the remote temporal and spatial control of protein functions within living cells is a major goal long wished for in life sciences . it is only in the last couple of years that significant progress has been made in this exciting field of research by developing different methods to allow for the lightdependent control of protein and cellular functions . light as a trigger for cellular functions is of outstanding interest as it provides a noninvasive method for ( photo)stimulation which thus limits cellular damage to a minimum . furthermore , lightinduced manipulations allow for high temporal and spatial resolution . at an early stage , photolabile caged compounds , e.g. caged ions , and neurotransmitters such as ca and glutamate were used to noninvasively stimulate neuronal activity ( zemelman et  al . , 2003 ; shoham et  al . , generally , such systems rely on the irreversible photolysis of a photolabile caged complex that releases a signalling molecule after photostimulation . the photochromic azobenzene system ( sadovski et  al . , 2009 ) provides an attractive alternative to caged compounds because it can reversibly be switched between two states by using two different wavelengths of excitation allowing for a reversible and repeatable on / off switching of protein function achieved by coupling of a receptor ligand to an azobenzene derivative . illumination of the azobenzenecoupled ligand results in a cis / trans isomerization of the azobenze moiety thus placing the ligand in a position that allows binding to the respective receptor and hence its activation . to this end , different cellular receptors and channels such as the nicotinic acetylcholine receptor and several ionotrophic glutamate receptors could be lightdependently controlled ( lester et  al . , 1980 ; more direct control over protein functions can be exerted by attaching azobenzene derivatives to a protein backbone by crosslinking to a position that influences the respective protein function . in this way , photocontrol over skeletal muscle myosin ( umeki et  al . , 2004 ) and a kinesin atpase ( yamada et  al . , 2007 hence , reversible and irreversible photocaged ligands present powerful tools to achieve a remote control over various cellular functions ( goeldner and givens 2005 ; banghart et  al . , 2006 ; gorostiza and isacoff , 2008 ) . nevertheless , their use is still limited by several drawbacks : ( i ) freely diffusible photocaged ligands and crosslinked azobenzenes can never be completely selective in vivo for just a single protein target , and ( ii ) the photoreleased ligand will diffuse freely through the cell thus limiting spatial resolution . genetically encoded photochromic proteins represent a promising alternative to photocaged or coupled ligands . retinal is a wellknown naturally occurring photochromic switch which functions as lightsensitive chromophore of visual opsins ( rhodopsins ) found in the mammalian eye ( yoshizawa , 1984 ) . in bacteria , retinal photoswitches occur as lightdriven proton pumps ( bacteriorhodopsins ) ( lozier et  al . , 1975 ) . here , the retinal molecule ( much like the abovementioned azobenzene derivatives ) isomerizes between the cis and the trans isomer in a lightdependent manner . recently , lightgated retinal proteins such as the algal channelrhodopsin2 ( nagel et  al . , 2003 ) , engineered variants of the same ( lin et  al . , 2009 ) , as well as lightdriven chloride pumps of the halorhodopsin family ( schobert and lanyi , 1982 ) have found their way into the toolbox of neurobiologists ( zhang et  al . , 2007a ) . in particular , channelrhodopsins are nowadays widely employed in neurosciences to selectively and noninvasively control the firing of specific neurones ( boyden et  al . , 2005 ; , 2005 ; zhang et  al . , 2007b ; pulver et  al . , 2009 ) . other naturally occurring lightsensitive proteins can also be employed including the photoactivated adenylyl cyclases ( pacs ) of the unicellular flagellate euglena gracilis ( iseki et  al . , 2002 ) which represent potent alternative systems for the photocontrol of neuronal activity ( nagahama et  al . , 2007 ; schrderlang et  al . , 2007 photoactivated adenylyl cyclases are blue lightsensitive photoreceptors that bind a flavin adeninedinucleotide ( fad ) molecule as chromophore in two socalled bluf ( sensors of bluelight using fad ) domains ( gomelsky and klug , 2002 ) . these bluf domains are assumed to undergo a lightdependent conformational rearrangement that triggers the catalytic conversion of adenosine triphosphate to the secondary messenger molecule cyclic adenosinemonophosphate ( camp ) ( ntefidou et  al . , 2006 ) which in turn regulates various cellular functions ( yoshikawa et  al . , 2005 these naturally occurring lightgated systems attenuate the problem of spatial control over the cellular response , as they can be selectively