Ultrafast real-time visualization of active site flexibilityof flavoenzyme thymidylate synthase ThyXSergey P. Laptenoka,b, Latifa Bouzhir-Simaa,b, Jean-Christophe Lambrya,b, Hannu Myllykallioa,b, Ursula Liebla,b,and Marten H. Vosa,b,1

aLaboratory for Optics and Biosciences, Centre National de la Recherche Scientifique Ecole Polytechnique, 91128 Palaiseau, France; and bInstitut Nationalde la Santé et de la Recherche Médicale U696, 91128 Palaiseau, France

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved April 18, 2013 (received for review November 9, 2012)

In many bacteria the flavoenzyme thymidylate synthase ThyXproduces the DNA nucleotide deoxythymidine monophosphatefrom dUMP, using methylenetetrahydrofolate as carbon donorand NADPH as hydride donor. Because all three substrates bind inclose proximity to the catalytic flavin adenine dinucleotide group,substantial flexibility of the ThyX active site has been hypothe-sized. Using femtosecond time-resolved fluorescence spectros-copy, we have studied the conformational heterogeneity and theconformational interconversion dynamics in real time in ThyX fromthe hyperthermophilic bacterium Thermotoga maritima. The dy-namics of electron transfer to excited flavin adenine dinucleotidefrom a neighboring tyrosine residue are used as a sensitive probeof the functional dynamics of the active site. The fluorescence de-cay spanned a full three orders of magnitude, demonstrating a verywide range of conformations. In particular, at physiological temper-atures, multiple angstrom cofactor-residue displacements occur onthe picoseconds timescale. These experimental findings are sup-ported by molecular dynamics simulations. Binding of the dUMPsubstrate abolishes this flexibility and stabilizes the active site ina configuration where dUMP closely interacts with the flavin co-factor and very efficiently quenches fluorescence itself. Our resultsindicate a dynamic selected-fit mechanism where binding of thefirst substrate dUMP at high temperature stabilizes the enzymein a configuration favorable for interaction with the second sub-strate NADPH, and more generally have important implications forthe role of active site flexibility in enzymes interacting with multiplepoly-atom substrates and products. Moreover, our data provide thebasis for exploring the effect of inhibitor molecules on the activesite dynamics of ThyX and other multisubstrate flavoenzymes.

protein dynamics | flavoprotein | ultrafast fluorescence spectroscopy |quenching

Configurational flexibility is essential for enzyme functionduring catalysis. Binding of one or more substrates, accom-

modation of the transition state where the actual reaction takesplace, relaxation to the product state, and release of the product(s) is possible because different configurations of the enzyme arecontinuously sampled, by thermal or reaction-driven motions,on timescales ranging from femtoseconds to microseconds. Theconfigurational space sampled in a certain time range will de-pend on the local protein flexibility/energy landscape and thetemperature (1). During these configurational changes, distancesbetween constituents of the enzyme complex change. It has beenrecognized that the fastest (femtosecond to picosecond) local-ized motions exist alongside the slower motions that occur on the(typically millisecond) timescale of catalysis (2–4).Various experimental techniques allow monitoring changes in

interactions resulting from configurational changes. Long-rangemicro/millisecond domain motions in flexible proteins have beenstudied by NMR (5) and FRET techniques (6, 7). Molecular dy-namics simulations and NMR experiments indicate that more lo-calized structural fluctuations in these flexible regions occur on thepicoseconds-to-nanoseconds timescale (6, 8). These fluctuationscan only be directly investigated using very high time-resolution

optical techniques. In particular, the fluorescence properties ofintrinsic or external fluorophores can be exquisitely sensitive tothe protein environment. In flavoproteins, interaction of theflavin cofactor with the protein environment has been shown todiminish the lifetime of the flavin fluorescence from the intrinsicnanosecond timescale to the femtoseconds-to-picoseconds time-scale, due to quenching by photooxidation of nearby aromaticresidues (3, 9–15). In the present study, we use ultrafast fluo-rescence spectroscopy to investigate conformational flexibility ofthe flavoenzyme thymidylate synthase ThyX.ThyX is a homotetrameric enzyme discovered a decade ago (16),

