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Quantum chemical modeling of structure and absorption spectra of the chromophore in green fluorescent proteins
Chemical Physics 231 Ž1998. 13–25
Quantum chemical modeling of structure and absorption spectra of the chromophore in green fluorescent proteins Alexander A. Voityuk, Maria-Elisabeth Michel-Beyerle, Notker Rosch ¨
)
Institut fur D-85747 Garching, Germany ¨ Physikalische and Theoretische Chemie, Technische UniÕersitat ¨ Munchen, ¨ Received 6 February 1998
Abstract As a first step towards modeling the green fluorescent protein ŽGFP., we have carried out absorption spectra calculations on chromophores of both native and mutant proteins using the semiempirical method INDOrS. A number of protonated and deprotonated states of the GFP fluorophore were considered. We find predicted and observed absorbance energies in very good agreement. Based on a comparison of calculated and experimental absorption spectra, we suggest structures for the ground and excited states of the chromophore of GFP. We assign the absorption maximum of GFP at 477 nm to an H-bonded complex of the zwitterion ŽO Y , HN, O X . involving the phenolic oxygen of Tyr66 and its environment; the nitrogen of the heterocyclic ring is protonated in this complex. Another peak at 397 nm is due to excitation of the corresponding protonated form ŽHO Y , HN, O X .q. Calculated transitions in the mutant chromophores ŽTyr66™ Phe., ŽTyr66 ™ Trp., and ŽTyr66 ™ His. at 355, 433, and 387 nm, respectively, are close to the corresponding experimental values of 360, 436, and 382 nm, suggesting cationic forms with the nitrogen protonated to be responsible for the absorbance. Proton transfer to or from the phenolic hydroxyl group of the chromophore is shown to be crucial for understanding the absorption and emission spectra of GFP. q 1998 Elsevier Science B.V. All rights reserved.
1. Introduction The green fluorescent protein ŽGFP. and its mutants are the subject of widespread interest, in particular with respect to their applications in molecular biology and biotechnology. Since the fluorescence of GFP occurs in the absence of any cofactors, the protein can serve as a revolutionary marker of gene expression and as a tag for localizing proteins within living cells w1–5x. The DNA sequence coding for GFP can be fused to that of any protein of interest w1,3,6,7x. By expression of the modified gene, the )
Corresponding author.
protein is covalently linked to GFP and thus carries a fluorescent label. GFP was first described by Shimomura and Johnson in 1962 w8x. The cloning of cDNA and the determination of the primary structure of GFP by Ward, Prendergast and colleagues was a crucial step in the study of this protein w9x. GFP consists of 238 amino acids. The conformation of GFP is extremely stable and denaturation of the protein occurs only under harsh conditions w10x. The chemical structure of the GFP fluorophore was established in a landmark study w11x. The visible absorbance and fluorescence of the protein were assigned to a p-hydroxybenzylidene-imidazolidinone chromophore. This flu-
0301-0104r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 0 1 0 4 Ž 9 8 . 0 0 0 8 0 - 9
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A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
orophore is formed by post-translational cyclization of the polypeptide backbone between residues Ser65, Tyr66, and Gly67, and by a ,b-dehydrogenation of Tyr66 w11x. The autocatalytic mechanism of the fluorophore formation is not yet understood. ˚. Recently, a number of highly resolved Ž; 1.9 A crystal structures on GFP have been reported: those of the wild-type GFP dimer w12x and monomer w13x as well as those of several GFP mutants w14x, w15x in which either the chromophore orrand its environment have been altered. All these proteins exhibit a rather regular cylindrical shape formed by 11 bstrands. This ‘barrel’, capped on both top and bot˚ and a length of tom, has an outer diameter of ; 30 A ˚ The chromophore is located nearly in the ; 40 A. center of the barrel and is completely protected from the bulk solvent. Modeling of the residues 57–71 led to the conclusion that there is a strain between the central a-helix and the b-barrel w15x. The immediate environment of the fluorophore includes several water molecules as well as charged and polar amino acid residues. Hydrogen bonds between the chromophore and its surroundings in GFP seem to play an important role w12–15x. According to the X-ray data, a number of proton donorracceptor centers are found in the vicinity of the chromophore ŽFig. 1.. Two residues, His148 and Thr203, and a water molecule interact with the phenolic hydroxyl group HO Y of Tyr66 ŽHO Y , N, O X . and may cause its deprotonation. Arg96 and Gln94 form hydrogen bonds with the carbonyl oxygen of the heterocyclic ring ŽO X .. Similar to His, the heterocyclic ring of the chromophore may be positively charged or neutral depending on its local environment and on the electrostatic potential created by the protein. The protonated nitrogen of the ring can be stabilized by the hydroxyl group of Ser65, the carboxyl group of Glu222, or water molecules located in the vicinity. According to X-ray data, the wild-type protein and the S65T mutant have different hydrogen bonding networks between the chromophore and its environment w13–15x. It should be noted that in the case of ionic H bonds, wX–H–Yxq between neutral units X and Y, or wX–H–Yxy between negatively charged units Xy and Yy, the equilibrium position of the proton can lie midway between X and Y w16x; however, the proton location is considerably affected by the environment. Recently, it has been shown that
Fig. 1. The chromophore and its environment in GFP based on the X-ray structure of GFP determined in Ref. w12x.
proton transfer in systems with ionic H bonds proceeds with very low activation barriers w17,18x. The absorption spectra of the native protein exhibit two excitation peaks w1–3x at 395 and 475 nm Ž397 and 477 nm according to the latest experiment w19,20x.. Based on the spectroscopic data it was proposed that the wild-type protein exists in two different forms: a dominant one ŽA. which is responsible for the absorption at 395 nm and another stable form ŽB. featuring the 475 absorption peak w1,3,5,10,19x. Recently, structural studies of GFP and its mutants have shown that in fact there exist two different stable conformations of GFP w13,15x; the equilibrium between these states seems to be governed by an H-bond network. Upon denaturation, the spectrum of GFP undergoes large changes w10,21x which clearly indicate that there also exist different forms of the chromophore in solution. Mutations of the fluorophore and its environment are also found to have a substantial effect on the absorption and fluorescence spectra w1–3,22–24x. Spectroscopic properties of molecular systems are known to be changed significantly by proton transfer w25–28x. Steady-state and picosecond time-resolved spectroscopy has re-
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
cently been applied to study native and mutant GFP w19,20x. It was shown that excited state proton transfer plays a key role in GFP emission w19,20,29x. Optical studies of individual GFP molecules have been recently reported w30x. Since the chromophore in GFP is completely protected from bulk water w12–14x p K a values of its subunits cannot be used to predict which center is protonated within the protein w31x. Moreover, low activation barriers for the proton transfer between the chromophore and its surroundings can lead to rapid changes between different states. Therefore, to assign spectroscopic features of GFP and its mutants, detailed knowledge on the electronic structure and spectra of the chromophore tautomers is needed. Despite strong experimental efforts, several important questions have yet to be answered: Which forms of the chromophore are responsible for the absorption of native and mutant GFP? Which geometrical structures correspond to these states? How are absorption spectra affected by mutation of amino acids either involved in the chromophore or located in its immediate vicinity? The goal of the present study is to answer these questions using quantum chemical calculations.
2. Models The chromophore of GFP and its environment are shown in Fig. 1. As a model we will consider a system containing a heterocyclic ring composed of the three amino acid residues Ser–Tyr–Gly. To cut the connection to the backbone of the protein, the amino group of Ser and the carboxyl group of Gly are substituted by hydrogen atoms. As mentioned above, it is difficult to predict which centers of the chromophore are protonated in
15
Scheme 2.
the protein; therefore, various possibilities should be taken into account. In principle, the following eight structures of the chromophore can exist in GFP. In the first structure, anion ŽO Y , N, O X .y, three atoms, O Y , N and O X can accept a proton from the environment Žsee arrows in Scheme 1.. Protonation of these centers in the anion results in the three neutral structures ŽHO Y , N, O X ., ŽO Y , HN, O X ., and ŽO Y , N, HO X ., respectively. Further protonation of these neutral species leads to the three cationic systems ŽHO Y , HN, O X .q, ŽHO Y , N, HO X .q, and ŽO Y , HN, HO X .q. Finally, protonation of all three c e n te r s in th e c h r o m o p h o r e y ie ld s ŽHO Y , HN, HO X . 2q. Besides the wild-type ŽWT. chromophore, we studied also three of its mutants. The mutations of tyrosine ŽTyr. to phenylalanine ŽPhe., tryptophan ŽTrp., or histidine ŽHis. in the chromophore were modeled by the systems shown in Scheme 2.
3. Method
Scheme 1.
The absorption spectra of the model systems were calculated with the spectroscopic INDO scheme, INDOrS, w32,33x as implemented in the program ZINDO w34x. The INDOrS technique has been shown
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A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
constraints using the method PM3 w39x implemented in the program SIBIQ w40x. For comparison, AM1 w41x calculations for some systems were also performed. The PM3 and AM1 methods are well-established semiempirical tools to calculate formation enthalpies and ground-state structures of organic species: average absolute errors are about 4.5 ˚ for bond kcalrmol for formation enthalpies, 0.02 A lengths, and 38 for angles w42x.
4. Geometries and relative stability
Fig. 2. Model for the hydrated anionic chromophore.
to be very successful in simulating spectroscopic properties of large organic compounds and biomolecules w35,36x. Standard parameters were used: two-center two-electron integrals were calculated using the Mataga–Nishimoto formula and the resonance integrals were computed with the scaling factors f s s 1.0 and f p s 0.585 w32x. An INDOrS calculation consists of two parts: a ground-state SCF calculation is followed by a configuration interaction calculation which yields excitation energies and oscillator strengths Žcomputed in the one-center approximation using the dipole length operator.. All single excitations from the 10 highest occupied molecular orbitals to the 20 lowest virtual orbitals were included in the configuration interaction. The effect of the environment on the spectra was estimated within a model w37x based on the self-consistent reaction field approach w38x. Since a straightforward polarizable-continuum model can lead to considerable errors for a system interacting with its environment via hydrogen bonds w37x, the chromophore in water was modeled by an ‘embedded cluster’ containing the chromophore and 6 water molecules Žtwo of them bound to each of the three centers O Y , O X , and N, Fig. 2.. We optimized the geometry of the various model systems without any
The calculated structures of the anion ŽO Y , N, O X .y ŽScheme 2. exhibit two low-energy conformations, with and without an intramolecular hydrogen bond between the hydroxyl group of Ser65 and N of the heterocyclic ring. The structure with the H bond is slightly more stable Žby 0.5 kcalrmol. than the other conformer. The phenol ring of Tyr and the heterocycle lie in the same plane. ŽIt is convenient to characterize the planarity of chromophores under consideration by the dihedral angle d s /C 1C 2 C 3 C 4 ; see Scheme 3.. Of the three neutral tautomers formed by protonation of the anionic form, the tautomer ŽHO Y , N, O X ,. was calculated to be ; 18 kcalrmol more stable than the zwitterion ŽO Y , HN, O X . which in turn is only slightly more stable, by 0.7 kcalrmol, than ŽO Y , N, HO X .. Similar relative energies of these three tautomers are predicted by the semiempirical method AM1. In the gas phase, zwitterions are known to be less stable than conventional forms; however, in a polar environment, zwitterions become much more favored w43x. Since ŽO Y , HN, O X . should exist in mutant GFP, as we will discuss below, the energy difference between ŽHO Y , N, O X . and the zwitterion should be recovered by environment effects, e.g. by specific interactions due to H bonds and by the electrostatic potential generated by the entire protein.
