Structural Determinants of Improved Fluorescence in a Family ofBacteriophytochrome-Based Infrared Fluorescent Proteins: Insightsfrom Continuum Electrostatic Calculations and Molecular DynamicsSimulationsMikolaj Feliks,†,‡,§ Celine Lafaye,†,‡,§ Xiaokun Shu,∥,⊥ Antoine Royant,*,†,‡,§,# and Martin Field*,†,‡,§

†Universite Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38044 Grenoble, France‡CNRS, IBS, F-38044 Grenoble, France§CEA, IBS, F-38044 Grenoble, France∥Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United States⊥Cardiovascular Research Institute, University of California, San Francisco, California 94158, United States#European Synchrotron Radiation Facility, F-38043 Grenoble, France

*S Supporting Information

ABSTRACT: Using X-ray crystallography, continuum elec-trostatic calculations, and molecular dynamics simulations, wehave studied the structure, protonation behavior, and dynamicsof the biliverdin chromophore and its molecular environmentin a series of genetically engineered infrared fluorescentproteins (IFPs) based on the chromophore-binding domain ofthe Deinococcus radiodurans bacteriophytochrome. Our studysuggests that the experimentally observed enhancement offluorescent properties results from the improved rigidity andplanarity of the biliverdin chromophore, in particular of thefirst two pyrrole rings neighboring the covalent linkage to theprotein. We propose that the increases in the levels of bothmotion and bending of the chromophore out of planarity favor the decrease in fluorescence. The chromophore-binding pocket insome of the studied proteins, in particular the weakly fluorescent parent protein, is shown to be readily accessible to watermolecules from the solvent. These waters entering the chromophore region form hydrogen bond networks that affect theotherwise planar conformation of the first three rings of the chromophore. On the basis of our simulations, the enhancement offluorescence in IFPs can be achieved either by reducing the mobility of water molecules in the vicinity of the chromophore or bylimiting the interactions of the nearby protein residues with the chromophore. Finally, simulations performed at both low andneutral pH values highlight differences in the dynamics of the chromophore and shed light on the mechanism of fluorescence lossat low pH.

Phytochromes are photoreceptors used by green plants,algae, bacteria, and fungi to absorb light and convert it into

physiological signals.1,2 They can absorb light in the red and far-red regions of the electromagnetic spectrum. Efforts have beenmade to genetically engineer alternatives to the naturalphytochromes that possess improved fluorescent properties.3,4

These properties include an increase in the quantum yield, anincrease in the extinction coefficient, and a shift of thefluorescence maximum closer to the infrared region of thespectrum. Engineering of infrared fluorescent proteins has nowbecome a new, emerging field.5

The chromophore-binding domain of the bacteriophyto-chrome from Deinococcus radiodurans (DrCBD) has attractedmuch attention.6 Significantly, a single-point mutant of thisdomain has been shown to be fluorescent in the near-infraredrange7 and has been called IFP1.0 (for “first version of an

infrared fluorescent protein”). A directed evolution approachsubsequently led to the design of a protein with significantlyimproved infrared fluorescence, IFP1.4.3 Another variant,IFP2.0, was later obtained with a higher binding affinity forthe chromophore biliverdin.4 The evolution tree of IFP1.4,shown in Figure 1, contains three intermediate proteins, IFP1.1,IFP1.2, and IFP1.3. Proteins from DrCBD to IFP1.2 occur asdimers, whereas proteins from IFP1.3 to IFP2.0 aremonomeric.To date, only a few attempts, both experimental and

computational, have been made to understand at the molecularlevel the origins of fluorescence in different proteins derived

Received: March 31, 2016Revised: July 18, 2016Published: July 29, 2016

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© 2016 American Chemical Society 4263 DOI: 10.1021/acs.biochem.6b00295Biochemistry 2016, 55, 4263−4274

from the bacterial phytochrome. The structure of DrCBD wasfirst determined in 2005 by Forest and co-workers at 2.5 Åresolution and represented the first structure of any fragment ofa phytochrome.8 The resolution of this DrCBD structure waslater improved to 1.8 Å because of a surface mutation.9 As afollow-up, three different structures of IFP1.0 were determinedat resolutions as high as 1.24 Å, revealing that the crucialHis207 residue could adopt two orthogonal orientations of itsimidazole group.10 Finally, the same group determined thestructure of IFP1.4 at 1.65 Å resolution, revealing that theaddition of mutations decreased structural heterogeneity in thechromophore-binding cavity and induced a relative movementof two secondary elements of the protein.11 These observationsagreed with the structure of IFP2.0 at 1.14 Å resolution that wedetermined shortly before this latter work.4

Other IFPs that have been investigated include the brightWi-Phy, designed by the Forest group, which differs by onlytwo mutations from its parent DrCBD and for which thestructure has been determined at 1.75 Å resolution.10 Anaturally monomeric fragment of a phytochrome from aBradyrhizobium species was also forced to evolve into thedefinitively monomeric IFPs, namely, mIFP12 and its markedlyblue-shifted variant iBlueberry.13 A distinct series of IFPs withemission maxima ranging from 670 to 720 nm14 have beendeveloped by Verkhusha and co-workers from the bacter-iophytochromes RpBphP2 and RpBphP6 from Rhodopseudo-monas palestris.6 So far, little direct structural information aboutthese proteins has been obtained.15,16 Likewise, a moleculardynamics study of a modeled structure of iRFP, derived fromRpBphP2, suggested that the improved fluorescent propertiesof the biliverdin chromophore were due to an increased tilt ofits terminal ring, a reduction in the number of water moleculeswith which it interacts, and a significant decrease in flexibility.17

Finally, another bacteriophytochrome from R. palestris,RpBphP1, was forced to evolve into the bright blue-shiftedinfrared FP BphP1-P1, for which a structure could beobtained.18 It is noteworthy that the mechanism of the blueshift in iBlueberry and BphP1-FP is identical and is due to thebinding of the chromophore to a cysteine residue of a differentdomain than for other IFPs.