expressed , e.g. in specific types of neurones . however , the problem remains that the function of a specific cellular protein can not be probed selectively in vivo . both channelrhodopsinmediated synaptic membrane depolarization and pactriggered control of the intracellular camp levels will affect cellular control mechanisms at different levels and initiate different physiological responses at the same time . thus , a generally applicable strategy to engineer optical control into any type of target protein in any type of organism including bacteria , plants and animals still represents a tempting goal . light , oxygen , voltage ( lov ) bluelight photoreceptor proteins ( christie et  al . , 1999 ) exert a lighttriggered control mechanism present in the three kingdoms of life . lov domains belong to the per , arndt , sim ( pas ) family of sensor domains ( taylor and zhulin , 1999 ) which is one of the most widely spread sensor module in nature found in all three kingdoms of life . the family includes apart from bluelight photoreceptors , other ligandbinding sensors suitable to detect changes in oxygen levels and redox potential ( taylor and zhulin , 1999 ) . lov photoreceptors , like pas sensor domains themselves , are found in the archaea , the bacteria , in lower and higher plants as well as in fungi ( u. krauss , b.q . minh , a. losi , w. grtner , t. eggert , a. von haeseler and k.e . recently , even the possibility of lov signalling modules to occur in animals including humans was highlighted ( van der horst et  al . , 2007 ) . in nature , lov photoreceptors are fused mostly nterminally to a wide array of different effector domains ( crosson et  al . , 2003 ; losi , 2006 ) . in plants , fusion partners include ser / thr kinases where a lightdependent autophosphorylation is initiated in response to bluelight illumination ( huala et  al . , 1997 ) . in the bacterial kingdom the lov sensor module is found in two to multicomponent systems together with other pas domains , hiskinases , antisigma factors , helix  turn  helix dnabinding domains , phosphatases , phosphodiesterases and guanylate cyclases ( losi , 2004 ; losi and grtner , 2008 ) . this broad spectrum of coupled effector domains which exert an array of different in vivo functionalities already suggests that sensor signal transduction mechanisms may be successfully transferred to many structurally and functionally different protein effector domains . the functional engineering of a lightactivated protein requires ( i ) an understanding of the primary photochemical events that lead to effector domain activation , ( ii ) knowledge of the signal propagation mechanism from the site of photon capture through the sensor core to the effector domain , and ( iii ) an effector domain whose activity can be controlled by a structural perturbation transmitted from the coupled sensor domain . for lov domains , issues ( i ) and ( ii ) are well resolved . thus , the question arises whether lov can be employed as a universal light switch to control protein functions . the fundamental principle underlying this approach the chimeric protein must consist of a photoreceptor ( or sensor ) domain ( in blue ) harbouring a chromophore which functions as the lightsensitive  switch. upon illumination with light of appropriate wavelength the chromophore absorbs light energy and undergoes electronic excitation which is accompanied by a conformational rearrangement in the sensor domain protein backbone around the chromophore . this change is further transmitted via relay effects ( e.g. via a linker polypeptide ) into an  effector domain ( in green ) possessing enzymatic activity . hence , the light signal propagates to the effectors ' active site ( as ) causing a structural pertubation which may in turn result in increased or decreased activity , altered substrate specificity or enantioselectivity . photon capture within the lightsensitive fmn chromophore bound noncovalently in the lov domain dark state ( lov447 ) results in the formation of the singlet exited state of fmn on a picosecond timescale . the singlet excited state in part relaxes back to the ground state by emitting photons in form of fluorescence and decays via intersystem crossing to the fmn triplet state ( lov660 ) . the triplet in turn gives rise to the socalled signalling state ( lov390 ) on a microsecond timescale . in the signalling state , a covalent bond is formed between a highly conserved cysteine residue in the protein core and the carbon ( c4 )  atom of the flavin isoalloxazine ring ( fig .  