Scheme 3.
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
The structures of ŽO Y , N, HO X . and ŽO Y , HN, O X . are essentially planar, d F 18. While the lowest-energy tautomer ŽHO Y , N, O X . has a nonplanar geometry, d s 268, this conformation is only 0.2 kcalrmol more stable than the corresponding planar structure. Using X-ray atomic coordinates w12,14x we found that the chromophore in GFP is essentially planar: d s 0.58 and d s 3.28 in wild-type GFP and in the mutant S65T, respectively. Thus, a planar structure of the chromophore was also adopted for ŽHO Y , N, O X .. PM3 predicts ŽHO Y , HN, O X .q to be the most stable tautomer of the three positively charged species obtained by double protonation of the anionic chromophore; the relative energies for ŽHO Y , N, HO X .q and ŽO Y , HN, HO X .q are 15.7 and 22.8 kcalrmol, respectively. As for the corresponding neutral species, the m ost stable cationic form , ŽHO Y , HN, O X .q is not planar Ž d s 118. while ŽHO Y , N, HO X .q and ŽO Y , HN, HO X .q exhibit essentially planar structures. The formation of ŽHO Y , HO X , HN. 2q in GFP appears to be unlikely because of its relatively small proton affinity Ž132 kcalrmol according to PM3.; thus, proton transfer from this form to a water molecule is exothermic with a reaction enthalpy of about y23 kcalrmol. Substitution of Tyr66 by Phe results in the mutant Tyr ™ Phe ŽScheme 2.. A nonplanar structure with d s 318 is calculated for this neutral chromophore by PM3. This mutant exhibits two protonated forms, ŽHN, O X .q and ŽN, HO X .q, both essentially planar; the tautomer ŽHN, O X .q is 15 kcalrmol more stable than ŽN, HO X .q. The mutant chromophore Tyr ™ Trp ŽScheme 2. has a more extended p-electron system and its neutral as well as its N- and O X -protonated forms are all almost planar. As expected, the lowest-energy tautomer is ŽHN, O X .q, lying 12 kcalrmol below its counterpart ŽN, HO X .q. The two mutations discussed so far reduce the number of relevant forms of the chromophore. By contrast, the mutation Tyr ™ His ŽScheme 2. increases the number of possible protonated species to be considered. Indeed, the imidazole ring of His features two electron donor atoms, Nd and N´ , that can be protonated whereas Tyr possesses only one such center, O Y . Two neutral and five positively charged protonated species are essentially planar; the deviation of the dihedral angle from planarity is less than 28. Accord-
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ing to PM3 as well as AM1 data, the centers Nd and N´ in His have very similar proton affinities and, consequently, the corresponding tautomers are almost equally stable Žwith heats of formation within 2 kcalrmol.. For example, the neutral species ŽNd , HN´ , N, O X . is 1.5 kcalrmol more stable than ŽHNd , N´ , N, O X .. Protonation of these forms leads to th re e sp e c ie s o f sim ila r e n e rg ie s, ŽNd , HN´ , HN, O X .q, ŽHNd , N´ , HN, O X .q, and ŽHNd , HN´ , N, O X .q, with relative energies of 0.0, 0.9, and 3.8 kcalrmol, respectively. Tautomers with th e c a rb o n y l o x y g e n p ro to n a te d , e .g . ŽNd , HN´ , N, HO X .q, are of considerably higher energy Žby ; 15 kcalrmol..
5. Absorption spectra As found experimentally w3,10,19,20,23,24x, the chromophore in GFP can be characterized by the position and intensity of the long-wavelength absorption band which can be altered considerably depending on pH, solvent, mutations, etc. For simulated absorption spectra of various forms of the chromophore we found only one strong absorption band in the visible region, well separated from other peaks. The maximum exhibits p ™ p ) character and arises due to an almost pure HOMO™ LUMO transition. The next higher excitation peaks lie in the UV region. 5.1. Chromophore in aqueous solution We start by considering the spectra for denatured GFP. The relevant state of the chromophore in aqueous solution is well determined by the pH of the solution. Upon denaturation, GFP loses its fluorescence and changes its absorption spectrum w10–12x. Native GFP has absorption maxima at 397 and 477 nm. Its denaturation by treatment with acid ŽpH - 4. results in an absorption peak at 383–384 nm, while protein denaturation with base ŽpH ) 12. leads to a maximum at 447 nm w10x. Since the p K a value of the phenolic hydroxyl of Tyr is ; 10 and the p K a value of imidazole in His is ; 6 w44x, the chromophore exists predominantly as the solvated anion ŽO Y , N, O X .y at pH ) 12 and as the solvated cation ŽHO Y , HN, O X .q at pH - 4. To simulate solvation
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A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
Table 1 Modeling of solvation effects on the chromophore absorption spectrum Basic ŽpH ) 10. ŽO Y , N, O X .y
Acidic ŽpH - 4. ŽHO Y , HN, O X .q
Exp.a
447
384
Models: Ža. Free chromophore Žb. Chromophoreq 6H 2 O Žc. ŽChromophore. solv. Žd. ŽChromophoreq 6H 2 O. solv.
448 443 429 435
401 405 395 397
Solution Species
Transition wavelength l Žin nm. of various models: Ža. free chromophore, Žb. chromophore with two water molecules per center O Y , O X , and N, Žc. same as Ža. with a reaction field cavity Ž ´ s 80., Žd. same as Žb. with a reaction field cavity Žsee text for details.. a Ref. w9x.
in water, calculations were carried out for the three models listed in Table 1. In the free state, i.e. in absence of a solvent, absorption peaks of ŽO Y , N, O X .y and ŽHO Y , HN, O X .q are calculated at 448 and 401 nm, respectively. H bonds in the clusters containing the chromophore and specifically bound water molecules have hardly any effect on the calculated spectra as absorption peaks of the anionic and cationic aqua complexes are shifted by only 4–5 nm relative to the corresponding free forms. The most noticeable alteration of the excitation energy is obtained within the continuum model for the anionic chromophore in polar medium Žwater, ´ s 80.; its excitation maximum is blue-shifted by 19 nm ŽTable 1.. This blue-shift is found to be reduced to 13 nm in the solvated cluster of the anion shown in Fig. 2. The corresponding absorption shifts calculated for the cation amount to only 6 and 4 nm, respectively. Thus, for the solvated anion, our most elaborate model predicts an absorption peak Ž l s 435 nm, f s 1.27. in good agreement with the observed absorption band at l s 447 nm. According to our calculations, the solvated cation has an absorption maximum Ž l s 397 nm, f s 0.85. which corresponds well to the experimental value of l s 384 nm. It is worth noting that calculated solvation effects on transition energies are rather small. 5.2. NatiÕe GFP chromophore One of the important questions open so far is how protonation of the chromophore affects the absorption spectrum. Table 2 lists the calculated absorption
energies and oscillator strengths of various protonated forms of the WT-chromophore. From these data one deduces that all three types of protonation of the anionic chromophore dramatically change the absorption energy. The most significant alteration in the spectrum, a blue-shift by more than 100 nm and a remarkable reduction of the absorption intensity, is induced by protonation of the phenolic oxygen O Y . As mentioned in Section 4, the dihedral angle between both rings in the chromophore can be changed. According to our calculation, a distortion of the planar structure of the chromophore results in a significant reduction of the long-wavelength absorption intensity, for example, for ŽHO Y , N, O X . we found the oscillator strengths to be 1.05, 0.68, 0.10, and 0.01 for conformations with dihedral angles Table 2 Calculated transition energies hn , corresponding wavelengths l, and oscillator strengths f of absorption peaks for various protonated forms of the free chromophore State
l Žnm.
hn ŽeV.
f
ŽO Y , N, O X .y with H bonda ŽO Y , N, O X .y without H bond a ŽHO Y , N, O X . ŽO Y , N, HO X . ŽO Y , HN, O X . ŽHO Y , HN, O X .q ŽHO Y , N, HO X .q ŽO Y , HN, HO X .q ŽHO Y , HN, HO X . 2q
448 445 325 377 507 401 428 402 422
2.77 2.79 3.81 3.29 2.45 3.10 2.90 3.08 2.94
1.24 1.26 1.05 1.22 0.87 0.70 1.22 0.61 1.32
a
Intramolecular hydrogen bond between the hydroxyl group of Ser and the nitrogen center of the heterocycle.
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
d s 08, 268, 458, and 908, respectively. Protonation of the carbonyl oxygen O X of the anion also leads to a blue-shift, by 71 nm, of the absorption peak in ŽHO X ., however, essentially without intensity change. Contrasting with these two cases, protonation of N in the heterocyclic ring which results in a zwitterion ŽO Y , HN, O X . leads to a noticeable redshift of the absorption band and a considerable reduction of the corresponding oscillator strength ŽTable 2.. Three different cations ŽHO Y , HN, O X .q, ŽO Y , HN, HO X .q, and ŽHO Y , N, HO X .q can be formed by double protonation of the anionic chromophore. Calculated differences in absorption energies among these cationic systems are considerably smaller than among the neutral species. Thus, changes in the absorption spectrum of the anionic chromophore by mono-protonation resulting in the neutral moieties are more pronounced than by double protonation producing the cationic species. As can be seen from Table 2, protonation of O Y or O X in the zwitterion ŽO Y , HN, O X . induces a blue-shift of the absorption band by 100 nm. This may have been anticipated by comparison with the anion and the neutral forms ŽHO Y , N, O X . and ŽO Y , N, HO X .. However, the cation ŽHO Y , N, HO X .q exhibits a red-shifted band compared with those of the parent molecules ŽHO Y , N, O X . and ŽO Y , N, HO X .. Interestingly, absorbance of the triple-protonated form, ŽHO Y , HN, HO X . 2q, is similar to that of the anion ŽO Y , N, O X . y ŽTable 2.. 5.3. Mutations of the GFP chromophore The calculated bands of the mutants are presented in Table 3 and compared to observed excitation energies. The mutations Tyr66 ™ Phe, Tyr66™ Trp or Tyr66™ His simplify the absorption spectrum of GFP by eliminating the long-wavelength maximum at 477 nm w4x. The mutation Tyr66 ™ Phe results in the loss of the phenolic hydroxyl group and, consequently, electronic states of the chromophore corresponding to deprotonated O Y do not exist in this mutant. The neutral compound and its cationic forms ŽHN, O X .q and ŽN, HO X .q are to some extent comparable to the states ŽHO Y , N, O X ., ŽHO Y , HN, O X .q, and ŽHO Y , N, HO X .q of the WT-chromophore, respectively. With respect to the related states of these
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Table 3 Calculated transition energies hn , corresponding wavelengths l, and oscillator strengths f of absorption peaks for various protonated forms of the free chromophore State
l Žnm.