In this paper, we have combined continuum electrostaticcalculations and molecular dynamics simulations to study theprotonation behavior and dynamics of the weakly fluorescentbacteriophytochrome and its six fluorescent mutants. We haveused the available crystal structures of the bacteriophyto-chrome-based proteins as well as our newly determined, 1.11 Åresolution structure of IFP1.4, which provides an increasedlevel of detail compared to that of the previously determinedstructure. The objective of our study has been to identifystructural factors responsible for the gradually improvingfluorescent properties within the given set of mutants byfocusing on the dynamics of the monomeric versions of all theproteins. The knowledge of these structural factors wouldprovide a rational basis for the future design of fluorescentproteins based on the biliverdin chromophore (or anotherbilin) with improved fluorescent properties.5

■ METHODSProtein Expression and Purification. The sequences of

IFP1.1, IFP1.2, IFP1.4, and IFP2.0 with a C-terminal six-His tagwere inserted into the modified pBad expression vectorcontaining the heme-oxygenase-1 gene.3 Recombinant proteinswere expressed and purified as described previously.4 Thepurity of the protein solutions was confirmed by sodiumdodecyl sulfate−polyacrylamide gel electrophoresis. Finalconcentrations were determined by UV−visible absorptionspectroscopy using calculated molar absorption coefficients at280 nm of 32555 M−1 cm−1 for IFP1.1 and IFP1.2, 35915 M−1

cm−1 for IFP1.4, and 34045 M−1 cm−1 for IFP2.0.Crystallization of IFP1.4. Mutagenesis of residue 307 has

been shown to improve crystal quality.9 We managed tocrystallize the E307Y mutant of IFP1.4 using the hanging-dropmethod at 293 K at a concentration of 14 mg/mL in a solutionconsisting of 26% PEG 400 and 0.1 M sodium acetate (pH5.0), i.e., under conditions similar to those used for IFP2.0.4

Crystals grew in 6 days, compared to 1 day for IFP2.0.X-ray Diffraction Data Collection and Structure

Refinement. Prior to diffraction experiments, crystals wereflash-cooled in liquid nitrogen because PEG 400 at thisconcentration is a cryoprotectant. X-ray diffraction experimentswere performed at the European Synchrotron Radiation Facility(Grenoble, France). Data were collected at 100 K at awavelength of 0.976 Å on beamline ID29.19 Diffraction datasets were processed using XDS,20 and intensities were scaledand reduced with AIMLESS.21 The crystal belongs to the C2space group and diffracted to 1.11 Å resolution. The solventcontent is 46%. Structural refinement was conducted withRefmac5 using anisotropic B factors for all atoms.22 As alreadydescribed for IFP2.0, the thioether bond between Cys24 andthe chromophore biliverdin is particularly sensitive to X-rays.Structural and experimental data have been deposited in theProtein Data Bank (PDB) as entry 5AJG. Crystallographic datastatistics can be found in Table 2.

pKa Measurements. The pKa values of the various IFPswere assessed using the measurement of their fluorescence insolutions at increasing pH values using phosphate-citrate buffer(from pH 2.6 to 7.2). Each buffer solution was obtained bymixing various amounts of 0.1 M citric acid and 0.2 M dibasicsodium phosphate.23 All samples (4 μL of concentrated IFP at1 mg/mL with 33 μL of buffer at a given pH) were loaded ontoa 96-well plate and placed in a fluorescence plate reader(Synergy H4, BioTek). Fluorescence was excited at 640 nm,and the fluorescence emission signal was integrated between

Figure 1. Evolution tree of D. radiodurans bacteriophytochrome-basedinfrared fluorescent proteins. Fluorescence quantum yields areindicated in parentheses as previously determined.3,4,10 Reversedmutations in IFP2.0 are underlined.

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660 and 800 nm. Each curve was measured in triplicate andnormalized against the value at pH 6.0, corresponding tomaximal emission for each variant.Preparation of the Protein Models. The protein models

used in our study were prepared on the basis of the highest-resolution crystal structure, when available [DrCBD, IFP1.0,IFP1.4, and IFP2.0 (see Table 1 for the known structures of

IFPs)]. IFP1.1 and IFP1.2, for which no structures are available,were modeled using the crystal structure of IFP1.0. TheMODELLER24 package was used to perform the mutations aswell as to repair unresolved atoms or side chains. All structuresmissing a loop between residues 127 and 152 were completedusing the loop present in the structure of DrCBD. DrCBD,IFP1.0, IFP1.1, and IFP1.2 are dimers in solution, and theirgeometries were generated by applying the appropriatesymmetry operations in pDynamo.25 For electrostatic calcu-lations, we used both the dimeric and monomeric geometries.For molecular dynamics simulations, only the monomericgeometries were used.The initial preparation of the protein models was conducted

in CHARMM.26,27 Molecular mechanics parameters for theprotein were taken from the CHARMM27 force field.28 Theparameters for the biliverdin chromophore in its fullyprotonated form were taken from the literature.29,30 Theparameters for the chromophore deprotonated at the B pyrrolering were refined on the basis of the parameters of the fullyprotonated chromophore. The refinement procedure isdescribed in section S3 of the Supporting Information. Duringthe initial preparation of the models, all titratable proteinresidues were set to their standard protonation states at pH 7;i.e., aspartates and glutamates were deprotonated, histidinesdoubly protonated, and all other residues protonated. The sidechains of the mutated and repaired residues, the added loopfragment, and all hydrogen atoms were subsequently geometry-optimized in CHARMM. During the optimizations, thecoordinates of the other parts of the proteins were kept fixedat their crystallographic positions.Determining the Protonation States of Titratable