2 ) . from this longest living species in the lov photocycle the protein returns to the dark state ( lov447 ) within seconds , hours or days depending on the type of lov protein ( losi , 2007 ) . presumably , the signalling state must trigger a conformational change in the lov domain which then relays the light signal to fused effector domains . however , the molecular mechanism of this signal propagation is still somewhat controversial as crystal structures solved for the dark and light state of the protein did not display major differences ( crosson and moffat , 2001 ; 2002 ; fedorov et  al . , 2003 ; halavaty and moffat , 2007 ) . recently , solution nmr spectroscopy was used to analyse a plant lov2 sensor domain responsible for phototropin activation . hereby , gardner 's group suggested that the unfolding or dissociation of a helicalsegment located cterminally to the lov core and termed jhelix triggers phototropin autophosphorylation ( harper et  al . . different possible mechanisms of activation were suggested with the lov domain acting either as a darkstate inhibitor or as a lightstate activator ( harper et  al . , 2004 ) . for bacterial lov proteins , no lightdependent unfolding of helices , 2007 ; mglich and moffat , 2007 ) , although the respective bacterial lov proteins apparently possess cterminally fused helical polypeptides ( krauss et  al . , 2005 ; recently , structural and in vitro functional data suggested that the signal propagation in some bacterial lov proteins might occur via a rotational movement of the cterminal jhelices ( mglich and moffat , 2007 ; mglich et  al . , 2009 ) . thus , the mechanisms of effector domain activation by lovs currently appear to be different in both plant and bacterial systems . however , in both bacterial and plant lov systems , photon capture in the lov domain results in a structural perturbation that transmits conformational changes through the lov sensor core , either via unfolding or via rotation of the jhelical linker located at the cterminus of the lov domain . this structural perturbation is then relayed into the effector domain resulting in changes or alterations of cellular activities like phosphorylation , protein the figure shows the lov core structure of the avena sativa phototropin1 lov2 domain ( aslov2 , pdb entry : 2v1a ) in the dark state ( upper part ) with the lightsensitive flavin chromophore in stick representation . the inset shows a closeup view of the fmn binding pocket in dark state , with a noncovalently bound fmn molecule in close vicinity of the photoactive cystein ( c450 ) . the lower part of the figure visualizes the conformational change suggested to occur in the signalling state after bluelight illumination . a covalent bond is formed between the photoactive cysteine ( c450 ) ( see inset , lower part of the figure ) and the flavin isoalloxazine ring . this in turn triggers unfolding ( or dissociation ) of the cterminally located jhelix which results in the activation of plant phototropin ( harper et  al . please note that the mechanistic model is based on nmr data of aslov2 whereas no unfolding of the jhelix is observed in the lightstate crystal structure of aslov2 . do we understand this signal transduction mechanism well enough to engineer lightswitching abilities into any target effector protein using lov sensor domains as the light trigger ? the first such system described used a plant lov domain fused to the wellcharacterized escherichia coli tryptophane repressor protein ( trpr ) ( strickland et  al . , 2008 ) . an allosterically regulated  leverarm formed from shared helices on both the lov domain ( jhelix ) and the trpr side allowed for lightdependent control over dna binding to the trpr helix  turn  helix dna binding site ( strickland et  al . , 2008 ) . more recently , a similar design strategy was used to construct a lightcontrolled histidine kinase by joining the hiskinase domain of the pas sensor fixl of bradyrhizobium japonicum with the lov domain of the bacillus subtilis ytva photoreceptor protein ( mglich et  al . , 2009 ) . again , shared helical linkers were employed as signal propagating elements between the sensor core and the respective effector domain . fixl contains a haembinding pas sensor domain that structurally resembles the fmnbinding lov domain of ytva ( mglich and moffat , 2007 ) . for the lovtap construct , a helix  turn helix dnabinding domain was employed that is found in nature fused to pas sensors ( aravind et  al . , thus far , just a single example exists which demonstrates lightdependent control over a completely sensorunrelated protein . here , the extensively characterized enzyme , dihydrofolate reductase ( dhfr ) from e. coli , was used as target effector module to be engineered with a plant lov domain allowing for allosteric control over dhfr biocatalytic activity ( lee et  al . , 2008 ) . dhfr is an enzyme that catalyses the reduction of 5,6dihydrofolate ( dhf ) to 5,6,7,8tetrahydrofolate ( thf ) using nadph as a cofactor . the enzyme plays a central role to maintain the cellular levels of tetrahydrofolate and its derivatives which are essential for purine and thymidylate biosynthesis . therefore , this enzyme has been an important target for the development of antibacterial agents , anticancer drugs and other therapeutics ( volpato and pelletier , 2009 ) . extensive kinetic studies of e. coli dhfr and various mutants along with xray crystallography , nmr spectroscopy and computational studies have revealed the correlation between protein dynamics and catalytic function ( sawaya and kraut , 1997 ; schnell et  al . , 2004 ; hammesschiffer and benkovic , 2006 ) and figure 3a depicts the structure of dhfr showing the ligand binding site as well as the location of important loop regions that are involved in enzyme catalysis . a. structures of aslov2 ( pdb entry : 2v0u ) and dhfr ( pdb entry : 1rx2 ) , illustrating surfaceexposed sca sites ( red ) , the chromophore fmn ( orange ) , nadph ( magenta ) and folate ( green ) . the sca method identifies networks of statistically correlated amino acid residues in the lov domain as well as in dhfr . the network links the fmn binding pocket of the lov domain to n and cterminal helices ( shown in red ) that undergo significant conformational changes upon light activation ( see fig .  3 ) . the design strategy was to insert the lightsensing domain into one of the surface positions ( fg loop , in red ) in the dhfr network so that the light signal can be transmitted to the dhfr active site though the network .  b. schematic representation of the lov2dhfr chimeric constructs . to functionally couple the lightinduced signal with the dhfr catalysis , site a ( amino acids 120122 ) is an allosteric site within the coupled network of dhfr , while site b ( amino acids 8689 ) is a control site that is not correlated with the active site .  c. lightdependent enzyme activity in the lov2dhfr chimera . hydride transfer rates ( khyd ) were measured by a stoppedflow kinetic method under dark ( shown as hatched bars ) and lightexposed conditions ( blue bars ) . among the chimeras , light dependence was observed only when the lov domain is attached to a specific position ( a120 ) . the construct with a point mutation ( c450s ) that locks the lov domain into the dark state or a chimera lacking the jhelix did not show the light dependence . the kinetics of the dark recovery of the hydride transfer rate exhibits a single exponential decay ( right part of c ) , which is comparable to the kinetics of the fmnthiol covalent adduct decay . an evolutionary approach using statistical coupling analysis ( sca ) also has identified networks of coevolving amino acids in various proteins ( hatley et  al . , 2003 ; statistical coupling analysis for a multiple sequence alignment of 1104 pas domains ( hefti et  al . , 2004 ) ( the general folding class lov domains belong to ) revealed a spatially contiguous network of amino acids that linked the chromophore binding site to surfaceexposed residues at the n and cterminal regions of the domain . statistical coupling analysis of 418 members of the dhfr family also probed for network residues within dhfr and identified the fg loop , one of the functional loops in the dhfrcoupled network , as a potential surface allosteric site ( lee et  al . the strategy for engineering a new allosteric protein then was to connect the networks of these two protein modules through a covalent linkage joining their surface sites such that light can modulate the dhfr activity through the response of a lightsensing lov domain from plant phototropin ( avena sativa lov2 ) ( salomon et  al . the computational sca method enabled the prediction of allosteric surface sites where the perturbation of a regulatory element ( input domain ) will control protein function at the remote site ( output domain ) . several sets of chimeric proteins were generated in which the lov2 domain is inserted via its n and cterminal helical extensions into dhfr at two different surface sites ( sites a and b , fig . site a chimeras have the lov domain insertion at an allosteric site within the coupled network ( a120a122 , fg loop ) , and site b chimeras served as a control with another surface site ( b86b89 , ce loop ) that is similarly distant but statistically uncorrelated with the active site . the engineered lov2dhfr chimera exhibited lightdependent enzyme activity based on the functional communication between the lightsensing protein module and dhfr . dark recovery kinetics based on a 447  nm absorbance and enzyme activity showed that the intrinsic features of each domain are structurally and functionally intact in the lov2dhfr chimera . the light switch worked only when the lightsensing domain is attached to a specific position within the network of dhfr . in the presence of light , the hydride transfer rate measured by a stoppedflow kinetic method exhibited a twofold increase of enzyme activity compared with the dark condition . the photocycle between light and dark state generated a reversible