which is essential for de novo synthesis of the DNA precursor 2′-deoxythymidine-5′-monophosphate (dTMP) in a large number ofbacterial systems. ThyX shows no structural homology to thymi-dylate synthase ThyA, which is used in most eukaryotes (17). Be-cause the ThyX pathway is used by a number of pathogenic bacteriaand absent in humans, ThyX is considered a promising antimicrobialtarget (16, 18); it catalyses carbon transfer from N5,N10-methylene-5,6,7,8-tetrahydrofolate (MTHF or CH2H4folate) to deoxyuridinemonophosphate (dUMP) using NADPH as a hydride donor andconsequently has three substrates (dUMP,MTHF, NADPH) withthe flavin adenine dinucleotide cofactor shuttling between thefully oxidized (FAD) and fully reduced (FADH2) forms. dUMPbinds in close interaction with the flavin group, displacing a nearbyTyr residue (19). A very recent study shows that folate derivativesmay bind to the opposite side of the flavin cofactor with respect todU(20). The binding site of NADPH has not been determined bystructural studies, but inhibition studies indicate that folate andNADPH binding sites may partially coincide (21). Furthermore,reduction of flavin by NADPH appears gated by the presence ofdUMP (21), further pointing at substrate-induced, functionalstructural rearrangements. Steady-state crystallographic studies ofthis enzyme also indicate substantial flexibility of the active site: inthe substrate-free structure the flavin group and its close environ-ment appear disordered (19); for the folate-bound form, multipleflavin configurations have been suggested (20).Using a newly developed, ultrafast fluorescence spectrometer

with full spectral resolution, we performed studies on the dynamicsof FAD fluorescence in wild-type and genetically modified ThyXenzymes from several bacterial species. In the present work wefocus on the enzyme from the hyperthermophilic bacterium Ther-motogamaritima that allows studies over a wide temperature range.We identified a close-lying tyrosine as well as the substrate dUMPitself as fluorescence quenchers. The observed fluorescence decayrevealed the presence of a wide range of conformations, whose

Author contributions: H.M., U.E.L., and M.H.V. designed research; S.P.L., L.B.-S., and J.-C.L.performed research; S.P.L. contributed new reagents/analytic tools; S.P.L., J.-C.L., andM.H.V. analyzed data; and S.P.L., H.M., U.E.L., and M.H.V. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: marten.vos@polytechnique.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218729110/-/DCSupplemental.

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interconversion accelerates at the physiological temperature of thishyperthermophilic enzyme. Binding of dUMP was found to stabi-lize the active site in a configuration allowing close interactionbetween dUMP and FAD and favorable for interaction withNADPH.Our data have important implications for the role of activesite flexibility in multisubstrate enzymes and ultimately permit ex-ploring the effect of inhibitor molecules on the active site dynamics.

ResultsThe absorption spectrum of ThyX from T. maritima (TmThyX;Fig. S1A) is very similar to that published previously (22), and thefluorescence spectrum (Fig. S1C) typical for oxidized flavins. Theenzyme contains four FAD binding sites (closest center-to-centerdistance 27 Å). Because in principle, resonance energy transferbetween identical molecules within the protein (homo-FRET) cantake place, potentially complicating the analysis, we measuredfluorescence anisotropy. The anisotropy was close to the theo-retical maximum of 0.4, implying that no homo-FRET occurs.This finding is consistent with an estimation of energy transfertimes of >3 ns using the Förster formalism and the observation(see below) that quenching takes place on faster timescales.Transient fluorescence spectra and kinetics at the emission

maximum are shown in Fig. 1; kinetics at different wavelengthsare shown in Fig. S2, and they show a highly multiphasic fluo-rescence decay, spanning timescales from ∼1 ps to ∼1 ns.However, all decay occurs substantially faster than the intrinsicnanosecond (∼3 ns) decay of FAD (23). The most probableorigin of this quenching is electron transfer (ET) from close-byaromatic residues to the excited flavin cofactor. According to thecrystal structure of TmThyX (19), Tyr-91, a widely conservedresidue (24), is the closest aromatic residue to the FAD cofactor,and therefore likely to act as main fluorescence quencher. Totest this hypothesis, we investigated the Y91F mutant protein,

designed to prevent electron transfer while maximally preservingthe structural environment of the flavin.The overall fluorescence decay kinetics of the mutant ThyX

