J. Phys. Chem. B 2007, 111, 10807-10812
10807
Optical Absorption of a Green Fluorescent Protein Variant: Environment Effects in a Density Functional Study Carlo Camilloni,†,‡ Davide Provasi,*,†,‡ Guido Tiana,†,‡ and Ricardo A. Broglia†,‡,§ Department of Physics, UniVersity of Milano, Via Celoria 16, 20133 Milan, Italy, INFN, Milan Section, Milan, Italy, and The Niels Bohr Institute, UniVersity of Copenhagen, BlegdamsVej 17, DK-2100 Copenhagen, Denmark ReceiVed: March 30, 2007; In Final Form: July 8, 2007
We present an ab initio study of the optical absorption properties of a particularly interesting fluorescent protein (E2GFP), whose complex photophysics still escapes elucidation. In particular, we focus on the role of the protein environment, showing that the effects of both nearby residues and the external field due to residues not accounted for explicitly are needed to properly reproduce the experimental data. The spectra calculated taking such contributions into account provide for the first time a robust identification of the states relevant for the photophysics of this system.
1. Introduction Time-dependent density functional theory (TDDFT) is an invaluable technique to study how the optical properties of molecules depend on their chemical and conformational modifications. The comparison of such calculations with experimental absorption spectra allows, in principle, to capture all the relevant structural information on the nature of the different states of the system. A particularly challenging task in this field is the study of protein chromophores. Although the chromophore is usually composed of a few tens of atoms, and is thus within the computational limits of TDDFT, its optical properties are likely to be affected by its environment, that is by the protein matrix. For example, it is long known that mutations in the sequence of the green fluorescent protein (GFP) can be used to produce mutants that show different optical spectra.1-4 This evidence suggests that it is not possible to account for the properties of protein chromophores neglecting the polarization effects of the rest of the protein. On the other hand, the computational effort and the memory requirements of TDDFT calculations scale quadratically with the system size for finite systems, and, consequently, it is not feasible to perform a full ab initio calculation for a protein composed of 227 amino acids, as in the case of GFP. The basic idea of the present work is to investigate what ingredients beyond the ionic and electronic degrees of freedom of the chromophore are needed to correctly describe the absorption spectrum of a mutated form of GFP. One method of choice in such cases is a hybrid approach, in which the environment is included as a classical field, while the ground-state potential energy surface and the optical excitation energies of the chromophore are described within TDDFT. Such a model is expected to be adequate for cases where the excited state is strictly localized on the chromophore. In other cases, the atoms below a certain distance from the * Corresponding author. E-mail:
[email protected]; phone: +3902-5031765. † University of Milano. ‡ INFN. § University of Copenhagen.
chromophore are included in the first-principle description, and it is supposed that contributions of the residues beyond such a threshold can be ignored. While this approach looks reasonable to model the effects of solvation, it is clear that a complex protein matrix can contribute significantly to the electric-field inhomogeneities around the chromophore. We will show that this is indeed the case for a fluorescent protein (E2GFP), where the inclusion of both residues in contact with the chromophore and the long-range electrostatic contributions of the whole protein is needed to reproduce the absorption properties. The rest of the paper is organized as follows: A brief description of the E2GFP is given in Section 2. The next section outlines the different approximations done in the calculation of the optical spectrum of this protein, while the corresponding results are described in Section 4. A comparison with experimental data is given in Section 5, and some conclusions are drawn in Section 6. 