Tyr66™ Phe Neutral ŽHN, O X .q ŽN, HO X .q Tyr66™ Trp Neutral ŽHN, O X .q ŽN, HO X .q Tyr66 ™His ŽHNd , N´ , N, O X . ŽNd , HN´ , N, O X . ŽHNd , N´ , HN, O X .q ŽNd , HN´ , HN, O X .q ŽHNd , N´ , N, HO X .q ŽNd , HN´ , N, HO X .q
360 Žexp.a . 296 355 388 436 Žexp.a . 337 433 445 382 Žexp.a . 340 325 409 387 427 410
a
hn ŽeV.
F
4.19 3.50 3.20
0.87 0.62 1.06
3.68 2.86 2.79
0.79 0.53 0.90
3.65 3.81 3.03 3.21 2.90 3.03
0.88 0.49 0.62 0.44 1.06 0.90
Ref. w3x.
counterparts, the excitation peaks of the mutant species are blue-shifted by ; 30–45 nm; the oscillator strengths change insignificantly, particularly if strength ratios are compared. The best agreement between experimental and calculated excitation energies is obtained for the state ŽHN, O X .q of the mutant chromophore Ž360 nm vs. 355 nm.. Similar results are obtained for the mutation Tyr66 ™ Trp. As for mutation Tyr66 ™ Phe, neutral and two protonated states ŽHN, O X .q and ŽN, HO X .q of the mutant can be related to the states ŽH O Y , N , O X ., ŽH O Y , H N , O X .q , a n d ŽHO Y , N, HO X .q of the WT-chromophore, respectively. The p-electron system of Trp is more extended than that of Phe. This difference is responsible for the red-shift of the absorbance of Tyr66 ™ Trp compared to that of Tyr66 ™ Phe. The excitation energies of the neutral and of the cationic forms ŽHN, O X .q and ŽN, HO X .q of the Trp-containing chromophore are red-shifted by 12, 32, and 17 nm, respectively, relative to those of the related states of the native chromophore Žsee Tables 2 and 3.. Close agreement is found between the absorption energy calculated for ŽHN, O X .q of this mutant, 433 nm, and the observed band, 436 nm. The calculated excitation energies of mutant Tyr66™ His are also listed in Table 3. As men-
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A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
tioned previously, the two N centers of the imidazole ring of His exhibit similar proton affinities and, therefore, the neutral species ŽHNd , N´ , N, O X . and ŽNd , HN´ , N, O X . as well as the positively charged m o ie tie s ŽH N d , N ´ , H N , O X .q and ŽNd , HN´ , HN, O X .q have almost the same energy Žwithin 2 kcalrmol.. Nevertheless, the absorption spectra of these tautomers differ remarkably. Our calculations predict the excitation maxima of Nd-protonated species to be red-shifted by 15–20 nm relative to their N´-protonated counterparts. Also, the ŽHNd .-type tautomers have a more intense absorbance than the related ŽHN´ . moieties. Comparing the calculated excitation energies to the experimental band maximum at 382 nm one concludes that the most likely state of the chromophore is ŽNd , HN´ , HN, O X .q with a computed absorption at 387 nm. 5.4. Proton affinities Based on the calculated transition energies for various protonated forms of the chromophore, one can predict variations of its proton affinity ŽPA. by excitation using a simple relation between energies ŽFig. 3., similar to the Forster cycle w25,26x. The PA ¨ difference of a system in the excited state M ) and its ground state M equals the difference in the transition energies of M and its protonated form HMq: PA Ž M ) . s PA Ž M . q hn Ž M . y hn Ž HMq . . Thus, if the absorbance of M is blue-shifted upon protonation, hn ŽM. - hn ŽHMq. , then the proton affinity PAŽM ) . of the excited chromophore is smaller than that of the ground state, hence ŽHMq. )
Table 4 Calculated proton affinities PA for various forms of the native chromophore of GFP PAŽO Y . GS ES
PAŽO X . D
GS ES
PAŽN. D
GS ES
D
ŽO Y , N, O X .y 323 300 y23 305 294 y11 306 315 9 ŽHO Y , N, O X . 210 231 y21 225 242 17 ŽO Y , HN, O X . 243 228 y15 220 206 y14 ŽO Y , N, HO X . 228 237 9 221 226 5 Comparison of the three protonation sites O Y , O X and N in the ground ŽGS. and singlet excited state ŽES. as well as the corresponding difference D. Energies in kcalrmol.
is more acidic than HMq. By the same token, a red-shifted absorption maximum of HMq compared to the transition energy of M implies that the basicity of the chromophore increases by excitation. Therefore, the considerable blue-shift of the absorption band of ŽHO Y , N, O X . with respect to that of the parent anion ŽTable 2. indicates the hydroxyl group of Tyr to be much more acidic in the excited state of the chromophore than in the ground state. The absolute PA values calculated for the ground states and the lowest singlet states of the various forms of the chromophore are compared in Table 4. The difference in the transition energies of the anion and ŽHO Y , N, O X . by ; 1 eV translates into a decreased proton affinity of O Y by 23 kcalrmol due to excitation. Conversely, the proton affinity of the heterocycle nitrogen increases by excitation ŽTable 4.. The most pronounced increase of PAŽN. Ž17 kcalrmol. is found for the neutral form ŽHO Y , N, O X .. Interestingly, whereas the tautomer ŽHO Y , N, O X . is the most stable one in the ground state Žsee Section 4., its lowest singlet excited state lies 13 kcalrmol above that of the zwitterion ŽO Y , HN, O X ..
6. Discussion 6.1. Analysis of HOMO and LUMO
Fig. 3. Forster cycle for deriving the proton affinity PA) of an ¨ excited state.
As mentioned above, the chromophore absorption peak is due to the HOMO™ LUMO electronic transition. The highest occupied and the lowest virtual molecular orbitals of the anion are sketched in Fig. 4. One notes that the HOMO is localized to a
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
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6.2. Assignment of spectra
Fig. 4. Sketches of HOMO and LUMO of the anionic form of the chromophore.
considerable extent Ž67%. at the phenol ring of Tyr; this MO is nonbonding with respect to both intraand inter-ring atomic orbital interactions. Since the phenol contribution drops to only 25% in the LUMO, the HOMO™ LUMO transition is accompanied by a charge transfer in the chromophore. Thereby, electron density is withdrawn from both the carbon atoms of the phenol ring and C a of Tyr and redistributed to the heterocyclic ring and C b of Tyr. Since the electron density at O Y is larger in the ground state than in the excited state, one expects protonation of O Y to increase the transition energy due to a more pronounced stabilization of the HOMO, as is indeed the case ŽTable 2.. Conversely, protonation of N, carrying a more negative charge in the excited state, induces a relative stabilization of the LUMO and consequently, a decrease of the HOMO–LUMO energy gap resulting in a red-shifted absorbance, again in line with the calculated excitation energy changes ŽTable 2.. Concomitantly, electronic excitation increases the acidity of the phenol hydroxyl group due to the decreasing electronic density on O Y as well as the basicity of N because of the increasing negative charge of the latter center. The corresponding quantitative estimates are given Table 4 and were discussed in Section 5.4. Thus, the proton-induced changes of the excitation energy and the proton affinity can be rationalized by a simple frontier orbitals analysis of the chromophore.
Based on the results presented in Tables 1–3 we shall now discuss the absorption spectra of GFP. Native GFP shows two peaks at 397 and 477 nm caused by two forms of the protein with different protonation states of the chromophore w13,15,19x. The first peak is usually assigned to the neutral form ŽHO Y , N, O X ., whereas the second peak is ascribed to the anion ŽO Y , N, O X .y w30x. However, this interpretation is not supported by results of the present calculations. Let us consider this point in more detail. First of all, a good agreement found between observed and calculated spectra of the anionic and cationic forms of the chromophore in aqueous solution ŽTable 1. suggests a strategy whereby one compares calculated and experimental transition energies of the chromophore in order to identify spectroscopically relevant tautomers. The calculated absorption of the neutral form ŽHO Y , N, O X . is in the UV region; however, protonation of the second center in the chromophore shifts the absorption peak to the visible region Žsee Table 2.. Three conceivable structures ŽHO Y , HN, O X .q, ŽHO Y , N, HO X .q, and ŽO Y , HN, HO X . q should be considered. Although th e c a lc u la te d a b s o r p tio n p e a k o f ŽHO Y , HN, HO X . 2q lies at 422 nm, formation of the dication seems to be unlikely in the protein or in aqueous solution Žsee Section 4.. The most likely tautomer exhibiting the observed absorbance at 397 nm is ŽHO Y , HN, O X .q Žcalculated at 401 nm, Table 2.. In fact, protonation of O Y as well as of the heterocyclic nitrogen in the chromophore of GFP seems to be quite reasonable as will be discussed in the following. Furthermore, deprotonation of the phenolic hydroxyl of ŽHO Y , HN, O X .q is found to be followed by a considerable red-shifted absorbance in line with experiment. Although the predicted absorption energy of ŽO Y , HN, HO X .q agrees equally well with experiment, such an assignment is hardly possible. Experimentally, the proton affinity of phenolic oxygen is well known to be considerably larger than that of a carbonyl oxygen w45x. According to our calculation ŽTable 4. the proton affinity PAŽO Y . of ŽO Y , HN, O X . is 23 kcalrmol larger than PAŽO X .. Finally, there is no experimental evidence on proto-
22
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nation of the carbonyl oxygen in proteins. The last two arguments also apply to the tautomer ŽHO Y , N, HO X .q which we therefore also exclude. Moreover, deprotonation of either O Y or O X of this tautomer results in blue-shifted absorbance which is inconsistent with experimental data w19,20,30x. Thus, the absorption peak at 397 nm observed in the W T -p ro tein is assig n ed to th e catio n ŽHO Y , HN, O X .q rather than to the neutral form ŽHO Y , N, O X . as assumed previously. Thus, we can conclude that the spectroscopically relevant form of the chromophore in WT-GFP is the cation ŽHO Y , HN, O X .q with the protonated heterocyclic nitrogen atom. Independent support for the protonation of the nitrogen center in the heterocycle of the chromophore can be obtained from a comparison of experimental and calculated spectra of the mutant chromophores ŽTable 3.. The mutant chromophore Tyr66 ™ Phe and Tyr66™ Trp can exist only in three forms ŽN, O X ., ŽHN, O X .q, and ŽN, HO X .q. This makes the assignment of their absorption peaks easier compared to those for the WT-chromophore. The absorbance of the neutral forms of Tyr66™ Phe and Tyr66 ™ Trp are to be expected in the UV region, at 296 and 337 nm ŽTable 3., respectively, at variance with experimental data Ž360 and 436 nm, respectively.. Therefore, we exclude neutral species as relevant. On the other hand, very good agreement with experiment is found for both mutants in the form ŽHN, O X .q. The calculated absorption energy of the species ŽN, HO X .q does not agree as well with experiment as that of ŽHN, O X .q. Again, due to the considerable difference in proton affinities PAŽN. ) PAŽO X ., formation of ŽN, HO X .q seems to be unlikely Žsee Section 4.. As to the mutant Tyr66 ™ His, two points are worth noting. First, best agreement between calculated and observed absorption energies is found for the cation ŽHN´ , HN, O X .q with the heterocyclic nitrogen protonated. Second, although the cations Ž HN ´ , HN, O X . q and ŽHNd , HN, O X .q are of almost equally stable, their spectroscopic properties differ remarkably. This finding may be taken into account when designing new GFPs. The experimental absorption peak at 477 nm found for native GFP lies between the excitation maxima at 401 and 507 nm calculated for the cation