protein are much slower than those of WT (Fig. 1B; note thelogarithmic scaling after 10 ps), implying that ET from Tyr-91 isindeed the dominant quenching process. However, in both cases,a fast decay in the order of ∼1 ps is present (see below), which weassign to relaxation processes in the excited state. Furthermore,the decay of the mutant protein still takes place faster than thatof FAD in solution, indicating that aromatic residues other thanTyr-91 also contribute to quenching. The shape of the fluores-cence spectra was found to be constant after ∼200 ps and to besignificantly different from that of FAD in solution (Fig. S3A).This observation further confirms that free FAD does not con-tribute to the signal.Analysis of the full data set in terms ofmultiexponential decay of

both the WT and the Y91F mutant protein requires at least fourdecay rates (Fig. S3A and B). This analysis indicates that red shiftsoccur concomitant with fluorescence decay on the timescale <200ps, and presumably reflect charge relaxation processes of the flavinenvironment (25). The nonexponentiality of the decay can beassigned to a distribution of conformations of the donor–acceptorpairs that do not or only partly interconvert during the excitedstate lifetime. The fact that the nonexponentiality is also foundin the mutant protein implies that this distribution is not uniquelydue to heterogeneity specifically of the Tyr-91 conformation.

Temperature Studies. To obtain detailed insight into the confor-mationalflexibility of the active site, wemeasured thefluorescenceproperties of the enzyme as a function of temperature. Here, wetake advantage of the fact that the ThyX enzyme from thehyperthermophile T. maritima allows studies over a wide range oftemperatures, up to 70 °C, into the physiological range of the en-zyme (at higher temperatures the isolated enzyme starts to pre-cipitate). Fig. S1C shows that the total fluorescence decreases withincreasing temperature, and that this decrease is fully reversibleupon cooling. The fluorescence decrease rather than increase athigher temperatures indicates that FAD is not released at hightemperature (up to 70 °C), as has been observed in flavodoxin (26).As shown in Fig. 2, the decrease of the total fluorescence is the

result of the acceleration of the fluorescence decay kinetics. Ina multiexponential global analysis, at all temperatures at leastfour components (eight fit parameters in total) are required tosatisfactorily fit the data. It was possible to describe the data atall temperatures with the same set of four rate constants (Fig. S3C and D). In this case, the amplitudes of the two longest decaycomponents decreased and those of the shortest decay compo-nents increased with temperature. Qualitatively, the result of thisanalysis implies that with increasing temperature the distribu-tion of fluorescence decays shifts toward the shorter-lived side,without the faster decays themselves becoming faster.The high number of exponentials (and fit parameters) re-

quired to fit the data suggests that a continuous distribution ofrates, rather than four distinct rates, may provide a more ade-quate and simpler description. To explore this possibility, weattempted to fit the integrated fluorescence decay with a powerlaw. For all temperatures, a satisfactory fit was obtained (at t >2 ps)using Eq. S1, which contains only three fit parameters: the am-plitude I0, mean lifetime τ0, and distribution width parameters q.The corresponding lifetime distributions are shown in Fig. 3C.The average lifetime τ0 (Fig. 3A) is seen to gradually decreasewith temperature. Overall, the relative width of the distribution(Fig. 3B) decreases at higher temperatures and in particularat 70 °C, which is in the physiological temperature range forT. maritima. Through its influence on the electronic coupling, theET rate k critically depends on the edge-to-edge distance r be-tween the flavin cofactor and the quenching residue as k= k0e−βr(3, 27). Assuming ET between Tyr and excited FAD is near

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Fig. 1. (A) Transient fluorescence spectra of WT TmThyX measured at 20 °C.The feature at ∼460 nm in the 0-ps spectrum is due to Raman scattering ofwater. (B) Kinetics at 520 nm ofWT and Y91F ThyX. The time axis is linear until10 ps and logarithmic thereafter. Solid lines are fits with a four-parameterexponential decay; the dashed line with power law.