2. The E2GFP Mutant The GFP is widely used as a fluorescent marker in molecular biology, and intense research efforts have been devoted toward understanding the molecular mechanisms of its optical properties in order to improve practical usefulness. The GFP chromophore p-hydroxy-benzylidene-imidazolinone5,6 is formed by cyclization of a Ser-Tyr-Gly tripeptide and dehydrogenation of the central Tyr. In its folded state, the protein backbone forms a β-barrel that shields the chromophore from the solvent, preventing the quenching of fluorescence. Within the β-barrel, the chromophore is surrounded by ionizable residues, a fact that is unusual for the center of a protein,1,7 where neutral hydrophobic amino acids are usually found. Wild-type GFP absorbs blue light (3.13 eV), with a weaker peak at 2.61 eV, and emits green light (2.44 eV) with a high quantum yield.8,9 It should be mentioned that the relative strength of the two absorption peaks depends sensibly on physicochemical factors (such as pH, temperature, and ionic strength).9,10 This fact suggests that the chromophore has different stable forms: it has been proposed that the larger 3.13 eV peak is due to a neutral state (historically called the A state)
10.1021/jp072511e CCC: $37.00 © 2007 American Chemical Society Published on Web 08/22/2007
10808 J. Phys. Chem. B, Vol. 111, No. 36, 2007 and that the minor peak at 2.61 eV is caused by an ionized chromophore (state B).11 Different modifications of the sequence have been studied, generating a series of mutants. Except for a mutation (F64L) that helps the folding of the protein at room temperature, all others only modify the optical properties of the protein and involve residues of the chromophore or those in close contact with it. The mutation of the Ser in the chromophore with a Thr completely suppresses the 3.13 eV peak, amplifying the 2.61 eV peak and red-shifting it to 2.53 eV.12,14 The molecular basis of these effects is complex: structural data shows two major differences in the wild-type structure compared with that of S65T. Thr203 has two conformers in the wild-type protein, the most populated of which is not stable in the mutated form: its side chain rotates to a position where it no longer forms hydrogen bonds with the Tyr66 oxygen atom, and therefore it cannot stabilize a negative charge on the chromophore. A similar effect is seen for the His148 side chain, which also moves away from the Tyr66 hydroxyl in wild-type. To shift to longer wavelengths of the emission peak, π-π stacking of the aromatic ring of the chromophore Tyr has been obtained by mutating the Thr203 into His, Trp, or Tyr.3,13 The stacking reduces the energy of the emission to about 20 nm (and yields a yellow fluorescent protein). In this work we study a mutant of the GFP (containing the mutations T203Y, S65T, and F64L, and called E2GFP in ref 15) that features interesting and complex photophysics, with the presence of fluorescent “bright” states and “dark” states in which no fluorescence is present. Interestingly, it has been found that such states can be reversibly populated and depopulated via photoconversion at specific wavelengths:16,17 upon irradiation at 2.60 eV, one observes an almost complete loss of fluorescence that can be restored using light at 3.54 eV. No clear-cut indication has been found regarding the molecular dynamics involved in this interesting reversible photoconversion. However, the particular shape of the chromophore and fluorescence correlation spectroscopy experiments on the F64L and S65T mutants point to cis-trans photoisomerization as a possible mechanism,18 as this structural modification is a wellknown mechanism for radiationless decay in organic molecules19 (e.g., in the photocycles of retinal in bacteriorhodopsin and of the photoactive yellow protein chromophore). Only indirect evidence has been reported so far to experimentally confirm such a hypothesis. Nonetheless, slight differences in the absorption spectrum of bright and dark states can be detected experimentally,16 and therefore the study of such optical spectra provides direct information on the structural properties of the relevant Kohn-Sham states. Such study is the object of this work. 3. Models and Theoretical Framework 3.1. Structural Optimization. No experimental structural information is available for the E2GFP mutant. Therefore, the structures employed in the present calculations have been obtained from the experimental data available for the wild-type GFP and a subsequent structural refinement of the whole protein by classical molecular dynamics.16,20 The resulting structure contains the chromophore in the neutral state and cis configuration (state Acis, see Figure 1); an intermediate state (I) can be obtained by removing the proton from Tyr66 and thence the anionic state (Bcis) relaxing the geometry.
Camilloni et al.
Figure 1. Ionic cis configuration of the neutral state of the E2GFP chromophore. The atoms included in the model called “dressed chromophore” in the text are represented. Hydrogen atoms that link the quantum part to the classical atoms of the rest of the protein are shown in light gray.