ŽHO Y , HN, O X .q and the zwitterion ŽO Y , HN, O X ., respectively ŽTable 2.. In ŽHO Y , HN, O X .q a proton is bound to O Y with an interatomic distance RŽO Y – ˚ whereas in the zwitterion this proton is H. of ; 1 A, removed from the chromophore, formally to infinite distance. In GFP the proton is transferred from O Y of the chromophore to its immediate environment containing water molecules, His148, and Thr203 Žsee Fig. 1.. To estimate the effect of the proton location on the absorption of the chromophore, the aqua com plexes wŽ H O Y , H N , O X . q P H 2 O x and wŽO Y , HN, O X . P H 3 Oqx were calculated. The formation of hydrogen bonds between the chromophore and surrounding water molecules does not induce significant alterations of the spectrum. In fact, both the formation of aqua complexes of the chromophore ŽTable 1. as well as the intramolecular hydrogen bond between N of the heterocycle and the hydroxyl group of Ser65 ŽTable 2. affect the absorption spectrum only insignificantly. In line with these results, the solvent interaction via a hydrogen bond H 2 O PPP HO Y in wŽHO Y , HN, O X .qP H 2 Ox causes a relatively small change in the spectrum Ž l s 410 nm, f s 0.74. as compared to the free cation ŽHO Y , HN, O X .q Ž l s 401 nm, f s 0.70.. However, interaction of a hydronium ion H 3 Oq with the zwitterion ŽO Y , HN, O X . leads to a considerable shift of the absorption peak w46x. A decrease of the distance ˚ Žthe correRŽO Y –O. to 3.0, 2.7, 2.6, and 2.5 A, sponding distances O Y –Hq are about 1.9, 1.6, 1.5, ˚ respectively. results in blue-shifted maxand 1.4 A, ima at l s 493, 481, 475, and 460 nm, respectively. Based on the results of these calculations we conclude that the observed absorption of GFP at 477 nm can be assigned to the H-bonded complex of the zwitterion with a protonated group Že.g., H 3 Oq. at ˚ Note that a an O Y –O distance of ; 2.6–2.7 A. recent structure analysis w14x yielded the following distances between O Y of the chromophore and its ˚ immediate environment: O Y –OŽThr203. s 2.6 A, ˚ O Y –OŽH 2 O. s 2.6 A. Thus, from the comparison between calculated and experimental absorption energies for the WTchromophore and its mutants, we have suggested a consistent interpretation of the observed spectra implying the heterocyclic nitrogen to be protonated in all relevant species. This conclusion is in line with available crystal data for GFP. On the bases of
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
atomic coordinates of WT-GFP w12x one finds two water molecules in the vicinity of the nitrogen cen˚ respectively. The ter, at distances of 3.3 and 3.5 A, corresponding electrostatic contacts may stabilize the protonated heterocyclic nitrogen. Moreover, in the S65T mutant w14x the distance between the hetero˚ short cyclic nitrogen and Og of Thr65 is only 2.8 A, enough to indicate a strong hydrogen bond between N and Og . 6.3. Mechanism of fluorescence Recently, a mechanism of absorption and fluorescence of GFP has been proposed on the basis of experimental data w19x. Our results just discussed support this mechanism albeit in the following modified form ŽFig. 5.. The two spectroscopically relevant forms of the chromophore of WT-GFP are the cation ŽHO Y , HN, O X .q and the zwitterion ŽO Y , HN, O X . rather than the neutral form ŽHO Y , N, O X . and the anion ŽO Y , HN, O X .y, respectively. ŽHO Y , HN, O X .q exhibits an absorption maximum at 397 nm. The peak at 477 nm corresponds to the absorption of the H bonded complex wŽHq PPP ŽO Y , HN, O X .x of the zwitterion. According to experiment, these two forms in their electronic ground states should be in equilibrium w19,20x. Considerable alterations of the proton affinity of the chromophore by its electronic excitation ŽSection 5.4. lead to the following changes in the system. Due to a significant increase of the acidity of the HO Y
Fig. 5. Experimental and calculated Žin parentheses. absorption and emission transitions in GFP. wHq PPP O Y , HNx designates an H-bonded complex between the zwitterionic form of the chromophore and its environment Žsee text..
23
group in ŽHO Y , HN, O X .q by excitation, the proton of the hydroxyl group is transferred to the chromophore environment, resulting in the excited state of ŽO Y , HN, O X . and a corresponding rearrangement of the chromophore environment, as proposed earlier w19x. Thus, the chromophore in the excited state exists only in the zwitterionic form. From the significant decrease of the proton affinity PAŽO Y . after excitation one expects a weakening of the hydrogen bond between O Y and its environment and, therefore, an increase of the corresponding distance Hq PPP O Y . In turn, this lengthening of the hydrogen bond results in a red-shifted emission. Experimentally, the chromophore fluorescence occurs at 510 nm in good agreement with the transition energy of 507 nm calculated for the zwitterion. After the fluorescence emission the chromophore remains in its zwitterionic form for a relatively long time, enabling an equilibrium to be established between the two forms of the ground state ŽFig. 5.. This slow interconversion between the two conformations of GFP in the ground state seems to account for the rapid loss of the absorption intensity at 397 nm upon exposure of GFP to light at that wavelength w19,20x. 6.4. Effect of mutations of the chromophore enÕironment Due to different hydrogen bonding networks between the chromophore and its environment, two forms of the chromophore have been found in WTGFP w13x. It has been established experimentally w3,22x that the absorption peak at 477 nm is eliminated when Thr203 is replaced by Ile. Therefore, Thr203 should play an important role in stabilizing the deprotonated phenolic ring of the chromophore ŽFig. 1.. On the other hand, mutation of Glu222 to Gly as well as replacing of Ser65 by Gly, Ala, Cys, Val, or Thr all cause the loss of the absorption peak at 397 nm w3,4x. A less drastic effect, namely inversion of the intensity ratio of the two peaks, is observed when Ile167 is mutated to Thr w5x. These effects seem to be hardly predictable computationally. In fact, one can easily estimate that a mutation induced change of only 2 kcalrmol in the relative stabilization of the two states in equilibrium entails a considerable varia-
24
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
tion of the intensity ratio of their absorption maxima Žby a factor of ; 30.. For comparison, we mention that the interaction energy of two unscreened unit ˚ changes by 2 kcalrmol if charges, separated by 5 A, ˚ the distance between them changes by only 0.15 A. Therefore, it is not too surprising that mutations of the chromophore environment can result in the apparent suppression of one of the two excitation maxima. For example, the mutation Ser65 ™ Thr Žwhereby an atom H in Ser65 is replaced by a CH 3 group. shifts the equilibrium between both forms, so that only one absorption maximum at 488 nm can be observed. Even changes in the solvent lead to alterations of the ratio of the absorption peaks w15x. At present, it does not seem possible to compute the relative energies of two GFP conformations with an accuracy of 1 kcalrmol or better and, therefore, to predict the effect of these types of mutations on the ratio of two maxima in the absorption spectra of GFP. However, the effect of mutations leading to a change of the chromophore, e.g. ŽTyr66™ Trp., and therefore to new absorption and emission spectra, may well be predicted with the methods used in the present investigations as shown above.
7. Conclusions Using the semiempirical methods PM3 and INDOrS, we have studied the electronic structure and absorption spectra of native and mutant GFP chromophores as well as their geometries, relative stabilities, and proton affinities. Several forms of the native and mutant chromophores are found to be nonplanar outside the protein environment; however, the corresponding planar structures are only slightly higher in energy Žwithin ; 1 kcalrmol. and, consequently, can be stabilized in GFP due to steric and electrostatic interactions with the chromophore environment. Although hydrogen bonds between the chromophore and water molecules do not significantly affect its spectroscopic properties, proton transfer between the chromophore and its surroundings results in considerable alterations of spectra. The good agreement between computed and observed absorbance of the WT-chromophore and three of its mutants suggests that the structure of important states of the chromophore in GFPs can be identified
by comparing experimental and simulated absorption spectra. In this way, the absorption maximum of WT-GFP at 477 nm has been assigned to the H-bonded complex between the zwitterion ŽO Y , HN, O X . and its environment. Another peak at 397 nm is ascribed to the excitation of the cationic form ŽHO Y , HN, O X .q. Due to the change of the proton affinity of the chromophore by electronic excitation, there is only one important excited state, ŽO Y , HN, O X . ) , responsible for the emission of native GFP. Our results obtained for the native chromophore and its three mutants ŽTyr ™ Phe., ŽTyr ™ Trp., and ŽTyr ™ His. indicate that the heterocyclic nitrogen is protonated both in the relevant ground states and the corresponding excited states. At variance with previous ad hoc suggestions, we conclude that protonation of the heterocyclic nitrogen in the chromophores is a crucial factor which determines absorption and fluorescence of GFP.
Acknowledgements This work has been supported by the Deutsche Forschungsgemeinschaft via SFB 377 and by the Fonds der Chemischen Industrie.
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A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25 w13x K. Brejc, T.K. Sixma, P.A. Kitts, S.R. Kain, R.Y. Tsien, M. Ormo, ¨ S.J. Remington, Proc. Natl. Acad. Sci. USA 94 Ž1997. 2306. w14x M. Ormo, ¨ A. Cubitt, K. Kallio, L. Gross, R. Tsien, S.J. Remington, Science 273 Ž1996. 1392. w15x G.J. Palm, A. Zdanov, G.A. Gaitanaris, R. Stauber, G.N. Pavlakis, A. Wlodawer, Nature Struct. Biol. 4 Ž1997. 365. w16x D.A. Smith ŽEd.., Modeling the Hydrogen Bond. Am. Chem. Soc., Washington, DC, 1994, ACS Symp. Ser. No. 569. w17x S. Scheiner, Acc. Chem. Res. 27 Ž1994. 402. w18x S. Scheiner, T. Kar, J. Am. Chem. Soc. 117 Ž1995. 6970. w19x M. Chattoraj, B.A. King, G.U. Bublitz, S.G. Boxer, Proc. Natl. Acad. Sci. USA 93 Ž1996. 8362. w20x H. Lossau, A. Kummer, R. Heinecke, F. Pollinger-Dammer, ¨ C. Kompa, G. Bieser, T. Jonsson, C.M. Silva, M.M. Yang, D.C. Youvan, M.E. Michel-Beyerle, Chem. Phys. 213 Ž1996. 1. w21x W.W. Ward, C.W. Cody, R.C. Hart, M.J. Cormier, Photochem. Photobiol. 31 Ž1980. 611. w22x T. Ehring, D.J. O’Kanne, F.G. Prendergast, FEBS Lett. 367 Ž1995. 163. w23x S. Dalagrave, R.E. Hawtin, C.M. Silva, M.M. Yang, D.C. Youvan, BiorTechnology 13 Ž1995. 151. w24x R. Mitra, C.M. Silva, D.C. Youvan, Gene 173 Ž1996. 13. w25x T. Forster, Naturwissenschaften 36 Ž1949. 186. ¨ w26x T. Forster, Z. Electrochem. 54 Ž1950. 42. ¨ w27x M.J. Kasha, J. Chem. Soc., Faraday Trans. II 82 Ž1986. 2379. w28x P.F. Barbara, P.K. Walsh, L.E. Bruce, J. Phys. Chem. 93 Ž1989. 29. w29x D.C. Youvan, M.E. Michel-Beyerle, Nature Biotechnol. 14 Ž1996. 1219.