change in the enzyme activity and the single exponential measure of the enzyme activity ( khyd ) from the lightexcited chimera to the dark species matched the rate of thermal relaxation of the fmn . in the case of lov2dhfr , the kinetic results showed that the signal initiated by light absorption by the fmn chromophore within the hydrophobic binding pocket of the lov domain is propagated through the structure to cause conformational changes at the opposite site of the sensor domain , particularly by the destabilization of the cterminal jhelix , that in the natural phototropin system is implicated in the signalprogation mechanism ( harper et  al . , 2003 ) . in turn these conformational changes act to affect the dhfr conformation , leading to an enzyme activity altered by the light and dark state of the lov domain . the construct lacking the jhelix or a variant with a c450s mutation in the lov2 domain did not show this light dependence . hereby , the mutation of c450 abolishes the lov photocycle and thus traps the sensor in a dark state conformation ( salomon et  al . , 2000 ) . in addition , the switch did not operate when the lov domain was connected to the other functional loop ( ce loop ) that is not in the network , clearly suggesting the presence of unique network(s ) in proteins through which the signal is transmitted . obviously , these results demonstrate that the lightdependent activity of the lov2dhfr results from the allosteric communication between lightsensing domain and dhfr . this novel approach of connecting sca implicated sectors from two different proteins may furnish a general tool for the creation of a new allosteric proteins from any sensorunrelated protein . do we understand this signal transduction mechanism well enough to engineer lightswitching abilities into any target effector protein using lov sensor domains as the light trigger ? the first such system described used a plant lov domain fused to the wellcharacterized escherichia coli tryptophane repressor protein ( trpr ) ( strickland et  al . , 2008 ) . an allosterically regulated  leverarm formed from shared helices on both the lov domain ( jhelix ) and the trpr side allowed for lightdependent control over dna binding to the trpr helix  turn  helix dna binding site ( strickland et  al . , 2008 ) . more recently , a similar design strategy was used to construct a lightcontrolled histidine kinase by joining the hiskinase domain of the pas sensor fixl of bradyrhizobium japonicum with the lov domain of the bacillus subtilis ytva photoreceptor protein ( mglich et  al . , 2009 ) . again , shared helical linkers were employed as signal propagating elements between the sensor core and the respective effector domain . fixl contains a haembinding pas sensor domain that structurally resembles the fmnbinding lov domain of ytva ( mglich and moffat , 2007 ) . for the lovtap construct , a helix  turn helix dnabinding domain was employed that is found in nature fused to pas sensors ( aravind et  al . , thus far , just a single example exists which demonstrates lightdependent control over a completely sensorunrelated protein . here , the extensively characterized enzyme , dihydrofolate reductase ( dhfr ) from e. coli , was used as target effector module to be engineered with a plant lov domain allowing for allosteric control over dhfr biocatalytic activity ( lee et  al . , 2008 ) . dhfr is an enzyme that catalyses the reduction of 5,6dihydrofolate ( dhf ) to 5,6,7,8tetrahydrofolate ( thf ) using nadph as a cofactor . the enzyme plays a central role to maintain the cellular levels of tetrahydrofolate and its derivatives which are essential for purine and thymidylate biosynthesis . therefore , this enzyme has been an important target for the development of antibacterial agents , anticancer drugs and other therapeutics ( volpato and pelletier , 2009 ) . extensive kinetic studies of e. coli dhfr and various mutants along with xray crystallography , nmr spectroscopy and computational studies have revealed the correlation between protein dynamics and catalytic function ( sawaya and kraut , 1997 ; schnell et  al . , 2004 ; hammesschiffer and benkovic , 2006 ) and figure 3a depicts the structure of dhfr showing the ligand binding site as well as the location of important loop regions that are involved in enzyme catalysis . a. structures of aslov2 ( pdb entry : 2v0u ) and dhfr ( pdb entry : 1rx2 ) , illustrating surfaceexposed sca sites ( red ) , the chromophore fmn ( orange ) , nadph ( magenta ) and folate ( green ) . the sca method identifies networks of statistically correlated amino acid residues in the lov domain as well as in dhfr . the network links the fmn binding pocket of the lov domain to n and cterminal helices ( shown in red ) that undergo significant conformational changes upon light activation ( see fig .  