Residues. In the next step, we performed Poisson−Boltzmannelectrostatic calculations combined with a Monte Carlo (MC)titration to evaluate protonation states of titratable residues inall models. The electrostatic calculations were performed using

pDynamo,25,31 interfaced to the external solver of the Poisson−Boltzmann equation, MEAD,32 and to the Monte Carlosampling program, GMCT.33 Protonation state energies andtitration curves were calculated using pDynamo’s ownroutines.31 The following parameters were set for thePoisson−Boltzmann continuum electrostatic model. Chargesand radii for the protein and chromophore atoms were takenfrom the CHARMM27 force field28 and literature,29,30

respectively. To construct the volume of the protein, an ion-exclusion layer of 2.0 Å and a solvent probe of 1.4 Å were used.The dielectric constants of the protein (εp) and the solvent (εs)were set to 4 and 80, respectively. The solvent was assigned anionic strength (I) of 100 mM. The calculations were performedat 300 K. Electrostatic potentials were calculated using fourgrids, each consisting of 1213 nodes, with focusing steps atresolutions from 2.0 to 0.25 Å. The protonation probabilities oftitratable residues were estimated for pH values ranging from 0to 14. For every pH step, the MC calculation consisted of 100equilibration scans and 3000 production scans. The chromo-phore was treated as a nontitratable site, and its protonationstate was fixed either to the fully protonated form or to theform deprotonated at the B pyrrole ring, depending on theparticular calculation. The carboxylic groups at rings B and C ofthe chromophore were treated separately as titratable sites. Theparameters of glutamate (model pKa and atomic charges) wereused for the treatment of these groups.

Molecular Dynamics Simulations. Before the moleculardynamics simulations, the proteins were assigned protonationstates according to the previous electrostatic calculations. Foreach protein, we performed simulations with the fullyprotonated chromophore at either low or neutral pH (4 or 7,respectively). Additionally, the simulations were repeated withthe chromophore deprotonated at the B pyrrole ring at bothpH values (see the Supporting Information for details). Thesimulations were performed using NAMD.34 We used customTcl scripts and the VMD35 package to prepare the models forthe simulations as well as to analyze the resulting trajectories. Inthe first step, each protein structure was solvated in arectangular cell of explicit water molecules, using the TIP3Pwater model. A 9 Å wide buffer of solvent molecules was usedbetween the protein and the boundary of the periodic cell.Counterions, chloride and sodium, were added appropriately tobalance the non-zero charge of each of the proteins. The finalmodels consisted of 29 × 103 to 31 × 103 atoms, depending onthe protein. For each protein, the simulation was preceded by1000 steps of geometry optimization of the entire system usingperiodic boundary conditions. The initial temperature was setto 20 K. Both the heating stage and the consecutive productionsimulation were performed in the NPT ensemble, under apressure of 1 bar. During the heating stage, the temperature ofthe system was increased in steps of 10 K/ns from 20 to 300 K.At the end of each heating window, 10000 equilibration stepswere performed. In the last window, the number of such stepswas doubled. The production run was performed at a finaltemperature of 300 K for 10 ns, using a time step of 2 fs. Bondsinvolving hydrogen atoms were constrained using the SHAKEmethod36 to decrease the computational cost of the simulation.During the simulations, we monitored the most importantgeometrical parameters of the system. These included, forexample, the six dihedral angles of the chromophore and theroot-mean-square deviation (rmsd) of its atoms. To ensure thatthe system had reached equilibrium, we traced the root-mean-square deviation of the chromophore atoms (see Figure S5 of

Table 1. Protein Structures, Crystallographic and Computer-Generated, Used To Construct the Continuum Electrostaticand Molecular Dynamics Models

proteinPDBentry

resolution(Å) source year comments

DrCBD 2O9C 1.45 Wagner9 2007 −IFP1.0 3S7O 1.24 Auldridge10 2012 −IFP1.1 − − this work − generated

fromIFP1.0

IFP1.2 − − this work − generatedfromIFP1.0

IFP1.4 5AJG 1.11 this work 2015 −IFP2.0 4CQH 1.14 Yu4 2014 −

Other Available Structures of Phytochromes Not Used in This WorkDrCBD 1ZTU 2.50 Wagner8 2005 lower

resolutionIFP1.4 4O8G 1.65 Bhattacharya11 2014 lower

resolution

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the Supporting Information for the rmsd evolution in differentproteins). For the important dihedral angles of thechromophore, we also plotted the distributions and tabulatedthe fitted normal distribution parameters based on thecomplete simulations, as well as on their first and secondhalves (see Figures S9−S12 and Tables S1 and S2).

■ RESULTS AND DISCUSSIONStructure of IFP1.4.We determined the structure of IFP1.4

at 1.11 Å resolution and compared it to that of the parentDrCBD, to that of the closest progenitor for which a structurehas been determined, IFP1.0, and to that of its descendantIFP2.0 (Table 2). For DrCBD and IFP1.0, we chose the

structures with the highest resolution, 2O9C9 and 3S7O,10

respectively. We previously determined the 1.14 Å structure ofIFP2.0 (PDB entry 4CQH).4 Residual density on thechromophore showed that the two enantiomers alreadyobserved in IFP1.010 and the 1.65 Å structure of IFP1.410 arealso present in our structure; only the enantiomer with thehighest conformation occupancy was modeled.Comparison of IFP1.4 with IFP1.0 primarily shows that

His207 has two orthogonal orientations of the imidazole ring,as already observed in a lower-resolution structure of IFP1.0.10

This conformational difference appears only to induce themovement of loop 197−206 and that of Tyr263 toward thechromophore (Figure 2a). Comparison of IFP1.4 with IFP2.0(with an rmsd value of all Cα atoms of only 0.27 Å) shows thatthe chromophore environment has hardly changed (Figure 2b).The largest differences occur around residue 207 (His207 inIFP1.4 and Thr207 in IFP2.0), whereas there are only minoreffects in the vicinity of the reversion mutation V186M and thequasi-isosteric mutation F198Y. Indeed, the V186M mutation,

which was introduced between IFP1.0 and IFP1.1, has beenshown to affect only the absorption and fluorescence emissionmaxima, but not the fluorescence quantum yield,11 therebyhighlighting the importance of the A288V mutation.4 Finally,comparison of the DrCBD and IFP1.0 structures shows that theorientation of the side chain of residue 207 has effects on thepositioning of Tyr263 and the loop bearing residues 204−206(Figure S2).

pKas of IFPs. We determined the pKas of IFP1.1, IFP1.2,IFP1.4, and IFP2.0. IFP1.1 and IFP1.2 have the highest pKavalues (4.8), followed by IFP1.4 (4.6) and IFP2.0 (4.2) (FigureS3). The low pKa of IFP2.0 corresponds to the mutation of theionizable residue His207 into a threonine, while the decreasedpKa of IFP1.4 indicates a better shielding of the chromophorefrom bulk solvent.

