural water molecule is present in all available 
 crystal structures and plays an important role in amino acid recognition . 

the side chains for residue 
 l274 m were treated as flexible , while all other side chains were kept 
 rigid .
the unnatural amino acid ligands were constructed using chembiodraw3d , 
 and the molecular geometry was optimized using the mmff94 force field .
the ligand input files 
 were prepared for docking using autodock tools as 
 well .
lamarkian genetic algorithm was used for docking with the following 
 parameters : number of runs : 75 , ga_pop_size 150 , ga_num_evals 250  000  000 , 
 ga_num_generations 27  000 were set , all other parameters were 
 kept default .
each coumarin lysine yielded a low energy cluster with binding 
 scores of 9.63 kj / mol , 6.18 kj / mol , and 6.14 
 kj / mol for 1 , 2 , and 3 , respectively .
 the site - specific 
 incorporation of three new coumarin lysine analogues 
 into proteins was achieved in bacterial and mammalian cells using 
 an engineered pyrrolysyl - trna synthetase system . 
 the genetically encoded 
 coumarin lysines were successfully applied as fluorescent cellular 
 probes for protein localization and for the optical activation of 
 protein function . as a proof - of - principle , 
 photoregulation of firefly 
 luciferase was achieved in live cells by caging a key lysine residue , 
 and excellent off to on light - switching ratios were observed . 
 furthermore , 
 two - photon and single - photon optochemical control of egfp maturation 
 was demonstrated , enabling the use of different , potentially orthogonal 
 excitation wavelengths ( 365 , 405 , and 760 nm ) for the sequential activation 
 of protein function in live cells . 
 these results demonstrate that 
 coumarin lysines are a new and valuable class of optical probes that 
 can be used for the investigation and regulation of protein structure , 
 dynamics , function , and localization in live cells . 
 the small size 
 of coumarin , the site - specific incorporation , the application as both 
 a light - activated caging group and as a fluorescent probe , and the 
 broad range of excitation wavelengths are advantageous over other 
 genetically encoded photocontrol systems and provide a precise and 
 multifunctional tool for cellular biology . 
Introduction
Results and Discussion
Summary
Experimental Section