3 ) . the design strategy was to insert the lightsensing domain into one of the surface positions ( fg loop , in red ) in the dhfr network so that the light signal can be transmitted to the dhfr active site though the network .  b. schematic representation of the lov2dhfr chimeric constructs . to functionally couple the lightinduced signal with the dhfr catalysis , site a ( amino acids 120122 ) is an allosteric site within the coupled network of dhfr , while site b ( amino acids 8689 ) is a control site that is not correlated with the active site .  c. lightdependent enzyme activity in the lov2dhfr chimera . hydride transfer rates ( khyd ) were measured by a stoppedflow kinetic method under dark ( shown as hatched bars ) and lightexposed conditions ( blue bars ) . among the chimeras , light dependence was observed only when the lov domain is attached to a specific position ( a120 ) . the construct with a point mutation ( c450s ) that locks the lov domain into the dark state or a chimera lacking the jhelix did not show the light dependence . the kinetics of the dark recovery of the hydride transfer rate exhibits a single exponential decay ( right part of c ) , which is comparable to the kinetics of the fmnthiol covalent adduct decay . an evolutionary approach using statistical coupling analysis ( sca ) also has identified networks of coevolving amino acids in various proteins ( hatley et  al . , 2003 ; peterson et  al . , 2004 ) . statistical coupling analysis for a multiple sequence alignment of 1104 pas domains ( hefti et  al . , 2004 ) ( the general folding class lov domains belong to ) revealed a spatially contiguous network of amino acids that linked the chromophore binding site to surfaceexposed residues at the n and cterminal regions of the domain . statistical coupling analysis of 418 members of the dhfr family also probed for network residues within dhfr and identified the fg loop , one of the functional loops in the dhfrcoupled network , as a potential surface allosteric site ( lee et  al . , 2008 ) . the strategy for engineering a new allosteric protein then was to connect the networks of these two protein modules through a covalent linkage joining their surface sites such that light can modulate the dhfr activity through the response of a lightsensing lov domain from plant phototropin ( avena sativa lov2 ) ( salomon et  al . the computational sca method enabled the prediction of allosteric surface sites where the perturbation of a regulatory element ( input domain ) will control protein function at the remote site ( output domain ) . several sets of chimeric proteins were generated in which the lov2 domain is inserted via its n and cterminal helical extensions into dhfr at two different surface sites ( sites a and b , fig . site a chimeras have the lov domain insertion at an allosteric site within the coupled network ( a120a122 , fg loop ) , and site b chimeras served as a control with another surface site ( b86b89 , ce loop ) that is similarly distant but statistically uncorrelated with the active site . the engineered lov2dhfr chimera exhibited lightdependent enzyme activity based on the functional communication between the lightsensing protein module and dhfr . dark recovery kinetics based on a 447  nm absorbance and enzyme activity showed that the intrinsic features of each domain are structurally and functionally intact in the lov2dhfr chimera . the light switch worked only when the lightsensing domain is attached to a specific position within the network of dhfr . in the presence of light , the hydride transfer rate measured by a stoppedflow kinetic method exhibited a twofold increase of enzyme activity compared with the dark condition . the photocycle between light and dark state generated a reversible change in the enzyme activity and the single exponential measure of the enzyme activity ( khyd ) from the lightexcited chimera to the dark species matched the rate of thermal relaxation of the fmn . in the case of lov2dhfr , the kinetic results showed that the signal initiated by light absorption by the fmn chromophore within the hydrophobic binding pocket of the lov domain is propagated through the structure to cause conformational changes at the opposite site of the sensor domain , particularly by the destabilization of the cterminal jhelix , that in the natural phototropin system is implicated in the signalprogation mechanism ( harper et  al . , 2003 ) . in turn these conformational changes act to affect the dhfr conformation , leading to an enzyme activity altered by the light and dark state of the lov domain . the construct lacking the jhelix or a variant with a c450s mutation in the lov2 domain did not show this light dependence . hereby , the mutation of c450 abolishes the lov photocycle and thus traps the sensor in a dark state conformation ( salomon et  al . , 2000 ) . in addition , the switch did not operate