Protonation States of Biliverdin. For each of the studiedproteins, we calculated the probabilities of protonation states oftitratable residues. In addition to titratable protein groups, thebacterial phytochrome binds the biliverdin chromophore(Figure 3), which could also be a titratable group. In principle,the chromophore can bind or release one proton at each of thefour pyrrole rings. However, there is a consensus in theliterature about the protonation state of the chromophore.Raman spectroscopy and other experiments suggest that thebacteriophytochrome contains predominantly the fully proto-nated form of biliverdin.17,37 Theoretical studies using time-dependent density functional theory indicate that thechromophore likely exists in its fully protonated form,38,39 asit was found that the calculated absorption spectrum of thedeprotonated chromophore was shifted by 15−60 nm incomparison to the experimentally observed one. On the otherhand, light- and X-ray-induced deprotonation of the biliverdin

Table 2. Data Collection and Refinement Statisticsa forIFP1.4

Data Collectionspace group C2cell dimensions

a, b, c (Å) 95.4, 53.0, 66.2α, β, γ (deg) 90, 90.9, 90

resolution (Å) 38.4−1.11 (1.13−1.11)Rmerge 3.4 (62.3)I/σI 17.6 (2.0)completeness (%) 98.5 (92.1)multiplicity 4.3 (3.7)

Refinementresolution (Å) 27.3−1.11 (1.14−1.11)no. of reflections 121711Rwork, Rfree 0.146 (0.227), 0.168 (0.245)no. of atoms

protein 2525biliverdin 43water/ion 263

B factor (Å2)protein 17.1biliverdin 10.0water/ion 25.8

root-mean-square deviationbond lengths (Å) 0.014bond angles (deg) 1.94

aValues in parentheses are for the highest-resolution shell.

Figure 2. Comparison between the chromophore environment of thenewly determined structure of IFP1.4 and those of (a) IFP1.0 (PDBentry 3S7O) and (b) IFP2.0 (PDB entry 4CQH). Color code: IFP1.0,magenta; IFP1.4, cyan; IFP2.0, yellow. This figure was prepared withPyMOL.46

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chromophore have been experimentally confirmed,40,41 re-inforcing the notion that one of the pyrrole protons may bequite labile. In this paper, we focus primarily on the situation inwhich the chromophore is protonated at all pyrrole rings in thewhole spectrum of pH. For the sake of completeness, however,we have also examined the less probable scenario in which thechromophore is deprotonated at one of the pyrrole rings atneutral pH. We chose the B-deprotonated form based on thecomparison of gas-phase energies and geometries of differentprotonation forms of the chromophore. The rationale behindour approach was that the fluorescence of IFP1.4,3 as well asthe other proteins (Figure S3), was shown to be highly pH-dependent. The fluorescence is at a maximum in the pH rangeof 5.5−6.5 but decreases at pH <4. Therefore, we tried to assesswhether the observable loss of fluorescence might be related tothe changes in the protonation of biliverdin. We report on theseconsiderations in sections S2 and S3 of the SupportingInformation.Protonation States of the Proteins. From the calculated

titration curves (Figure S8 of the Supporting Information), itcan be seen that the probability profiles for both the fullyprotonated and B-deprotonated chromophores usually alignclosely. Thus, we can conclude that the protonation state of thechromophore affects the protonation states of other sites onlyslightly. The only major exception is His260, whoseprotonation state is strongly influenced by the protonationstate of the chromophore (Figure S8), because this residue islocated in the direct vicinity of rings B and C. If thechromophore is treated in its fully protonated form, His260always remains ε-protonated in all proteins, regardless of thepH. However, if the chromophore is deprotonated at ring B,the situation becomes more complicated, because His260 cannow be either fully protonated or ε-protonated, depending onthe pH and the particular protein variant. The highestprobability of occurrence of the fully protonated His260 isfor IFP2.0 and the lowest for IFP1.4. Moreover, with thechromophore fixed to its B-deprotonated form, His260 displaysa highly irregular titration behavior, for which a single pKa valuecannot be defined, as seen from the titration curve (Figure S8).Another interesting residue is His207, because the D207H

mutation in DrCBD results in the fluorescent IFP1.0. His207exhibits a pKa value between 4 and 6 depending on the protein

variant and the protonation state of the chromophore. It is hasalready been shown for IFP1.43 that fluorescence ceases below4 but reaches its maximum at around pH 6, and this has beenconfirmed for IFP1.1, IFP1.2, and IFP2.0 (Figure S3). Therange of pH values between these two values is associated witha sharp increase in fluorescence. Therefore, the protonationstate of His207 must control the fluorescence of thechromophore. Although the protonatable side chain ofHis207 is oriented toward the solvent, its different protonationstates may result in different hydrogen bond networks thatinteract with the chromophore. As we will show in themolecular dynamics section, changing the protonation states ofboth histidines, His260 and His207, leads to differentinteraction patterns between water molecules and the regionof the chromophore-binding pocket.Because some of the proteins, namely, DrCBD and variants

IFP1.0−IFP1.2, occur as dimers, we also performed com-parative electrostatic calculations using the geometries of thecomplete dimers. From these calculations, we conclude that theaddition of the second monomer does not noticeably changethe protonation behavior of the residues in the first monomer.Exceptions are limited to a few residues at the interfacebetween the two monomers (Figures S7 and S8). For example,in the monomer, Glu306 has a high probability of beingprotonated at low pH values, but in the dimer, it always remainsdeprotonated because of interactions with the nearby Arg141from the second monomer. Another example is His138, whichin the dimer becomes ε-protonated, because it interacts closelywith Arg100 from the other monomer.