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w30x R.M. Dickson, A.B. Cubitt, R.Y. Tsien, W.E. Moerner, Nature ŽLondon. 388 Ž1997. 355. w31x A. Warshel, J. Aquist, Annu. Rev. Biophys. Chem. 20 Ž1991. 267. w32x J. Ridley, M.C. Zerner, Theor. Chim. Acta 32 Ž1973. 111. w33x M.C. Zerner, G. Loew, U. Muller-Westerhof, J. Am. Chem. ¨ Soc. 102 Ž1980. 589. w34x M.C. Zerner, ZINDO. A comprehensive semiempirical SCFrCI package, 1995. w35x S. Canuto, M.C. Zerner, J. Am. Chem. Soc. 112 Ž1990. 2114. w36x M.A. Thompson, M.C. Zerner, J. Am. Chem. Soc. 113 Ž1991. 8210. w37x M. Karelson, M.C. Zerner, J. Phys. Chem. 96 Ž1992. 6949. w38x S. Miertus, J. Tomasi, J. Chem. Phys. 65 Ž1982. 239. w39x J.J.P. Stewart, J. Comput. Chem. 10 Ž1989. 209–221. w40x A.A. Voityuk, SIBIQ 2.4. A program for semiempirical calculations, 1995. w41x M.J.S. Dewar, E.G. Zoebisch, E. Healy, J.J.P. Stewart, J. Am. Chem. Soc. 107 Ž1985. 3902. w42x J.J.P. Stewart, J. Comput.-Aided Mol. Design 4 Ž1990. 1. w43x Y.-J. Zheng, R. Ornstein, J. Am. Chem. Soc. 118 Ž1996. 11237. w44x R.C. Weast ŽEd.., CRC Handbook of Chemistry and Physics, 61st ed., CRC Press, Boca Raton, FL, 1980. w45x S.G. Lias, J.E. Bartmess, J.F. Liebman, J.L. Holmes, R.D. Levin, W.G. Mallard, Gas Phase Ion and Neutral Thermochemistry, J. Phys. Chem. Ref. Data 17 Ž1988. 1–2, Suppl. 1. w46x A.A. Voityuk, M.E. Michel-Beyerle, N. Rosch, Chem. Phys. ¨ Lett. 272 Ž1997. 162.
Quantum chemical modeling of structure and absorption spectra of the chromophore in green fluorescent proteins Alexander A. Voityuk, Maria-Elisabeth Michel-Beyerle, Notker Rosch ¨
)
Institut fur D-85747 Garching, Germany ¨ Physikalische and Theoretische Chemie, Technische UniÕersitat ¨ Munchen, ¨ Received 6 February 1998
Abstract As a first step towards modeling the green fluorescent protein ŽGFP., we have carried out absorption spectra calculations on chromophores of both native and mutant proteins using the semiempirical method INDOrS. A number of protonated and deprotonated states of the GFP fluorophore were considered. We find predicted and observed absorbance energies in very good agreement. Based on a comparison of calculated and experimental absorption spectra, we suggest structures for the ground and excited states of the chromophore of GFP. We assign the absorption maximum of GFP at 477 nm to an H-bonded complex of the zwitterion ŽO Y , HN, O X . involving the phenolic oxygen of Tyr66 and its environment; the nitrogen of the heterocyclic ring is protonated in this complex. Another peak at 397 nm is due to excitation of the corresponding protonated form ŽHO Y , HN, O X .q. Calculated transitions in the mutant chromophores ŽTyr66™ Phe., ŽTyr66 ™ Trp., and ŽTyr66 ™ His. at 355, 433, and 387 nm, respectively, are close to the corresponding experimental values of 360, 436, and 382 nm, suggesting cationic forms with the nitrogen protonated to be responsible for the absorbance. Proton transfer to or from the phenolic hydroxyl group of the chromophore is shown to be crucial for understanding the absorption and emission spectra of GFP. q 1998 Elsevier Science B.V. All rights reserved.
1. Introduction The green fluorescent protein ŽGFP. and its mutants are the subject of widespread interest, in particular with respect to their applications in molecular biology and biotechnology. Since the fluorescence of GFP occurs in the absence of any cofactors, the protein can serve as a revolutionary marker of gene expression and as a tag for localizing proteins within living cells w1–5x. The DNA sequence coding for GFP can be fused to that of any protein of interest w1,3,6,7x. By expression of the modified gene, the )
Corresponding author.
protein is covalently linked to GFP and thus carries a fluorescent label. GFP was first described by Shimomura and Johnson in 1962 w8x. The cloning of cDNA and the determination of the primary structure of GFP by Ward, Prendergast and colleagues was a crucial step in the study of this protein w9x. GFP consists of 238 amino acids. The conformation of GFP is extremely stable and denaturation of the protein occurs only under harsh conditions w10x. The chemical structure of the GFP fluorophore was established in a landmark study w11x. The visible absorbance and fluorescence of the protein were assigned to a p-hydroxybenzylidene-imidazolidinone chromophore. This flu-
0301-0104r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 0 1 0 4 Ž 9 8 . 0 0 0 8 0 - 9
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orophore is formed by post-translational cyclization of the polypeptide backbone between residues Ser65, Tyr66, and Gly67, and by a ,b-dehydrogenation of Tyr66 w11x. The autocatalytic mechanism of the fluorophore formation is not yet understood. ˚. Recently, a number of highly resolved Ž; 1.9 A crystal structures on GFP have been reported: those of the wild-type GFP dimer w12x and monomer w13x as well as those of several GFP mutants w14x, w15x in which either the chromophore orrand its environment have been altered. All these proteins exhibit a rather regular cylindrical shape formed by 11 bstrands. This ‘barrel’, capped on both top and bot˚ and a length of tom, has an outer diameter of ; 30 A ˚ The chromophore is located nearly in the ; 40 A. center of the barrel and is completely protected from the bulk solvent. Modeling of the residues 57–71 led to the conclusion that there is a strain between the central a-helix and the b-barrel w15x. The immediate environment of the fluorophore includes several water molecules as well as charged and polar amino acid residues. Hydrogen bonds between the chromophore and its surroundings in GFP seem to play an important role w12–15x. According to the X-ray data, a number of proton donorracceptor centers are found in the vicinity of the chromophore ŽFig. 1.. Two residues, His148 and Thr203, and a water molecule interact with the phenolic hydroxyl group HO Y of Tyr66 ŽHO Y , N, O X . and may cause its deprotonation. Arg96 and Gln94 form hydrogen bonds with the carbonyl oxygen of the heterocyclic ring ŽO X .. Similar to His, the heterocyclic ring of the chromophore may be positively charged or neutral depending on its local environment and on the electrostatic potential created by the protein. The protonated nitrogen of the ring can be stabilized by the hydroxyl group of Ser65, the carboxyl group of Glu222, or water molecules located in the vicinity. According to X-ray data, the wild-type protein and the S65T mutant have different hydrogen bonding networks between the chromophore and its environment w13–15x. It should be noted that in the case of ionic H bonds, wX–H–Yxq between neutral units X and Y, or wX–H–Yxy between negatively charged units Xy and Yy, the equilibrium position of the proton can lie midway between X and Y w16x; however, the proton location is considerably affected by the environment. Recently, it has been shown that
Fig. 1. The chromophore and its environment in GFP based on the X-ray structure of GFP determined in Ref. w12x.
proton transfer in systems with ionic H bonds proceeds with very low activation barriers w17,18x. The absorption spectra of the native protein exhibit two excitation peaks w1–3x at 395 and 475 nm Ž397 and 477 nm according to the latest experiment w19,20x.. Based on the spectroscopic data it was proposed that the wild-type protein exists in two different forms: a dominant one ŽA. which is responsible for the absorption at 395 nm and another stable form ŽB. featuring the 475 absorption peak w1,3,5,10,19x. Recently, structural studies of GFP and its mutants have shown that in fact there exist two different stable conformations of GFP w13,15x; the equilibrium between these states seems to be governed by an H-bond network. Upon denaturation, the spectrum of GFP undergoes large changes w10,21x which clearly indicate that there also exist different forms of the chromophore in solution. Mutations of the fluorophore and its environment are also found to have a substantial effect on the absorption and fluorescence spectra w1–3,22–24x. Spectroscopic properties of molecular systems are known to be changed significantly by proton transfer w25–28x. Steady-state and picosecond time-resolved spectroscopy has re-
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
cently been applied to study native and mutant GFP w19,20x. It was shown that excited state proton transfer plays a key role in GFP emission w19,20,29x. Optical studies of individual GFP molecules have been recently reported w30x. Since the chromophore in GFP is completely protected from bulk water w12–14x p K a values of its subunits cannot be used to predict which center is protonated within the protein w31x. Moreover, low activation barriers for the proton transfer between the chromophore and its surroundings can lead to rapid changes between different states. Therefore, to assign spectroscopic features of GFP and its mutants, detailed knowledge on the electronic structure and spectra of the chromophore tautomers is needed. Despite strong experimental efforts, several important questions have yet to be answered: Which forms of the chromophore are responsible for the absorption of native and mutant GFP? Which geometrical structures correspond to these states? How are absorption spectra affected by mutation of amino acids either involved in the chromophore or located in its immediate vicinity? The goal of the present study is to answer these questions using quantum chemical calculations.
2. Models The chromophore of GFP and its environment are shown in Fig. 1. As a model we will consider a system containing a heterocyclic ring composed of the three amino acid residues Ser–Tyr–Gly. To cut the connection to the backbone of the protein, the amino group of Ser and the carboxyl group of Gly are substituted by hydrogen atoms. As mentioned above, it is difficult to predict which centers of the chromophore are protonated in
15
Scheme 2.
the protein; therefore, various possibilities should be taken into account. In principle, the following eight structures of the chromophore can exist in GFP. In the first structure, anion ŽO Y , N, O X .y, three atoms, O Y , N and O X can accept a proton from the environment Žsee arrows in Scheme 1.. Protonation of these centers in the anion results in the three neutral structures ŽHO Y , N, O X ., ŽO Y , HN, O X ., and ŽO Y , N, HO X ., respectively. Further protonation of these neutral species leads to the three cationic systems ŽHO Y , HN, O X .q, ŽHO Y , N, HO X .q, and ŽO Y , HN, HO X .q. Finally, protonation of all three c e n te r s in th e c h r o m o p h o r e y ie ld s ŽHO Y , HN, HO X . 2q. Besides the wild-type ŽWT. chromophore, we studied also three of its mutants. The mutations of tyrosine ŽTyr. to phenylalanine ŽPhe., tryptophan ŽTrp., or histidine ŽHis. in the chromophore were modeled by the systems shown in Scheme 2.
3. Method
Scheme 1.
The absorption spectra of the model systems were calculated with the spectroscopic INDO scheme, INDOrS, w32,33x as implemented in the program ZINDO w34x. The INDOrS technique has been shown
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A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
constraints using the method PM3 w39x implemented in the program SIBIQ w40x. For comparison, AM1 w41x calculations for some systems were also performed. The PM3 and AM1 methods are well-established semiempirical tools to calculate formation enthalpies and ground-state structures of organic species: average absolute errors are about 4.5 ˚ for bond kcalrmol for formation enthalpies, 0.02 A lengths, and 38 for angles w42x.