Figure 2. Ionic trans configuration of the neutral state of the E2GFP chromophore. The atoms included in the model called “dressed chromophore” in the text are represented.
Following the above discussion, we have also considered the corresponding states (trans states) in which the benzene ring of Tyr66 is rotated 180° around the bond that connects it to the rest of the chromophore (see Figure 2). After the preparation, all the states of the gas-phase chromophore have been optimized through Car-Parrinello molecular dynamics,21 within the generalized gradient approximation. At this stage, we consider only the residues forming the chromophore (i.e., 65-67). The backbone connecting them to the rest of the protein has been cut. In order to avoid cutting through peptide bonds, the carbonyl group was removed from Gly67, and the carbonyl group of the previous residue Leu64 was retained. Hydrogens were used to reproduce the right valence at the boundary. This model (hereafter referred to as “bare chromophore”) contains 39 atoms and 116 valence electrons. All calculations were performed with the BLYP exchangecorrelation energy functional,22,23 and the interaction of valence electrons with the ions was described using consistently generated norm-conserving pseudopotentials. The use of gradient-corrected functionals to study hydrogen-bond interactions has proven to well reproduce the physical properties of such systems.24
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The system is enclosed in a 16 × 14 × 14 Å3 box, and a cutoff (70 Ry) limits the number of plane waves to about 105. Convergence of the total energy and geometry details with respect to the basis cutoff has been carefully tested. To include the effects of the environment on the optical properties of the chromophore, we choose to also study a more refined model, also including in the first-principle description the residues and the water molecules closer to the chromophore. In particular, we choose to include all residues and crystallographic water molecules that could form hydrogen bonds with the chromophore (i.e., Ser205, Glu220, His148, Arg96, and Gln94). Thr203 was also included since it is involved in the Y203T mutation from the wild-type to the E2GFP. The model (hereafter called “dressed chromophore”) contains 100 atoms and 298 electrons. The details of the calculation were the same as above, except that the cell was enlarged to maintain a distance of at least 4 Å between the cell border and the system. To separately assess the role of the protein matrix on the optical properties of the chromophore, we also considered two other models where either the chromophore alone or the chromophore and its short-range environment are subject to the electrostatic field generated by the rest of the protein (we refer to them respectively as “bare chromophore with external field” and “dressed chromophore with external field”). Such external field was calculated using partial point charges obtained from the Gromos force field.26 The polarization due to the classically treated atoms on the quantum system is described by a Coulomb potential modified at short range. It is known27 that the functional form of this potential can be chosen so as to obtain the correct interaction properties and to prevent an unphysical escape of the electronic density toward the classical atoms (the so-called spill-out effect).25 3.2. Absorption Properties. The optical absorption is obtained by solving the time-dependent Kohn-Sham equations. Solving the Kohn-Sham equation in real time and representing the wave function on a uniform spatial grid (an algorithm first used in nuclear physics, whose details have been described several times28-32) allows for reliable control of convergence without the need to perform a sum over virtual empty KohnSham states.33 This approach therefore provides a natural way to overcome the severe basis set problems occurring in alternative methods. The real-time response to an impulsive perturbation is Fouriertransformed to get the dynamic polarizability in the entire range of interest. The optical absorption strength S(E) as a function of the photon energy E is then obtained from the imaginary part of the polarizability R(E) by the equation
S(E) )
2meE p2e2
ImR(E)
where me is the electron mass. We used a local density approximation (LDA) for the description of the ground state of the system and the corresponding adiabatic LDA for the exchange correlation kernel in the time-dependent Kohn-Sham equations. Use of gradient corrections is possible within this framework, but it has been shown4,34 that the optical absorption is quite insensitive to this change. The numerical parameters that need to be specified for the calculation are the mesh spacing (that is, 0.23 Å), the box shape (that is, a sum of 4 Å spheres centered over each atom), the time step (that is, 0.002 fs), and the number of time steps (that is, 12,000). Thus the total propagation time is T ) 24 fs. One
TABLE 1: Variation of Some Structural Parameters of the Chromophorea system
model
χ2
χ1
θ
Acis
bare chromophore bare chromophore + ext. field dressed chromophore bare chromophore bare chromophore + ext. field dressed chromophore
0.72 1.30 -1.81 0.72 10.4 4.26
1.76 -8.20 -0.51 179.99 177.95 175.65
129.00 134.67 133.64 128.95 134.21 139.29
Atrans
a Angle θ is the one formed by the CC bonds that join the aromatic rings, whereas dihedral angles χ2 and χ1 are the ones defined those bonds and the CN bonds on the aromatic rings themselves.