Molecular Dynamics Simulations. Because of the veryslight differences in the protonation behavior of titratableresidues between the monomers and dimers, we used thegeometries of the monomers for our molecular dynamicssimulations. We assumed that changes in protein dynamics dueto the interactions from the additional monomer would not besufficiently significant to justify the use of the complete dimers,which would be computationally more costly. Moreover, thedimerization interface and the region around the chromophore-binding pocket, on which we focus in this study, are located onopposite sides of the monomer.For each of the studied proteins, we performed simulations at

two values of pH, namely pH 4 and 7. Two separate situationswere considered because the fluorescence of IFPs was shown tocease at low pH and reach its maximum near neutral pH, mostlikely because of the ionization of different groups in theprotein. The pH value of a simulation was regulated byadjusting the protonation states of all titratable protein residuesin accordance with the previous electrostatic calculations. Theprotein-bound biliverdin was fixed to be in its fully protonatedform regardless of the pH. However, we have also studied theless likely case in which the chromophore is deprotonated at itsB pyrrole ring (see sections S2−S4 of the SupportingInformation).

Statistical Distribution of the Important Dihedral Angles.During the simulations, we traced the values of the six dihedralangles of the chromophore located between the pyrrole rings(see Figure 3 for the definitions and numbering of thedihedrals). Finally, we calculated and compared statisticaldistributions of the dihedrals in different protein variants.Figure 4 shows the plotted distributions, and Table 3 lists thefitted normal distribution parameters μ and σ, describing themean value of the dihedral and its spread, respectively,calculated from the last 5 ns of the 10 ns molecular dynamics

Figure 3. Close-up of the fully protonated biliverdin chromophore asseen in the binding pocket of DrCBD (crystal structure with addedand geometry-optimized hydrogens). Arrows indicate rotations aroundcentral bonds of the crucial dihedrals that define the planarity andrigidity of the chromophore. Green circles indicate individual pyrrolerings of the chromophore. The pyrrole water is shown in the middle.The dashed line indicates the point of covalent binding of thechromophore to Cys24.

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simulations. A value of μ closer to 0° (or −180° in the case of

the differently defined dihedral 6) indicates a more planar

dihedral, whereas a σ value closer to zero indicates a less

fluctuating and therefore more rigid dihedral. To maximize the

fluorescent properties, both values should ideally be zero (or, in

the case of dihedral 6, μ = −180°). For a comprehensive

analysis of the dynamic behavior of biliverdin in different

proteins, see section S1 of the Supporting Information. In whatfollows, we report only a few key observations.Overall, it is dihedral angles 1 and 2, closest to the covalent

bond between the chromophore and Cys24, that show themost consistent variation of planarity among the members ofthe family of proteins. At neutral pH, dihedral 2 in DrCBD istilted, on average, by ∼13° and becomes more planar in thefluorescent proteins until reaching 2° in IFP2.0. A similar,

Figure 4. Statistical distributions of the six dihedral angles of the chromophore for all proteins collected from the last 5 ns of the 10 ns moleculardynamics simulations. The simulations were performed at both neutral and acidic pH, and the chromophore was kept in its fully protonated form.See Figure 3 for the definitions of the dihedrals.

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although less pronounced, trend is seen for dihedral 1. Theobserved behavior of the average values of the other dihedrals isin general less systematic.Under the low-pH conditions, the chromophore dihedral

angles are, on balance, more tilted. The latter is especiallyevident for dihedral angles 1 and 2, for proteins DrCBD,IFP1.4, and IFP2.0. Otherwise, the differences in chromophoredynamics between the two pH situations are less straightfor-ward to distinguish, as seen from Figure 4 and Table 3. Finally,we note that there do not seem to be marked differences in therigidities of any of the dihedrals at either pH, except for acouple of extreme cases in the simulation under acidicconditions.Analysis of Interactions in the Cavity of the Chromo-

phore. In the previous section, we focused on the intrinsicdynamics of the chromophore molecule, embedded in theprotein. In this section, we analyze the interactions between thechromophore and its protein surrounding, with specialemphasis on the dynamics of water molecules in the vicinityof the chromophore, in particular the pyrrole water. We shallrestrict ourselves to the analysis of the protein models based oncrystal structures, namely, DrCBD, IFP1.0, IFP1.4, and IFP2.0,in the situation at neutral pH and with the fully protonatedchromophore, representing physiologically relevant conditions.Additionally, in section S4 of the Supporting Information, wediscuss a hypothetical scenario in which at neutral pH thechromophore is deprotonated at the B pyrrole ring.In the following, we focus on events that occur in the second

halves of each simulation, as these better represent theequilibrated state of the protein. However, we do also discussbriefly the geometrical changes that occur for each protein withrespect to their starting X-ray structures.The crystallographic structure of DrCBD shows a water

molecule locked by hydrogen bonds among rings A−C of thechromophore (Figure 5). During the equilibration stage, thispyrrole water moves away from its initial position and forms abridge between the carboxylic group of Asp207 and thecarbonyl oxygen of the A chromophore ring. The pyrrole water

shows recurring moves outward toward the solvent and inwardtoward the chromophore. These moves are possible, becausethe side chains of Asp207 and Tyr263 have moved away fromthe protein and point toward the solvent. In particular, the sidechain of Tyr263 shows a noticeable flexibility and can close thepocket of the chromophore for short periods. A network ofhydrogen bonds is seen connecting the inner pyrrole water,solvent waters, Asp207, and the carboxyl moiety of ring A. Thisnetwork allows the pyrrole water to leave its starting locationbetween the pyrrole rings and be replaced by another watermolecule from the solvent. Such water replacements recurduring the rest of the simulation. Nevertheless, the regionbetween the pyrrole rings is always occupied by at least onewater molecule connected to the outer solvent through a bridgeof hydrogen bonds with other water molecules. During thesimulation, a hydrogen bond is seen between the δ-nitrogen ofHis260 and the H_C hydrogen atom of ring A (see section S4of the Supporting Information for the labeling of the keychromophore atoms). The average distance between these twoatoms is 2.5 Å. The presence of the H_C···ε-nitrogen bondindicates a displacement of rings A and B from planarity.