4. Geometries and relative stability
Fig. 2. Model for the hydrated anionic chromophore.
to be very successful in simulating spectroscopic properties of large organic compounds and biomolecules w35,36x. Standard parameters were used: two-center two-electron integrals were calculated using the Mataga–Nishimoto formula and the resonance integrals were computed with the scaling factors f s s 1.0 and f p s 0.585 w32x. An INDOrS calculation consists of two parts: a ground-state SCF calculation is followed by a configuration interaction calculation which yields excitation energies and oscillator strengths Žcomputed in the one-center approximation using the dipole length operator.. All single excitations from the 10 highest occupied molecular orbitals to the 20 lowest virtual orbitals were included in the configuration interaction. The effect of the environment on the spectra was estimated within a model w37x based on the self-consistent reaction field approach w38x. Since a straightforward polarizable-continuum model can lead to considerable errors for a system interacting with its environment via hydrogen bonds w37x, the chromophore in water was modeled by an ‘embedded cluster’ containing the chromophore and 6 water molecules Žtwo of them bound to each of the three centers O Y , O X , and N, Fig. 2.. We optimized the geometry of the various model systems without any
The calculated structures of the anion ŽO Y , N, O X .y ŽScheme 2. exhibit two low-energy conformations, with and without an intramolecular hydrogen bond between the hydroxyl group of Ser65 and N of the heterocyclic ring. The structure with the H bond is slightly more stable Žby 0.5 kcalrmol. than the other conformer. The phenol ring of Tyr and the heterocycle lie in the same plane. ŽIt is convenient to characterize the planarity of chromophores under consideration by the dihedral angle d s /C 1C 2 C 3 C 4 ; see Scheme 3.. Of the three neutral tautomers formed by protonation of the anionic form, the tautomer ŽHO Y , N, O X ,. was calculated to be ; 18 kcalrmol more stable than the zwitterion ŽO Y , HN, O X . which in turn is only slightly more stable, by 0.7 kcalrmol, than ŽO Y , N, HO X .. Similar relative energies of these three tautomers are predicted by the semiempirical method AM1. In the gas phase, zwitterions are known to be less stable than conventional forms; however, in a polar environment, zwitterions become much more favored w43x. Since ŽO Y , HN, O X . should exist in mutant GFP, as we will discuss below, the energy difference between ŽHO Y , N, O X . and the zwitterion should be recovered by environment effects, e.g. by specific interactions due to H bonds and by the electrostatic potential generated by the entire protein.
Scheme 3.
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
The structures of ŽO Y , N, HO X . and ŽO Y , HN, O X . are essentially planar, d F 18. While the lowest-energy tautomer ŽHO Y , N, O X . has a nonplanar geometry, d s 268, this conformation is only 0.2 kcalrmol more stable than the corresponding planar structure. Using X-ray atomic coordinates w12,14x we found that the chromophore in GFP is essentially planar: d s 0.58 and d s 3.28 in wild-type GFP and in the mutant S65T, respectively. Thus, a planar structure of the chromophore was also adopted for ŽHO Y , N, O X .. PM3 predicts ŽHO Y , HN, O X .q to be the most stable tautomer of the three positively charged species obtained by double protonation of the anionic chromophore; the relative energies for ŽHO Y , N, HO X .q and ŽO Y , HN, HO X .q are 15.7 and 22.8 kcalrmol, respectively. As for the corresponding neutral species, the m ost stable cationic form , ŽHO Y , HN, O X .q is not planar Ž d s 118. while ŽHO Y , N, HO X .q and ŽO Y , HN, HO X .q exhibit essentially planar structures. The formation of ŽHO Y , HO X , HN. 2q in GFP appears to be unlikely because of its relatively small proton affinity Ž132 kcalrmol according to PM3.; thus, proton transfer from this form to a water molecule is exothermic with a reaction enthalpy of about y23 kcalrmol. Substitution of Tyr66 by Phe results in the mutant Tyr ™ Phe ŽScheme 2.. A nonplanar structure with d s 318 is calculated for this neutral chromophore by PM3. This mutant exhibits two protonated forms, ŽHN, O X .q and ŽN, HO X .q, both essentially planar; the tautomer ŽHN, O X .q is 15 kcalrmol more stable than ŽN, HO X .q. The mutant chromophore Tyr ™ Trp ŽScheme 2. has a more extended p-electron system and its neutral as well as its N- and O X -protonated forms are all almost planar. As expected, the lowest-energy tautomer is ŽHN, O X .q, lying 12 kcalrmol below its counterpart ŽN, HO X .q. The two mutations discussed so far reduce the number of relevant forms of the chromophore. By contrast, the mutation Tyr ™ His ŽScheme 2. increases the number of possible protonated species to be considered. Indeed, the imidazole ring of His features two electron donor atoms, Nd and N´ , that can be protonated whereas Tyr possesses only one such center, O Y . Two neutral and five positively charged protonated species are essentially planar; the deviation of the dihedral angle from planarity is less than 28. Accord-
17
ing to PM3 as well as AM1 data, the centers Nd and N´ in His have very similar proton affinities and, consequently, the corresponding tautomers are almost equally stable Žwith heats of formation within 2 kcalrmol.. For example, the neutral species ŽNd , HN´ , N, O X . is 1.5 kcalrmol more stable than ŽHNd , N´ , N, O X .. Protonation of these forms leads to th re e sp e c ie s o f sim ila r e n e rg ie s, ŽNd , HN´ , HN, O X .q, ŽHNd , N´ , HN, O X .q, and ŽHNd , HN´ , N, O X .q, with relative energies of 0.0, 0.9, and 3.8 kcalrmol, respectively. Tautomers with th e c a rb o n y l o x y g e n p ro to n a te d , e .g . ŽNd , HN´ , N, HO X .q, are of considerably higher energy Žby ; 15 kcalrmol..
5. Absorption spectra As found experimentally w3,10,19,20,23,24x, the chromophore in GFP can be characterized by the position and intensity of the long-wavelength absorption band which can be altered considerably depending on pH, solvent, mutations, etc. For simulated absorption spectra of various forms of the chromophore we found only one strong absorption band in the visible region, well separated from other peaks. The maximum exhibits p ™ p ) character and arises due to an almost pure HOMO™ LUMO transition. The next higher excitation peaks lie in the UV region. 5.1. Chromophore in aqueous solution We start by considering the spectra for denatured GFP. The relevant state of the chromophore in aqueous solution is well determined by the pH of the solution. Upon denaturation, GFP loses its fluorescence and changes its absorption spectrum w10–12x. Native GFP has absorption maxima at 397 and 477 nm. Its denaturation by treatment with acid ŽpH - 4. results in an absorption peak at 383–384 nm, while protein denaturation with base ŽpH ) 12. leads to a maximum at 447 nm w10x. Since the p K a value of the phenolic hydroxyl of Tyr is ; 10 and the p K a value of imidazole in His is ; 6 w44x, the chromophore exists predominantly as the solvated anion ŽO Y , N, O X .y at pH ) 12 and as the solvated cation ŽHO Y , HN, O X .q at pH - 4. To simulate solvation
18
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
Table 1 Modeling of solvation effects on the chromophore absorption spectrum Basic ŽpH ) 10. ŽO Y , N, O X .y
Acidic ŽpH - 4. ŽHO Y , HN, O X .q
Exp.a
447
384
Models: Ža. Free chromophore Žb. Chromophoreq 6H 2 O Žc. ŽChromophore. solv. Žd. ŽChromophoreq 6H 2 O. solv.
448 443 429 435
401 405 395 397
Solution Species
Transition wavelength l Žin nm. of various models: Ža. free chromophore, Žb. chromophore with two water molecules per center O Y , O X , and N, Žc. same as Ža. with a reaction field cavity Ž ´ s 80., Žd. same as Žb. with a reaction field cavity Žsee text for details.. a Ref. w9x.
in water, calculations were carried out for the three models listed in Table 1. In the free state, i.e. in absence of a solvent, absorption peaks of ŽO Y , N, O X .y and ŽHO Y , HN, O X .q are calculated at 448 and 401 nm, respectively. H bonds in the clusters containing the chromophore and specifically bound water molecules have hardly any effect on the calculated spectra as absorption peaks of the anionic and cationic aqua complexes are shifted by only 4–5 nm relative to the corresponding free forms. The most noticeable alteration of the excitation energy is obtained within the continuum model for the anionic chromophore in polar medium Žwater, ´ s 80.; its excitation maximum is blue-shifted by 19 nm ŽTable 1.. This blue-shift is found to be reduced to 13 nm in the solvated cluster of the anion shown in Fig. 2. The corresponding absorption shifts calculated for the cation amount to only 6 and 4 nm, respectively. Thus, for the solvated anion, our most elaborate model predicts an absorption peak Ž l s 435 nm, f s 1.27. in good agreement with the observed absorption band at l s 447 nm. According to our calculations, the solvated cation has an absorption maximum Ž l s 397 nm, f s 0.85. which corresponds well to the experimental value of l s 384 nm. It is worth noting that calculated solvation effects on transition energies are rather small. 5.2. NatiÕe GFP chromophore One of the important questions open so far is how protonation of the chromophore affects the absorption spectrum. Table 2 lists the calculated absorption
energies and oscillator strengths of various protonated forms of the WT-chromophore. From these data one deduces that all three types of protonation of the anionic chromophore dramatically change the absorption energy. The most significant alteration in the spectrum, a blue-shift by more than 100 nm and a remarkable reduction of the absorption intensity, is induced by protonation of the phenolic oxygen O Y . As mentioned in Section 4, the dihedral angle between both rings in the chromophore can be changed. According to our calculation, a distortion of the planar structure of the chromophore results in a significant reduction of the long-wavelength absorption intensity, for example, for ŽHO Y , N, O X . we found the oscillator strengths to be 1.05, 0.68, 0.10, and 0.01 for conformations with dihedral angles Table 2 Calculated transition energies hn , corresponding wavelengths l, and oscillator strengths f of absorption peaks for various protonated forms of the free chromophore State
l Žnm.
hn ŽeV.
f
ŽO Y , N, O X .y with H bonda ŽO Y , N, O X .y without H bond a ŽHO Y , N, O X . ŽO Y , N, HO X . ŽO Y , HN, O X . ŽHO Y , HN, O X .q ŽHO Y , N, HO X .q ŽO Y , HN, HO X .q ŽHO Y , HN, HO X . 2q
448 445 325 377 507 401 428 402 422
2.77 2.79 3.81 3.29 2.45 3.10 2.90 3.08 2.94
1.24 1.26 1.05 1.22 0.87 0.70 1.22 0.61 1.32
a
Intramolecular hydrogen bond between the hydroxyl group of Ser and the nitrogen center of the heterocycle.