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barrierless (3, 11), and β = 1.36 Å−1 (28), we can convert thelifetime distribution (Fig. 3C) to a distribution of distances fromwhich ET takes place (Fig. 3D). In view of uncertainty in k0, weshow the distribution as relative to a mean distance R0. At 20 °Cthe distribution is very large (FWHM >2 Å), implying strongconformational heterogeneity. We note that this distributionshould be considered qualitatively and does not necessarily di-rectly correspond to the static distribution of flavin–quencherpairs, because conformational flexibility within the fluorescencelifetime may bias the distribution toward shorter distances. Thispoint is illustrated in Fig. 3D. At higher temperatures, the dis-tance distribution shifts to shorter distances, and becomes nar-rower—in particular, close to the physiological temperaturerange (70 °C). Because warming should enhance the configura-tional distribution, the picture that emerges is that (i) at alltemperatures a very large distribution of configurations is pop-ulated, and that (ii) at higher temperatures the interconversionbetween these is accelerated so that the configurations that giverise to the fastest quenching rates are more easily reached withinthe timescale of fluorescence decay.

dUMP Binding. In the absence of the two other substrates, dUMPbinds close to oxidized FAD through aromatic stacking against itsisoalloxazine ring system (19). Fig. 4 shows that this binding leadsto dramatic quenching of FAD fluorescence: the dominant decayphase occurs with a time constant of ∼200 fs in this case, and theslower decay phases are strongly, although not completely, sup-pressed (∼90% decay within the first few picoseconds).The origin of this quenching may be either a direct interaction

between dUMP and the FAD cofactor, or a change in the in-teraction of FAD and the quenching residues, in particular Tyr-91.Indeed, the available TmThyX crystal structures suggest that upondUMP binding, Tyr-91 moves toward the FAD cofactor (19).To discriminate between these two possibilities, we investigatedthe Y91F mutant enzyme, which binds dUMP with comparableaffinity and shows a similar perturbation of the absorption spec-trum (Fig. S1B) as WT (see below). We observed (Fig. 4) that theeffect of dUMP binding on the fluorescence decay is very similarfor WT and Y91F TmThyX, which led us to conclude that it isdUMP itself that acts as quencher of flavin fluorescence. Theminor (∼10%), slower decay phases in the presence of dUMP (Fig.S3E) may reflect partial and/or heterogeneous dUMP binding.The strong quenching of FAD binding by dUMP can be used

as a sensitive probe to determine the affinity of TmThyX fordUMP. In agreement with the time-resolved data of Fig. 4, theoverall fluorescence was found to decrease ∼10-fold upon dUMPbinding to WT TmThyX. Analysis of the dUMP titration to 5-μMFAD binding sites yielded a Kd of 400 nM at 60 °C and in thepresence of 250 mM NaCl (Fig. S4). The apparent binding siteconcentration deduced from the binding curve was very close to

the FAD concentration, implying that flavin-devoid sites, whichare also present in ThyX in solution (SI Materials and Methods),do not bind dUMP in the same affinity range. This findingstrongly suggests that stacking with FAD is the major bindingdeterminant of dUMP. A similar affinity was found using thechange in the flavin absorption spectrum (Fig. S1A) as a measurefor dUMP binding, although the high dynamic range of thefluorescence measurements allows a more precise determination.Detailed binding studies will be presented elsewhere.We also used FAD fluorescence to investigate whether dUMP

binding influences the thermal stability of the complex. As men-tioned previously, in the absence of dUMP up to 70 °C FAD doesnot dissociate from the protein.Above 70 °C, the FAD fluorescencerises and redshifts, and at 90 °C an increase corresponding to ∼4%free FAD is observed (Fig. S5).When dUMP is bound, this increaseamounts to less than 0.5%, and no spectral shift is observed (Fig.S5). Although protein precipitation above 90 °C prohibited thedetermination of full dissociation curves of this hyperthermophilicenzyme, these data indicate that dUMP binding shifts denaturationto higher temperatures and thus thermally stabilizes the complex.

Molecular Dynamics Simulations. To investigate the molecularparameters underlying the experimentally observed conforma-tional flexibility, we performed molecular dynamics (MD) sim-ulations of WT TmThyX in the presence and absence of dUMPand at two temperatures (27 °C and 70 °C). The models that

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Fig. 3. Analysis of the temperature dependence of the fluorescence kineticsof WT TmThyX using Eq. S1. (A) Mean value τ0 of the lifetime distribution. (B)Heterogeneity factor q of the lifetime distribution. The solid lines are guidesfor the eye. (C) Lifetimes distribution. (D) Distribution of edge-to-edge dis-tances between FAD and electron donors normalized to the total integral.