Table 3. Distributions of the Six Dihedral Angles of the Chromophore, Collected from the Final 5 ns of the 10 ns MolecularDynamics Simulationsa

dihedral 1 dihedral 2 dihedral 3 dihedral 4 dihedral 5 dihedral 6

μ σ μ σ μ σ μ σ μ σ μ σ

(a) Neutral pHDrCBD 12.0 8.9 13.1 9.4 4.1 7.7 7.1 8.3 15.2 9.7 −139.2 8.5IFP1.0 11.1 8.2 9.6 9.0 1.7 8.2 5.9 8.0 9.0 9.5 −136.7 8.3IFP1.1 11.1 7.3 13.0 8.1 8.6 7.5 3.0 7.7 8.2 8.8 −131.4 8.2IFP1.2 11.4 7.3 13.5 8.5 7.8 7.5 3.4 7.8 7.7 8.8 −132.7 8.2IFP1.4 6.8 7.7 3.1 8.5 1.8 8.5 6.2 8.2 10.7 9.0 −137.3 8.6IFP2.0 7.7 7.5 2.0 8.2 7.1 8.8 9.6 8.0 13.6 9.0 −132.9 9.3

(b) Low pHDrCBD 14.6 6.9 15.9 8.1 4.8 7.5 7.3 7.9 18.7 9.2 −148.8 12.5IFP1.0 15.9 8.2 8.2 8.9 −8.1 8.5 5.6 8.9 14.2 9.1 −140.4 8.9IFP1.1 5.2 8.1 8.7 9.3 9.4 7.7 10.1 7.9 13.4 8.8 −130.9 8.4IFP1.2 6.8 7.5 7.8 8.5 5.6 8.9 2.2 8.6 8.2 8.9 −132.8 8.6IFP1.4 6.9 7.6 6.0 8.2 7.8 8.3 9.9 8.1 14.5 8.9 −133.9 8.0IFP2.0 10.7 7.7 4.3 8.7 −0.8 12.5 2.3 9.1 10.9 9.4 −136.8 9.0

aThe simulations were conducted at low or neutral pH. At both pH values, the chromophore was fixed to be fully protonated whereas the proteinresidues were protonated according to the results of continuum electrostatic calculations. The planarity and rigidity of the chromophore are definedby the normal distribution parameters, mean (μ) and variance (σ). For a given dihedral, a μ value closer to 0° (or −180° for dihedral 6) indicatesthat the fragment of the chromophore encompassing this dihedral is more planar. A lower value of σ indicates improved rigidity of the fragment.Bold font indicates a more favorable value, i.e., a more planar or rigid dihedral. See Figure 3 for the numbering of the dihedrals.

Figure 5. Environment of the so-called pyrrole water in IFP1.0. Thefour different rings of the chromophore are labeled A−D.

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In summary, the following events seem to be important inthe dynamics of water molecules in DrCBD. The side chains ofAsp207 and Tyr263 that make up the entrance to the cavity ofthe chromophore are quite flexible. The pyrrole water can bedisplaced from its crystallographic position and showsreasonable mobility inside the cavity. This water forms transienthydrogen bonds to His260 or to the carbonyl carbon atom atring A. At the same time, it maintains contact with other watermolecules from the bulk solvent through a chain of hydrogenbonds. Some solvent waters can find themselves between thepyrrole water and the carbonyl oxygen of the protein backboneat Asp207.To improve our understanding of water exchange between

the chromophore region and the solvent, we traced the paths ofindividual water molecules throughout the simulations. As seenin Figure 6a, DrCBD shows the highest mobility of watersamong the four analyzed proteins. The rather intensive waterexchange between DrCBD and the solvent is consistent withprevious experimental data for a variant of the bacteriophyto-chrome, whose crystal structure does not have the pyrrole waterbetween the rings of the chromophore.42 We note, however,that the structure with the absent pyrrole water was determinedat a significantly lower resolution of 2.2 Å. Moreover, in arecent study by Forest and co-workers, it was demonstratedthat the fluorescence can be improved after water moleculeshave been restricted from the vicinity of the chromophore.43

We next analyze the water migration in IFP1.0, which is thefirst protein in a series of fluorescent mutants of DrCBD. Thedifference between DrCBD and IFP1.0 is a single mutation ofAsp207 to histidine. His207 has a bulkier side chain that moreeffectively blocks access of water molecules from the solvent tothe cavity of the chromophore.In the crystallographic structure, a hydrogen bond is seen

between the carbonyl oxygen of chromophore ring A and the δ-hydrogen of His207. However, this bond easily breaks in theequilibrated structure, and His207 gains more flexibility. TheHis207 side chain rotates by 180° relative to its startingorientation, and the δ-hydrogen points toward the solvent. Thisnew orientation of His207 is maintained throughout the rest ofthe simulation. Tyr263 is less flexible than His207, and itsorientation does not generally change in IFP1.0. Both His207and Tyr263 seem to restrict interactions between the pyrrolewater and the outer waters. The pyrrole water in IFP1.0 islocked among rings A−C of the chromophore and forms twohydrogen bonds, first to the ε-nitrogen of His260 and secondto the carbonyl oxygen of ring A. These bonds remain intactuntil the end of the simulation.We do not observe any water exchange between the cavity

formed by the pyrrole rings and the outer solvent, and thepyrrole water is trapped between the rings (Figure 6b). Becausethe pyrrole water in IFP1.0 is practically locked at its initialposition, we also do not observe the hydrogen bond betweenthe δ-nitrogen of His260 and the H_C hydrogen atom of ring

Figure 6. Mobility of water molecules inside the chromophore-binding pocket in different proteins at pH 7 with the fully protonated chromophore.The binding pocket was defined as a region within 6 Å of the geometric center of the chromophore. Orange dots represent positions of watermolecules collected from a 10 ns simulation of each protein. The chromophore and its neighboring residues, Asp/His/Thr207 and His260, areshown in a ball-and-stick representation. The DrCBD protein shows both the highest mobility and level of exchange of water molecules with thesolvent. In IFP1.0 and IFP1.4, the pyrrole water is locked between the inner rings of the chromophore, because the bulky side chain of His207restricts the access of solvent waters to the chromophore-binding pocket. In IFP2.0, the mobility and exchange of water are nearly as intensive as inDrCBD, but the compact side chain of Thr207 does not destabilize the planar conformation of rings A and B of the chromophore, as the imidazoleof His207 does in IFP1.0 and IFP1.4.