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
d s 08, 268, 458, and 908, respectively. Protonation of the carbonyl oxygen O X of the anion also leads to a blue-shift, by 71 nm, of the absorption peak in ŽHO X ., however, essentially without intensity change. Contrasting with these two cases, protonation of N in the heterocyclic ring which results in a zwitterion ŽO Y , HN, O X . leads to a noticeable redshift of the absorption band and a considerable reduction of the corresponding oscillator strength ŽTable 2.. Three different cations ŽHO Y , HN, O X .q, ŽO Y , HN, HO X .q, and ŽHO Y , N, HO X .q can be formed by double protonation of the anionic chromophore. Calculated differences in absorption energies among these cationic systems are considerably smaller than among the neutral species. Thus, changes in the absorption spectrum of the anionic chromophore by mono-protonation resulting in the neutral moieties are more pronounced than by double protonation producing the cationic species. As can be seen from Table 2, protonation of O Y or O X in the zwitterion ŽO Y , HN, O X . induces a blue-shift of the absorption band by 100 nm. This may have been anticipated by comparison with the anion and the neutral forms ŽHO Y , N, O X . and ŽO Y , N, HO X .. However, the cation ŽHO Y , N, HO X .q exhibits a red-shifted band compared with those of the parent molecules ŽHO Y , N, O X . and ŽO Y , N, HO X .. Interestingly, absorbance of the triple-protonated form, ŽHO Y , HN, HO X . 2q, is similar to that of the anion ŽO Y , N, O X . y ŽTable 2.. 5.3. Mutations of the GFP chromophore The calculated bands of the mutants are presented in Table 3 and compared to observed excitation energies. The mutations Tyr66 ™ Phe, Tyr66™ Trp or Tyr66™ His simplify the absorption spectrum of GFP by eliminating the long-wavelength maximum at 477 nm w4x. The mutation Tyr66 ™ Phe results in the loss of the phenolic hydroxyl group and, consequently, electronic states of the chromophore corresponding to deprotonated O Y do not exist in this mutant. The neutral compound and its cationic forms ŽHN, O X .q and ŽN, HO X .q are to some extent comparable to the states ŽHO Y , N, O X ., ŽHO Y , HN, O X .q, and ŽHO Y , N, HO X .q of the WT-chromophore, respectively. With respect to the related states of these
19
Table 3 Calculated transition energies hn , corresponding wavelengths l, and oscillator strengths f of absorption peaks for various protonated forms of the free chromophore State
l Žnm.
Tyr66™ Phe Neutral ŽHN, O X .q ŽN, HO X .q Tyr66™ Trp Neutral ŽHN, O X .q ŽN, HO X .q Tyr66 ™His ŽHNd , N´ , N, O X . ŽNd , HN´ , N, O X . ŽHNd , N´ , HN, O X .q ŽNd , HN´ , HN, O X .q ŽHNd , N´ , N, HO X .q ŽNd , HN´ , N, HO X .q
360 Žexp.a . 296 355 388 436 Žexp.a . 337 433 445 382 Žexp.a . 340 325 409 387 427 410
a
hn ŽeV.
F
4.19 3.50 3.20
0.87 0.62 1.06
3.68 2.86 2.79
0.79 0.53 0.90
3.65 3.81 3.03 3.21 2.90 3.03
0.88 0.49 0.62 0.44 1.06 0.90
Ref. w3x.
counterparts, the excitation peaks of the mutant species are blue-shifted by ; 30–45 nm; the oscillator strengths change insignificantly, particularly if strength ratios are compared. The best agreement between experimental and calculated excitation energies is obtained for the state ŽHN, O X .q of the mutant chromophore Ž360 nm vs. 355 nm.. Similar results are obtained for the mutation Tyr66 ™ Trp. As for mutation Tyr66 ™ Phe, neutral and two protonated states ŽHN, O X .q and ŽN, HO X .q of the mutant can be related to the states ŽH O Y , N , O X ., ŽH O Y , H N , O X .q , a n d ŽHO Y , N, HO X .q of the WT-chromophore, respectively. The p-electron system of Trp is more extended than that of Phe. This difference is responsible for the red-shift of the absorbance of Tyr66 ™ Trp compared to that of Tyr66 ™ Phe. The excitation energies of the neutral and of the cationic forms ŽHN, O X .q and ŽN, HO X .q of the Trp-containing chromophore are red-shifted by 12, 32, and 17 nm, respectively, relative to those of the related states of the native chromophore Žsee Tables 2 and 3.. Close agreement is found between the absorption energy calculated for ŽHN, O X .q of this mutant, 433 nm, and the observed band, 436 nm. The calculated excitation energies of mutant Tyr66™ His are also listed in Table 3. As men-
20
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
tioned previously, the two N centers of the imidazole ring of His exhibit similar proton affinities and, therefore, the neutral species ŽHNd , N´ , N, O X . and ŽNd , HN´ , N, O X . as well as the positively charged m o ie tie s ŽH N d , N ´ , H N , O X .q and ŽNd , HN´ , HN, O X .q have almost the same energy Žwithin 2 kcalrmol.. Nevertheless, the absorption spectra of these tautomers differ remarkably. Our calculations predict the excitation maxima of Nd-protonated species to be red-shifted by 15–20 nm relative to their N´-protonated counterparts. Also, the ŽHNd .-type tautomers have a more intense absorbance than the related ŽHN´ . moieties. Comparing the calculated excitation energies to the experimental band maximum at 382 nm one concludes that the most likely state of the chromophore is ŽNd , HN´ , HN, O X .q with a computed absorption at 387 nm. 5.4. Proton affinities Based on the calculated transition energies for various protonated forms of the chromophore, one can predict variations of its proton affinity ŽPA. by excitation using a simple relation between energies ŽFig. 3., similar to the Forster cycle w25,26x. The PA ¨ difference of a system in the excited state M ) and its ground state M equals the difference in the transition energies of M and its protonated form HMq: PA Ž M ) . s PA Ž M . q hn Ž M . y hn Ž HMq . . Thus, if the absorbance of M is blue-shifted upon protonation, hn ŽM. - hn ŽHMq. , then the proton affinity PAŽM ) . of the excited chromophore is smaller than that of the ground state, hence ŽHMq. )
Table 4 Calculated proton affinities PA for various forms of the native chromophore of GFP PAŽO Y . GS ES
PAŽO X . D
GS ES
PAŽN. D
GS ES
D
ŽO Y , N, O X .y 323 300 y23 305 294 y11 306 315 9 ŽHO Y , N, O X . 210 231 y21 225 242 17 ŽO Y , HN, O X . 243 228 y15 220 206 y14 ŽO Y , N, HO X . 228 237 9 221 226 5 Comparison of the three protonation sites O Y , O X and N in the ground ŽGS. and singlet excited state ŽES. as well as the corresponding difference D. Energies in kcalrmol.
is more acidic than HMq. By the same token, a red-shifted absorption maximum of HMq compared to the transition energy of M implies that the basicity of the chromophore increases by excitation. Therefore, the considerable blue-shift of the absorption band of ŽHO Y , N, O X . with respect to that of the parent anion ŽTable 2. indicates the hydroxyl group of Tyr to be much more acidic in the excited state of the chromophore than in the ground state. The absolute PA values calculated for the ground states and the lowest singlet states of the various forms of the chromophore are compared in Table 4. The difference in the transition energies of the anion and ŽHO Y , N, O X . by ; 1 eV translates into a decreased proton affinity of O Y by 23 kcalrmol due to excitation. Conversely, the proton affinity of the heterocycle nitrogen increases by excitation ŽTable 4.. The most pronounced increase of PAŽN. Ž17 kcalrmol. is found for the neutral form ŽHO Y , N, O X .. Interestingly, whereas the tautomer ŽHO Y , N, O X . is the most stable one in the ground state Žsee Section 4., its lowest singlet excited state lies 13 kcalrmol above that of the zwitterion ŽO Y , HN, O X ..
6. Discussion 6.1. Analysis of HOMO and LUMO
Fig. 3. Forster cycle for deriving the proton affinity PA) of an ¨ excited state.
As mentioned above, the chromophore absorption peak is due to the HOMO™ LUMO electronic transition. The highest occupied and the lowest virtual molecular orbitals of the anion are sketched in Fig. 4. One notes that the HOMO is localized to a
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
21
6.2. Assignment of spectra
Fig. 4. Sketches of HOMO and LUMO of the anionic form of the chromophore.
considerable extent Ž67%. at the phenol ring of Tyr; this MO is nonbonding with respect to both intraand inter-ring atomic orbital interactions. Since the phenol contribution drops to only 25% in the LUMO, the HOMO™ LUMO transition is accompanied by a charge transfer in the chromophore. Thereby, electron density is withdrawn from both the carbon atoms of the phenol ring and C a of Tyr and redistributed to the heterocyclic ring and C b of Tyr. Since the electron density at O Y is larger in the ground state than in the excited state, one expects protonation of O Y to increase the transition energy due to a more pronounced stabilization of the HOMO, as is indeed the case ŽTable 2.. Conversely, protonation of N, carrying a more negative charge in the excited state, induces a relative stabilization of the LUMO and consequently, a decrease of the HOMO–LUMO energy gap resulting in a red-shifted absorbance, again in line with the calculated excitation energy changes ŽTable 2.. Concomitantly, electronic excitation increases the acidity of the phenol hydroxyl group due to the decreasing electronic density on O Y as well as the basicity of N because of the increasing negative charge of the latter center. The corresponding quantitative estimates are given Table 4 and were discussed in Section 5.4. Thus, the proton-induced changes of the excitation energy and the proton affinity can be rationalized by a simple frontier orbitals analysis of the chromophore.