technical point that should be mentioned is that the Fourier transform over the finite interval T gives peaks that are broadened by the time cutoff. In presenting our results, we remove the spurious oscillations associated with the time cutoff by multiplying by a filter function, amounting to a convolution in the frequency domain. To assess the effects of the protein matrix on the optical properties, we performed a TDDFT calculation of the excited states, including in the Kohn-Sham equations the static term generated by the point-wise contributions to the electrostatic potential, calculated as described above for the ground-state calculation. 4. Results and Discussion 4.1. Structural Effects of the Environment. In going from the cis to the trans configuration, different structural modifications take place. The rotation of the phenol ring removes the hydrogen bond that the water molecule makes with the chromophore (see Figures 1 and 2). This structural rearrangement also increases the distance of the chromophore with the Gln residue, thereby breaking one hydrogen bond between the environment and the chromophore imidazole ring. Also, the alignment of the phenols on the chromophore and the Thr203 changes from a configuration where the atoms of one ring are aligned with those of the other, to one where this alignment is lost. Some angles that give an overall idea of the chromophore conformation are reported in Table 1. Comparing the geometric gas-phase structure (bare chromophore) with more realistic models, one finds, as it has already been observed,34 that the planar structure is the most stable one only in the gas phase and that dressing the system breaks the planarity. No significant variation is found because of the inclusion of the external field. 4.2. Electronic Structure. 1. Bare Chromophore. In the cis configuration, the spectrum of the bare chromophore at low energy comprises two main peaks: one at 3.00 eV and one at approximately 3.46 eV (see Figure 3, left panel, solid black line); although these resonances are by no means restricted to a simple particle-hole character, one can gain physical insight by examining the Kohn-Sham orbitals and studying the noninteracting transitions. The transitions with large transition matrix elements are reported in Table 2 for the cis and trans states. In the bare chromophore (for both the cis and trans configurations), the highest occupied molecular orbital (HOMO) state is a π bonding, and the lowest unoccupied molecular orbital (LUMO) state is a π* antibonding state: both are delocalized on the whole chromophore. As in the case of the wild-type chromophore,34 the low-energy transition receives most of its strength from the π-π* transition between these single-particle states. The higher energy peak is mainly given by another π-π*
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Camilloni et al.
Figure 3. Comparison of the absorption spectra calculated for the first three models described in the text: (i) bare chromophore (black solid lines); (ii) the chromophore with the external field due to the remainder of the protein (red dash-dotted lines); and (iii) the dressed chromophore (blue dashed lines). Panel a shows spectra for the cis configuration, whereas the trans configuration is shown in panel b.