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A. This bond would displace the chromophore out of planarity,as seen during the simulation of DrCBD. Hence, the fragmentof the chromophore involving rings A and B is noticeably moreplanar and stable than in DrCBD.IFP1.4 is a significantly modified protein compared to the

parent protein (see Figure 1 for the directed evolution of IFPs).From the perspective of our work, the key structural differencesare mutations D207H, I208T, A288V, and V186M, becausethese occur in the direct vicinity of the chromophore andtherefore may most influence its dynamics.The geometry of IFP1.4 at the starting point of the

simulation resembles that of IFP1.0. Interestingly, throughoutthe simulation, His207 is somewhat more flexibile than inIFP1.0 and rotations of its side chain recur several times.During these rotations, the hydrogen bond between the δ-nitrogen of His207 and the carbonyl oxygen of ring A isrestored for short periods. On the basis of the simulations, wepropose that the bulky imidazoline group of His207 may havetwo opposing effects on the fluorescence of IFP1.4. On onehand, His207 restricts access of water molecules to the area ofthe chromophore. On the other hand, frequent rotation of theHis207 side chain may distort the otherwise planar geometry ofthe A and B pyrrole rings. However, the net effect of His207 inIFP1.4 seems to be beneficial for fluorescence.The pyrrole water is again seen trapped between the pyrrole

rings and isolated from the bulk solvent by Tyr263 and His207.Unlike in DrCBD, it is never substituted with nearby solventwaters. In the rotated conformation, the δ-hydrogen of His207forms a hydrogen bond to the carbonyl oxygen of the proteinbackbone at Pro204. The presence of the pyrrole waterbetween His260 and rings A and B prevents the formation ofthe His260···ring A hydrogen bond, which displaces these ringsout of planarity as seen in the simulations of DrCBD. However,because of the increased extent of rotation of the His207 sidechain, the pyrrole water is slightly more mobile inside the cavityof the chromophore. We also note that the tyrosyl group ofTyr263, which was quite flexible in the parent protein, adopts arather fixed conformation in IFP1.4. The exchange of watermolecules between the solvent and the protein, which was seenin DrCBD, does not generally occur here.Finally, IFP2.0 introduces a number of additional mutations

into IFP1.4 as well as reverse mutations (see Figure 1). The keychange that may have significant consequences on themigration of water molecules is the replacement of His207with threonine.The simulation of IFP2.0 shows a cycle in which the pyrrole

water escapes the pocket of the chromophore, a new pyrrolewater arrives after a short period, followed by the formation ofan extensive network of hydrogen bonds between the newpyrrole water and the solvent, and finally the removal of solventwaters from the pocket leaves only one water bound to thechromophore.At the beginning of the cycle, the pyrrole water is seen

among rings A−C of the chromophore, similar to the situationin IFP1.0 and IFP1.4. The inner hydrogen atoms of these ringsinteract with both the oxygen atom of the pyrrole water and thecarbonyl oxygen of the protein backbone at Thr207. Ahydrogen bond is established between His260 and one of thepyrrole water’s hydrogens. The orientation of Tyr263 isreminiscent of that in the previous proteins. The Tyr263 sidechain acts as an obstacle between the protein and the solvent.However, Tyr263 shows some degree of flexibility, which wasnot the case in IFP1.0 and IFP1.4. The tyrosyl group of Tyr263

can be displaced from its initial position where it could blocksolvent molecules from entering the cavity.Interestingly, such a brief displacement of Tyr263 in IFP2.0

can trigger the pyrrole water to escape from the cavity of thechromophore. Within <1 ns, a water molecule from the solvententers the empty cavity. The entry of a new pyrrole water isexpected, because empty cavities inside the protein cannot bestable for long periods. The new molecule adopts the sameorientation inside the cavity as the original molecule. After thearrival of the new pyrrole water, a hydrogen-bonded network ofwater molecules starts to form in the vicinity of rings C and D(Figure 6d). The side chain of Tyr263 moves farther from theentry to the cavity, which is now occupied by several watermolecules. We observe a rather intensive replacement of watermolecules within the newly formed network, including thepyrrole water, which is substituted several times during thesimulation. Approximately 2 ns after the formation of the newnetwork, the side chain of Tyr263 moves back to its initialposition. One water molecule is again trapped inside the cavity,and its contact with the solvent is now blocked.We note that during all observed events, the side chain of

Thr207 remains in its initial orientation. In this orientation, thehydroxyl group of Thr207 is oriented toward the proteinbackbone at Pro204 and the methyl group of threonine findsitself at the entry to the cavity between the pyrrole rings.Considering the fluorescence of the chromophore, the stabilityof Thr207 in IFP2.0 may be advantageous compared to that ofHis207 in IFP1.0 and IFP1.4, because frequent rotation of thehistidine side chain displaces rings A and B of the chromophoreout of planarity. Although the side chain of threonine is not asbulky as the side chain of histidine, because of its hydrophobicmethyl group, it may still limit access of solvent molecules tothe protein.In light of the previous simulations of IFP1.0 and IFP1.4, the

dynamics of water molecules in IFP2.0 is unexpected. Namely,IFP2.0, which is the most fluorescent protein, shows a ratherintensive exchange of water in the vicinity of the chromophore.In IFP1.0 and IFP1.4, water migration was not seen and thepyrrole water was permanently locked in the cavity of thechromophore. Given these observations, we propose that atleast two criteria have to be met to maximize the fluorescentproperties of a DrCBD-based protein. One is to reduce thelevel of migration of water molecules between the protein andthe solvent, which seems beneficial in proteins such as IFP1.0and IFP1.4. These proteins possess a bulky histidine residue atthe entry to the chromophore’s cavity. Water molecules areinvolved in rapidly changing networks of hydrogen bonds, andby interacting with the chromophore, they can displace it out ofplanarity. The second is to limit movement of the region of thechromopore comprising rings A and B. Threonine in place ofHis207 is better suited for this function, because it does notperform frequent rotation and its interaction with thechromophore is weaker. An ideal IFP protein would combinethe ability of IFP1.0 and IFP1.4 to restrict solvent moleculesfrom the chromophore with the ability of IFP2.0 to avoid thedistortion of rings A and B of the chromophore.