Based on the results presented in Tables 1–3 we shall now discuss the absorption spectra of GFP. Native GFP shows two peaks at 397 and 477 nm caused by two forms of the protein with different protonation states of the chromophore w13,15,19x. The first peak is usually assigned to the neutral form ŽHO Y , N, O X ., whereas the second peak is ascribed to the anion ŽO Y , N, O X .y w30x. However, this interpretation is not supported by results of the present calculations. Let us consider this point in more detail. First of all, a good agreement found between observed and calculated spectra of the anionic and cationic forms of the chromophore in aqueous solution ŽTable 1. suggests a strategy whereby one compares calculated and experimental transition energies of the chromophore in order to identify spectroscopically relevant tautomers. The calculated absorption of the neutral form ŽHO Y , N, O X . is in the UV region; however, protonation of the second center in the chromophore shifts the absorption peak to the visible region Žsee Table 2.. Three conceivable structures ŽHO Y , HN, O X .q, ŽHO Y , N, HO X .q, and ŽO Y , HN, HO X . q should be considered. Although th e c a lc u la te d a b s o r p tio n p e a k o f ŽHO Y , HN, HO X . 2q lies at 422 nm, formation of the dication seems to be unlikely in the protein or in aqueous solution Žsee Section 4.. The most likely tautomer exhibiting the observed absorbance at 397 nm is ŽHO Y , HN, O X .q Žcalculated at 401 nm, Table 2.. In fact, protonation of O Y as well as of the heterocyclic nitrogen in the chromophore of GFP seems to be quite reasonable as will be discussed in the following. Furthermore, deprotonation of the phenolic hydroxyl of ŽHO Y , HN, O X .q is found to be followed by a considerable red-shifted absorbance in line with experiment. Although the predicted absorption energy of ŽO Y , HN, HO X .q agrees equally well with experiment, such an assignment is hardly possible. Experimentally, the proton affinity of phenolic oxygen is well known to be considerably larger than that of a carbonyl oxygen w45x. According to our calculation ŽTable 4. the proton affinity PAŽO Y . of ŽO Y , HN, O X . is 23 kcalrmol larger than PAŽO X .. Finally, there is no experimental evidence on proto-
22
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
nation of the carbonyl oxygen in proteins. The last two arguments also apply to the tautomer ŽHO Y , N, HO X .q which we therefore also exclude. Moreover, deprotonation of either O Y or O X of this tautomer results in blue-shifted absorbance which is inconsistent with experimental data w19,20,30x. Thus, the absorption peak at 397 nm observed in the W T -p ro tein is assig n ed to th e catio n ŽHO Y , HN, O X .q rather than to the neutral form ŽHO Y , N, O X . as assumed previously. Thus, we can conclude that the spectroscopically relevant form of the chromophore in WT-GFP is the cation ŽHO Y , HN, O X .q with the protonated heterocyclic nitrogen atom. Independent support for the protonation of the nitrogen center in the heterocycle of the chromophore can be obtained from a comparison of experimental and calculated spectra of the mutant chromophores ŽTable 3.. The mutant chromophore Tyr66 ™ Phe and Tyr66™ Trp can exist only in three forms ŽN, O X ., ŽHN, O X .q, and ŽN, HO X .q. This makes the assignment of their absorption peaks easier compared to those for the WT-chromophore. The absorbance of the neutral forms of Tyr66™ Phe and Tyr66 ™ Trp are to be expected in the UV region, at 296 and 337 nm ŽTable 3., respectively, at variance with experimental data Ž360 and 436 nm, respectively.. Therefore, we exclude neutral species as relevant. On the other hand, very good agreement with experiment is found for both mutants in the form ŽHN, O X .q. The calculated absorption energy of the species ŽN, HO X .q does not agree as well with experiment as that of ŽHN, O X .q. Again, due to the considerable difference in proton affinities PAŽN. ) PAŽO X ., formation of ŽN, HO X .q seems to be unlikely Žsee Section 4.. As to the mutant Tyr66 ™ His, two points are worth noting. First, best agreement between calculated and observed absorption energies is found for the cation ŽHN´ , HN, O X .q with the heterocyclic nitrogen protonated. Second, although the cations Ž HN ´ , HN, O X . q and ŽHNd , HN, O X .q are of almost equally stable, their spectroscopic properties differ remarkably. This finding may be taken into account when designing new GFPs. The experimental absorption peak at 477 nm found for native GFP lies between the excitation maxima at 401 and 507 nm calculated for the cation
ŽHO Y , HN, O X .q and the zwitterion ŽO Y , HN, O X ., respectively ŽTable 2.. In ŽHO Y , HN, O X .q a proton is bound to O Y with an interatomic distance RŽO Y – ˚ whereas in the zwitterion this proton is H. of ; 1 A, removed from the chromophore, formally to infinite distance. In GFP the proton is transferred from O Y of the chromophore to its immediate environment containing water molecules, His148, and Thr203 Žsee Fig. 1.. To estimate the effect of the proton location on the absorption of the chromophore, the aqua com plexes wŽ H O Y , H N , O X . q P H 2 O x and wŽO Y , HN, O X . P H 3 Oqx were calculated. The formation of hydrogen bonds between the chromophore and surrounding water molecules does not induce significant alterations of the spectrum. In fact, both the formation of aqua complexes of the chromophore ŽTable 1. as well as the intramolecular hydrogen bond between N of the heterocycle and the hydroxyl group of Ser65 ŽTable 2. affect the absorption spectrum only insignificantly. In line with these results, the solvent interaction via a hydrogen bond H 2 O PPP HO Y in wŽHO Y , HN, O X .qP H 2 Ox causes a relatively small change in the spectrum Ž l s 410 nm, f s 0.74. as compared to the free cation ŽHO Y , HN, O X .q Ž l s 401 nm, f s 0.70.. However, interaction of a hydronium ion H 3 Oq with the zwitterion ŽO Y , HN, O X . leads to a considerable shift of the absorption peak w46x. A decrease of the distance ˚ Žthe correRŽO Y –O. to 3.0, 2.7, 2.6, and 2.5 A, sponding distances O Y –Hq are about 1.9, 1.6, 1.5, ˚ respectively. results in blue-shifted maxand 1.4 A, ima at l s 493, 481, 475, and 460 nm, respectively. Based on the results of these calculations we conclude that the observed absorption of GFP at 477 nm can be assigned to the H-bonded complex of the zwitterion with a protonated group Že.g., H 3 Oq. at ˚ Note that a an O Y –O distance of ; 2.6–2.7 A. recent structure analysis w14x yielded the following distances between O Y of the chromophore and its ˚ immediate environment: O Y –OŽThr203. s 2.6 A, ˚ O Y –OŽH 2 O. s 2.6 A. Thus, from the comparison between calculated and experimental absorption energies for the WTchromophore and its mutants, we have suggested a consistent interpretation of the observed spectra implying the heterocyclic nitrogen to be protonated in all relevant species. This conclusion is in line with available crystal data for GFP. On the bases of
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
atomic coordinates of WT-GFP w12x one finds two water molecules in the vicinity of the nitrogen cen˚ respectively. The ter, at distances of 3.3 and 3.5 A, corresponding electrostatic contacts may stabilize the protonated heterocyclic nitrogen. Moreover, in the S65T mutant w14x the distance between the hetero˚ short cyclic nitrogen and Og of Thr65 is only 2.8 A, enough to indicate a strong hydrogen bond between N and Og . 6.3. Mechanism of fluorescence Recently, a mechanism of absorption and fluorescence of GFP has been proposed on the basis of experimental data w19x. Our results just discussed support this mechanism albeit in the following modified form ŽFig. 5.. The two spectroscopically relevant forms of the chromophore of WT-GFP are the cation ŽHO Y , HN, O X .q and the zwitterion ŽO Y , HN, O X . rather than the neutral form ŽHO Y , N, O X . and the anion ŽO Y , HN, O X .y, respectively. ŽHO Y , HN, O X .q exhibits an absorption maximum at 397 nm. The peak at 477 nm corresponds to the absorption of the H bonded complex wŽHq PPP ŽO Y , HN, O X .x of the zwitterion. According to experiment, these two forms in their electronic ground states should be in equilibrium w19,20x. Considerable alterations of the proton affinity of the chromophore by its electronic excitation ŽSection 5.4. lead to the following changes in the system. Due to a significant increase of the acidity of the HO Y
Fig. 5. Experimental and calculated Žin parentheses. absorption and emission transitions in GFP. wHq PPP O Y , HNx designates an H-bonded complex between the zwitterionic form of the chromophore and its environment Žsee text..
23
group in ŽHO Y , HN, O X .q by excitation, the proton of the hydroxyl group is transferred to the chromophore environment, resulting in the excited state of ŽO Y , HN, O X . and a corresponding rearrangement of the chromophore environment, as proposed earlier w19x. Thus, the chromophore in the excited state exists only in the zwitterionic form. From the significant decrease of the proton affinity PAŽO Y . after excitation one expects a weakening of the hydrogen bond between O Y and its environment and, therefore, an increase of the corresponding distance Hq PPP O Y . In turn, this lengthening of the hydrogen bond results in a red-shifted emission. Experimentally, the chromophore fluorescence occurs at 510 nm in good agreement with the transition energy of 507 nm calculated for the zwitterion. After the fluorescence emission the chromophore remains in its zwitterionic form for a relatively long time, enabling an equilibrium to be established between the two forms of the ground state ŽFig. 5.. This slow interconversion between the two conformations of GFP in the ground state seems to account for the rapid loss of the absorption intensity at 397 nm upon exposure of GFP to light at that wavelength w19,20x. 6.4. Effect of mutations of the chromophore enÕironment Due to different hydrogen bonding networks between the chromophore and its environment, two forms of the chromophore have been found in WTGFP w13x. It has been established experimentally w3,22x that the absorption peak at 477 nm is eliminated when Thr203 is replaced by Ile. Therefore, Thr203 should play an important role in stabilizing the deprotonated phenolic ring of the chromophore ŽFig. 1.. On the other hand, mutation of Glu222 to Gly as well as replacing of Ser65 by Gly, Ala, Cys, Val, or Thr all cause the loss of the absorption peak at 397 nm w3,4x. A less drastic effect, namely inversion of the intensity ratio of the two peaks, is observed when Ile167 is mutated to Thr w5x. These effects seem to be hardly predictable computationally. In fact, one can easily estimate that a mutation induced change of only 2 kcalrmol in the relative stabilization of the two states in equilibrium entails a considerable varia-
24
A.A. Voityuk et al.r Chemical Physics 231 (1998) 13–25
tion of the intensity ratio of their absorption maxima Žby a factor of ; 30.. For comparison, we mention that the interaction energy of two unscreened unit ˚ changes by 2 kcalrmol if charges, separated by 5 A, ˚ the distance between them changes by only 0.15 A. Therefore, it is not too surprising that mutations of the chromophore environment can result in the apparent suppression of one of the two excitation maxima. For example, the mutation Ser65 ™ Thr Žwhereby an atom H in Ser65 is replaced by a CH 3 group. shifts the equilibrium between both forms, so that only one absorption maximum at 488 nm can be observed. Even changes in the solvent lead to alterations of the ratio of the absorption peaks w15x. At present, it does not seem possible to compute the relative energies of two GFP conformations with an accuracy of 1 kcalrmol or better and, therefore, to predict the effect of these types of mutations on the ratio of two maxima in the absorption spectra of GFP. However, the effect of mutations leading to a change of the chromophore, e.g. ŽTyr66™ Trp., and therefore to new absorption and emission spectra, may well be predicted with the methods used in the present investigations as shown above.
7. Conclusions Using the semiempirical methods PM3 and INDOrS, we have studied the electronic structure and absorption spectra of native and mutant GFP chromophores as well as their geometries, relative stabilities, and proton affinities. Several forms of the native and mutant chromophores are found to be nonplanar outside the protein environment; however, the corresponding planar structures are only slightly higher in energy Žwithin ; 1 kcalrmol. and, consequently, can be stabilized in GFP due to steric and electrostatic interactions with the chromophore environment. Although hydrogen bonds between the chromophore and water molecules do not significantly affect its spectroscopic properties, proton transfer between the chromophore and its surroundings results in considerable alterations of spectra. The good agreement between computed and observed absorbance of the WT-chromophore and three of its mutants suggests that the structure of important states of the chromophore in GFPs can be identified
by comparing experimental and simulated absorption spectra. In this way, the absorption maximum of WT-GFP at 477 nm has been assigned to the H-bonded complex between the zwitterion ŽO Y , HN, O X . and its environment. Another peak at 397 nm is ascribed to the excitation of the cationic form ŽHO Y , HN, O X .q. Due to the change of the proton affinity of the chromophore by electronic excitation, there is only one important excited state, ŽO Y , HN, O X . ) , responsible for the emission of native GFP. Our results obtained for the native chromophore and its three mutants ŽTyr ™ Phe., ŽTyr ™ Trp., and ŽTyr ™ His. indicate that the heterocyclic nitrogen is protonated both in the relevant ground states and the corresponding excited states. At variance with previous ad hoc suggestions, we conclude that protonation of the heterocyclic nitrogen in the chromophores is a crucial factor which determines absorption and fluorescence of GFP.
Acknowledgements This work has been supported by the Deutsche Forschungsgemeinschaft via SFB 377 and by the Fonds der Chemischen Industrie.
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