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included all four subunits were based on the X-ray structure ofdUMP-bound TmThyX that was obtained at −173 °C (19). Fig.S6 shows that after warming and equilibration of the model, thermsd of the backbone atoms of the model from the X-raystructure remained roughly constant and ∼1 Å for the 2.5-ns freedynamics trajectories. In the absence of dUMP, the deviation issignificantly larger for the trajectories at 70 °C than at 27 °C,indicating a larger conformational heterogeneity. Interestingly,such a difference is not observed in the absence of dUMP, in-dicating that this substrate effectively rigidifies the protein.We now focus on the vicinity of the FAD. Fig. 5 compares

time-averaged simulated structures of the four active sites;a corresponding full tetramer is shown in Fig. S7. Though thestructures in the presence of dUMP are similar to the X-raystructure (19), in the absence of dUMP the flavin is rotated in allsubunits by ∼50° with respect to the X-ray structure and showssubstantial orientational freedom (Fig. 5B; Fig. S8). Similarobservations were made with models based directly on the dUMP-devoid structure. Interestingly, these rotated conformations aresimilar to those observed recently in structural studies in some ofthe active sites of mutant TmThyX (20). As in the crystal struc-tures of the TmThyX WT proteins (19), in all subunits, the pres-ence of dUMP results in a shorter FAD–Tyr-91 distance.Fig. 6 shows thefluctuations of theFAD–Tyr-91 distance during

the free dynamics in each of the four subunits. The shortest edge-to-edge distance is thought to be relevant for the electronic cou-pling determining the electron transfer rate. Therefore, at eachinstant, this distance is plotted rather than the distance betweentwo fixed atoms. In the crystal structure the shortest distance isbetween the C6 atom of FAD and the Ce1 atom of Tyr-91. In oursimulations, other atom pairs, such as FAD N5-Tyr-91 Cδ1, alsooccur as shortest distance. The active site structures of Fig. 5 in-dicate that the distance fluctuations in the dUMP-devoid modelare rather the result of fluctuations of the flavin cofactor thanof Tyr-91.In the dUMP-devoid model, the FAD–Tyr-91 distance fluc-

tuates substantially in all four subunits, at 27 °C from ∼3 to ∼8 Å.Larger fluctuations of 2 Å or more appear infrequently (intervalsof several nanoseconds) on the simulation timescale, and thestatistics, especially at 27 °C, are clearly undersampled, in-dicating that substantial conformational changes also take placeon a longer timescale. These observations are in agreement withour analysis of the broad range of fluorescence decay times thatindicate that FAD–Tyr-91 distance changes take place on thetimescale of ∼1 ns or more. At 70 °C the fluctuations becomesubstantially more frequent, both the smaller, high-frequencyand the larger, low-frequency fluctuations.Interestingly, our simulations also indicate that the presence

of dUMP drastically reduces the distance fluctuations for both

temperatures. This finding implies that substrate binding leadsto a rigidification of the active site, and in particular of the FADcofactor positioning, and is generally consistent with the betterresolution of the active site in the X-ray crystallography studiesat cryogenic temperatures (19) and our finding of dUMP-induced thermal stabilization of the FAD–protein complex (Fig.S5). Similar analyses for other residues involved in substratebinding and catalysis also indicate substantial distance variationspecifically in the absence of dUMP. For instance Arg-147, Arg-174, and Ser-88, which all H-bond to dUMP, were all found to beable to intermittently H-bond to FAD in the absence of dUMP.

DiscussionIn this work, the quenching of flavin fluorescence in an enzymeinvolved in DNA synthesis, associated with MD simulations, wasused to probe the dynamic properties of the active site. Experi-mentally, we exploited the fact that photoexcited FAD (FAD*)can accept an electron from the aromatic residues Tyr and Trpon the femtosecond-to-nanosecond timescale, and showed thatin the case of TmThyX from, Tyr-91 is the principal electrondonor. It has been argued that the FAD*Tyr→FAD−Tyr°+ re-action occurs with a driving force in the range of 0.7–1 eV and ina near-activationless regime (3, 11). Indeed, the rates of thiselectron transfer reaction were found to be temperature in-dependent in flavoproteins with a much less heterogeneousfluorescence decay distribution than TmThyX (11, 13). More-over, in TmThyX, increase in temperature leads to a shift ofslow-decaying populations to fast-decaying populations ratherthan an increase of the rates itself. For these reasons we interpretthe temperature dependence observed in this work as originatingfrom changes in the distance-dependent electronic coupling be-tween the reactants, rather than from activation barriers. Wenote that temperature-dependent fluorescence decay times havebeen reported for the flavoprotein lipoamide dehydrogenase(29). The FAD cofactors in this protein have an extremely high