when the lov domain was connected to the other functional loop ( ce loop ) that is not in the network , clearly suggesting the presence of unique network(s ) in proteins through which the signal is transmitted . obviously , these results demonstrate that the lightdependent activity of the lov2dhfr results from the allosteric communication between lightsensing domain and dhfr . this novel approach of connecting sca implicated sectors from two different proteins may furnish a general tool for the creation of a new allosteric proteins from any sensorunrelated protein . one of the remaining open questions is how to modulate different levels of control over protein function by using a light signal . the detected effect in the lov2dhfr construct was only twofold ; theoretically it can be as high as hundreds fold ( yao et  al . , 2008 ) . this chimera also showed a fast transition from the lightinduced state to the dark state , which may not maximize the active catalytic population within the experimental time limit . since bacterial lov proteins are many times slower in their dark recovery kinetics compared with the plant photlov domains ( losi et  al . , 2002 ; cao et  al . , 2007 ) , the engineering of new bacterial lov domains with wide ranges of recovery kinetics as well as optimizing the linker composition should guide us to more optimal lightactivated allosteric protein switches . to this end we are currently investigating several bacterial lov domains as sensor switches for the control of dhfr . furthermore , we have constructed fusions of bacterial as well as plant lov domains with a nonallosteric regulated protein module , namely a lipase from b. subtilis ( eggert et  al . , 2001 ) . lipase constructs indeed retained both functionalities , namely bluelight activation of the lov domain and enzymatic activity ( fig . furthermore , we expect that subtle structural perturbations in the lipase , i.e. propagated from the sensor domain , may result also in an alteration of enzyme properties such as substrate specificity and ( enantio)selectivity . a and b. ( a ) illustration of the fusion strategy : a bacterial lov domain ( here of the b. subtilis ytva protein ) was fused nterminally to the lipase a of b. subtilis ( bsla ) resulting in the construct nlovbsla . the two protein parts are linked via a helical linker polypeptide naturally present in b. subtilis ytva . the fusion construct can be readily expressed in soluble form and retains both chromophore binding capacity in the lov domain as evidenced by flavin fluorescence ( left part of b ) and lipolytic activity of the lipase ( right part of b ) . here shown is the result of a standard lipase activity assay using pnitrophenylpalmitate ( pnpp ) as substrate ( + ) . the lipase cleaves the esterbond of pnpp resulting in the formation of the yellowcoloured pnitrophenylate anion indicative for enzymatic activity . c. observed light sensitivity of nlovbsla : flavinfluorescence was recorded in the proteins dark state ( black line ) as well as 30  s after bluelight illumination ( blue line ) . in the dark , the protein displays a bimodal fluorescence with maxima occurring at around 495 and 520  nm . upon formation of the signalling state , this fluorescence the inset shows the darkrecovery time trace of the fmn fluorescence peak recorded at 495  nm . the key question is whether a general strategy can be devised that allows one to choose any target enzyme , link it to a light activating domain at predetermined amino acid residues and thus create a lightcontrolled enzyme . one would hope to minimize the trial and error aspects of a combinatorial approach and instead rely on sca or other computational methods to predict the residues to be used for successful attachment . in the future , one can imagine light control encompassing not only biological regulation processes but also biocatalytic reactions and even complex reaction systems .
summarylight control over enzyme function represents a novel and exciting field of biocatalysis research . 
 bluelight photoreceptors of the light , oxygen , voltage ( lov ) family have recently been investigated for their applicability as photoactive switches . 
 we discuss here the primary photochemical events leading to light activation of lov domains as well as the proposed signal propagation mechanism to the respective effector domain . 
 furthermore , we describe the construction of lov fusions to different effector domains , namely a dihydrofolate reductase from escherichia coli and a lipase from bacillus subtilis . both fusion partners retained functionality , and alteration of enzyme activity by light 
 was also demonstrated . 
 hence , it appears that fusion of lov photoreceptors to functional enzyme target sites via appropriate linker structures may represent a straightforward strategy to design light controllable biocatalysts .