Stability of the Pyrrole Water in Different Simulations. Inthe last section, we summarized the dynamic behavior and fateof the pyrrole water in different proteins: DrCBD, IFP1.0,IFP1.4, and IFP2.0. As mentioned previously, the most water-accessible cavity of the chromophore is observed in DrCBD,followed by, unexpectedly, IFP2.0. The simulations suggest thatthe pyrrole water in DrCBD shows a high degree of mobility

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and can easily be replaced by solvent waters. Such replacementsrecur frequently throughout the simulation. Interestingly,IFP2.0 shows a limited migration of water during the firsthalf of the simulation. The pyrrole water is displaced from itsstarting position, and the cavity remains unoccupied for a shortperiod. However, in the second half of the simulation, theexchange of water molecules in IFP2.0 becomes nearly asintensive as in DrCBD. This observation suggests that limitingaccess of water molecules to the chromophore, which proved tobe successful in the development of Wi-Phy2 through theinsertion of a hydrophobic residue in the vicinity to thechromophore,43 may not be the only way to improve thefluorescent properties in IFPs. In IFP1.0, the bulky side chain ofHis207 blocks access of solvent waters to the protein, resultingin the pyrrole water being permanently trapped in the cavity. InIFP1.4, the His207 side chain is shown to be somewhat moreflexible than in IFP1.0. Nonetheless, the pyrrole water is againlocked inside the cavity of the chromophore, and no exchangeof water molecules is observed between the protein and thesolvent. On the basis of the simulations, we note that theflexibility of the Tyr263 side chain is also important for themigration of water molecules in IFPs.

■ CONCLUSIONSIn the work presented here, we have employed differentexperimental (X-ray crystallography) and modeling (electro-static calculations and molecular dynamics simulations)techniques to identify structural determinants of fluorescencein a series of infrared fluorescent proteins derived from the D.radiodurans phytochrome. As a basis for our study, we used anewly determined very high-resolution structure of IFP1.4,together with published high-resolution structures of othermutants in the family. We note that the collective effect on thefluorescence resulting from the introduced mutations cannot bepartitioned into individual contributions. Nonetheless, on thebasis of our crystal structure and modeling, we were able to gainsome insight into the reasons for the increase in fluorescence inthe studied series of IFPs.The simulations performed for different proteins suggest that

the gradual enhancement of fluorescent properties is likely tooriginate from the improved planarity of the chromophore. Onthe basis of the simulations, we propose that water moleculesentering the protein from the solvent are the main cause of theincreased level of motion of the chromophore. Theconformations of pyrrole rings A and B, adjacent to thecovalent linkage site, were shown to be most susceptible to themotion of water molecules. These waters destabilize theotherwise rigid and planar conformation of the chromophoreby forming different hydrogen bond networks between thechromophore and its molecular surroundings. The weaklyfluorescent DrCBD shows the most water-accessible cavity ofthe chromophore. In IFP1.0 and IFP1.4, the level of migrationof water molecules is significantly reduced. The structurallyimportant pyrrole water, which is normally located between thepyrrole rings of the chromophore, was found to be noticeablymore stable in these proteins than in DrCBD. However, theimidazoline group of His207 forms a transient hydrogen bondto the A pyrrole ring. The presence of this bond as well asfrequent rotation of the His207 side chain may have adestabilizing effect on the planarity of the chromophore.Interestingly, IFP2.0, which is the most fluorescent protein inthe studied family of IFPs, shows a rather intensive exchange ofwater between the protein and the solvent, second only to

DrCBD. On the other hand, the H207T mutation in IFP2.0 ishighly beneficial, because the compact side chain of threonineinteracts only weakly with the chromophore, which helpsmaintain the A and B pyrrole rings in a more planar orientation.Finally, it is conceivable that monomer−monomer inter-

actions within the dimer formed by certain mutants control thefluorescence efficiency to some extent,10,44 and future studiesshould focus on this specific aspect.In an attempt to capture the effect of pH on fluorescence, we

performed simulations at both low and neutral pH values. Ourmolecular dynamics simulations show that the dynamics of thechromophore is affected by a significant acidification of themedium. In the situation at low pH, the chromophore appearsto be generally more flexible and distorted than at neutral pH.However, the observed differences in the chromophoredynamics in relation to pH are rather small. Thus, it seemslikely that effects other than structural effects also contribute tothe loss of fluorescence at low pH, for example, proteindenaturation or an increased level of excited state protontransfer.45

Overall, our work is an initial attempt to provide a frameworkfor probing and improving the fluorescent properties ofproteins containing biliverdin, or another bilin, as achromophore.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.bio-chem.6b00295.

Sequence alignment, comparison of the chromophoreenvironment in DrCBD and IFP1.0, experimental pHdependence of infrared fluorescence, labeling of theimportant atoms of the chromophore, time evolution ofthe rmsd of the chromophore atoms, titration curves ofHis260, Asp207, His207, and protonatable groups at theinterface between the monomers, energy profiles usedduring the parametrization procedure, topology andparameter files for the B-deprotonated form of thechromophore, detailed discussion of the statisticaldistributions of the important dihedral angles, discussionof the gas-phase calculations, parametrization procedurefor the B-deprotonated chromophore, and analysis of thesimulations with the B-deprotonated chromophore atneutral pH (PDF)Movies showing water mobility during the simulations ofthe parent (AVI), IFP1.4 (AVI), and IFP2.0 (AVI)proteins.

■ AUTHOR INFORMATION

Corresponding Authors*E-mail: antoine.royant@ibs.fr.*E-mail: martin.field@ibs.fr.

FundingA. Royant, M. Feliks, and M. Field acknowledge financialsupport from the French National Research Agency (GrantsANR-11-JSV5-0009-01 and ANR-11-BSV5-0012).

NotesThe authors declare no competing financial interest.

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■ ACKNOWLEDGMENTS

The authors thank the two reviewers for valuable commentsthat helped to improve the manuscript.

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Biochemistry Article

DOI: 10.1021/acs.biochem.6b00295Biochemistry 2016, 55, 4263−4274

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