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Fig. 5. Superimposed structures of elements of the four active sites, timeaveraged over the last 500 ps of the free trajectories of MD simulations ofThyX in the presence (A) and absence (B) of dUMP. The superposition is onthe entire backbone of each subunit. The His53 residues associated with theactive sites belong to different subunits as the other elements. The carbonatoms are color-coded with the respective subunits.

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fluorescence quantum yield and are not quenched; here, theactivation of the fluorescence decay was due to distinct confor-mational changes that are static on the nanosecond timescale (29).The analysis in the present work by contrast highlights dynamicconformational changes occurring on the picosecond timescale.Our analysis is based on the widely used (3, 11, 14) notion that

excited-state quenching in flavoproteins is due to electron transferfrom aromatic residues. The validity of this notion has beendemonstrated for tryptophan quenchers (9, 30) and strongly sug-gested for tyrosine quenchers (31, 32). In both cases, the radicalpair can be subsequently stabilized by proton transfer from theresidue. Simultaneous proton and electron transfer, avoidinghigh-energy charge-separation intermediates, has been observedfor phenol oxidation reactions in aqueous solutions (33, 34).Though we cannot fully exclude that such simultaneous pro-cesses (and hence motions between Tyr-91 and potential protonacceptors) could play a role in FAD* quenching in ThyX, weconsider it unlikely in view of the evidence from other fla-voproteins and the fact that the high-driving force generated byFAD* formation (see above) energetically allows formation ofthe FAD−Tyr°+ intermediate by electron transfer only.We observed that not only aromatic residues, but also the sub-

strate dUMP quenches FAD fluorescence, strongly suggesting thatbound dUMP also donates an electron to FAD*. dUMP indeedbinds very close to FAD, and the isoalloxazine and uracil rings areactually in, or close to, van der Waals contact (distance ∼3.5 Å inthe crystal structure). The relevant redox properties of dUMPhavenot been determined, but the light-induced oxidation of uracil inthe presence of a photoreducing material has been reported (35),suggesting that the reaction is energetically possible. We will fur-ther investigate this hypothesis by transient absorption spectros-copy of the photoproducts formed in the ThyX–dUMP complex.The FAD* decay dynamics of ThyX are strikingly multiphasic,

spanning three orders of magnitude in time. We assign the largeand continuous lifetime distribution in ThyX fluorescence to con-formational heterogeneity in the active site; this assignment isstrongly supported by the MD simulation that shows large varia-tions of active site configurations. In itself, nonmonoexponentialdecay of the excited state of quenched flavin cofactors in fla-voproteins is frequent (15). Biexponential decay has been docu-mented spanning shorter time spans (14, 36, 37), and in flavodoxinmultiexponentiality has been associated predominantly with partialflavin dissociation from the protein (26). In blue light-sensing usingFAD (BLUF) photosensor proteins (13, 31, 32) and in flavin

reductase (3), broader and more heterogeneous lifetime dis-tributions have been interpreted in terms of ground-state hetero-geneity. The lifetime distribution in TmThyX is considerably largerthan in the abovementioned flavoproteins, and the associated do-nor–acceptor distance distribution is substantially wider (∼2 ÅFWHM at 20 °C; Fig. 3D) than the ∼1 Å FWHM distribution inYang et al. (3).Our results show that a very broad range of configurations is

accessible for TmThyX in the absence of substrate, and that thesedo not fully interconvert on the fluorescence timescale and at sub-catalytic temperatures. These findings can be considered consistentwith an early crystallographic study of substrate-free TmThyX,where fractions of the protein, and in particular the flavin ring,appear disordered, in contrast to the dUMP-containing protein(19), and appear general for ThyX enzymes (38, 39). More specif-ically for TmThyX, our MD simulations indicate the presence ofsubstantial variation in the distance between FAD and the Tyr-91quencher, with large changes (up to∼4Å) occurring infrequently onthe timescale of up to several nanoseconds. This flexibility is nolonger apparent in the presence of dUMP. Single-molecule studiesof flavin reductase have demonstrated that interconversion of thelifetime-distinguishable configurations for a substantial part takesplace on the timescale of milliseconds and beyond (3). Our tem-perature dependence studies of ThyX show that the distributiondoes not broaden at higher temperature, as could be expected frompure increase of accessible configurational space, but narrows andshifts to shorter effective distances. We assign this finding to ac-celeration of the interconversion rates of configurations on the sametimescale as fluorescence decay. This interpretation is supported byour MD simulations that indicate that substantial fluctuations indonor–acceptor distance do occur on the picosecond-to-nanosec-ond timescale, and that these, and in particular the less-frequentlarge amplitude fluctuations, occur substantially more frequent athigher, physiologically relevant, temperatures.We suggest that the flexibility in the active site of this enzyme, as

reflected in the picosecond–nanosecond multiangstrom conforma-tional fluctuations, is related to its capacity of binding multipledifferent aromatic substrates, of which at least two (NADPH/dUMPand MTHF/dUMP) simultaneously. The picture that emerges isthat the extremely rapid conformational sampling of the proteinallows dUMP to enter the active site and be accommodated close totheflavin. The binding of dUMP then stabilizes the flavin cofactor inthe active site, as indicated by our MD simulations, presumably in aconfiguration favorable for NADPH binding and subsequent flavinreduction. Thismechanism is in agreement with the reported drasticacceleration of flavin reduction by NADPH in the presence ofdUMP (21).We stress that our observations have further functionalconsequences. Indeed, low stereospecificity and a very high Km forNADPH have been observed for the TmThyX protein at 37 °C (40,41). Moreover, this protein turns over substantially faster at 65 °C(within the physiological range) than at 37 °C (41, 42).Our results indicate that a selected fit-type mechanism applies,

where the substrate binding does not necessarily change theenzyme configuration, but rather stabilizes a favorable configu-ration among many sampled. The accelerated and more exten-sive sampling at higher temperature does not substantiallyinfluence the equilibrium binding of the substrate, but may allowfaster exchange with the solvent of substrate and product.In conclusion, we exploited ultrafast time-resolved FAD fluo-

rescence quenching by a neighboring aromatic residue in ThyXand show that, in combination with temperature dependencestudies, it is a sensitive probe for conformational fluctuations.We have shown that the active site of ThyX, which has to ac-commodatemultiple substrates, is highly flexible and, in particular,samples configurations with very high speed, with large, multipleangstrom displacements occurring on the picoseconds timescale inthe physiological temperature range. We suggest that this struc-tural plasticity allows efficient binding of the dUMP substrate.

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Binding of dUMP, which acts as a very efficient FAD fluorescencequencher itself, arrests these movements and stabilizes the enzymein a configuration allowing FAD reduction by the NADPH sub-strate.We conjecture that the rapid large amplitudemotions of theactive site monitored in ThyX enzymes may be related to their ca-pacity of interacting with multiple different aromatic and relativelyrigid substrates and products. The method that we have exploredhere can be used to study the effect of other substrates and, inparticular, inhibitors, including potential antimicrobial agents (18),on the dynamics of the active site of ThyX. More generally, ourresults emphasize the important role of enzyme dynamics in theinteraction with the substrates. Whereas flavoenzymes provide anintrinsically well-suited tool for real-time monitoring of configura-tional evolution, extensive high-speed sampling of active site con-figurations on the intrinsic picosecond timescale of global protein

motions is likely to be a general feature for flexible protein domainsand important for understanding enzyme substrate reactivity (2).

Materials and MethodsProteins were expressed using a codon-optimized expression vector, andpurified following standard procedures. Time-resolved fluorescence experi-ments were performed using a recently developed spectrally resolved Kerr-gate femtosecond fluorometer (43). The models for the MD simulations(performed with CHARMM) in the presence and absence of dUMP shown inthe main text were based on the structure of the dUMP-containing protein(PDB ID code 1O26) (19); a model based on the substrateless structure (PDBID code 1O2A) gave similar results (Fig. S9). A detailed description of theexperimental procedures, data analysis, and simulation protocols is givenin SI Materials and Methods.

ACKNOWLEDGMENTS. This work was supported by the Agence National dela Recherche Grant ANR-09-PIRI-0019.

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