TABLE 2: Largest Transition Matrix Elements (in Debye) M ) ∑µ|〈p|xµ|h〉2 and Energy Differences (in eV) for the cis and trans States of the Bare Chromophore system
∆E
transition
M
Acis
2.18 5.10 3.23 4.71 3.62 2.18 5.11 4.35 3.21 3.71
HOMO-LUMO (HOMO-5)-(LUMO+1) (HOMO-3)-LUMO (HOMO-3)-(LUMO+1) (HOMO-5)-LUMO HOMO-LUMO (HOMO-5)-(LUMO+1) (HOMO-8)-LUMO (HOMO-3)-LUMO HOMO-(LUMO+1)
2.84 1.42 1.06 0.98 0.85 0.39 1.39 1.09 0.94 0.89
Atrans
transition between a double π state (HOMO-6, HOMO-5) and the π* (LUMO+1) state, both almost entirely localized on the phenyl ring. In the trans configuration, the low-energy peak is slightly red-shifted with respect to the cis configuration (2.95 eV), and part of its strength is transferred to the higher energy peak, whose energy remains almost unchanged (see Figure 3, right panel, solid black line). 2. Effect of Field on Bare Chromophore. From the considerations made above regarding the interactions of the chromophore with the environment, it is reasonable that the role of the environment cannot be captured only by a classical shielding effect due to the electrostatic interaction with the rest of the protein. As a matter of fact, the inclusion of such an electrostatic contribution matrix has the same effect on both structures: the low-energy peak is red-shifted, and the higher energy peak is pushed to still higher energy (3.62 eV in the cis case and 3.72 eV in the trans case) and increases its strength (Figure 3, dashdotted red lines). 3. Effect of the Near Residues. The inclusion of the nearby amino acids has a more conformation-dependent effect on the absorption spectra. In the cis state, three peaks can be observed: a strong one at 2.85 eV, and two smaller and closer ones at 3.35 and 3.66 eV (see Figure 3, left panel, blue dashed line). The effect is sensibly different on the trans state, in keeping with the different hydrogen bond topology and stacking with the environment: in this case, we have two strong peaks very close in energy (2.95 and 3.14 eV, ∆E ) 0.19 eV) and a smaller one at higher energy (3.61 eV).
Figure 4. Calculated absorption spectra including the effects of the nearby residues and the electrostatic contributions of the remainder of the protein (dressed chromophore with external field). Panel a shows spectra for the cis configurations, for the neutral (black solid line) and the anionic (red dashed line); the trans configurations are in panel b.
Again, one can gain direct insight on the electronic structure by examining how the single-particle states change in the presence of the environment. Although several states are present in the gap, slightly below Fermi energy one can identify two nearly degenerate π bonding states (states HOMO-7 and HOMO-6 in the cis ∆E ) -0.06 eV, and states HOMO-5 and HOMO-7 in the trans ∆E ) -0.19 eV) and, slightly above, a π antibonding state (state LUMO+1 in the cis, state LUMO+3 in the trans). In terms of the states of the bare model, the π bonding state on the chromophore and the π bonding on the Thr203 phenyl ring mix and produce two states that are delocalized on both the chromophore and Thr203. These two states correspond to the HOMO of the bare system. Since the phenyl rings of the chromophore and Thr203 are staggered in the trans conformation, the two resulting states have different energies, whereas the alignment in the cis conformation accounts for the degeneracy of the two states. For the cis configuration, moreover, the hydrogen bond network also delocalizes the state on Ser205 and Glu222. The phenyl ring of Thr203 provides another π bonding state (state HOMO-2 in the trans state, entirely localized on Thr203, and state HOMO-4 in the cis case, which also somewhat spreads on the phenyl of the chromophore) that lies in the gap, while the π antibonding state remains localized on the aromatic rings of the chromophore, with very little density on the environment. Thus, the main absorption peaks stem again from the states that correspond to the HOMO (that are now split) and the LUMO, and the additional low-energy peak comes from the π bonding state on the Thr and the LUMO. The region of the spectrum at higher energy (E ∼ 3.5 eV) is clearly more difficult to discuss because of the larger number of transitions that contribute to it. 4. Effect of the Field on the Dressed Model. The complete spectra, resulting from the response of the system subject to the static external field is shown in Figure 4. The comparison with the spectra of the same system without such a field (see blue dashed line in Figure 3) testifies that the long-range electrostatic contributions from the protein matrix have a remarkable effect on the response properties. We try to elucidate the effects of the external field starting from the unperturbed system and including perturbatively the contributions of the external field. Following the discussion above, we restrict our analysis to the four most relevant states.
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TABLE 3: Largest Transition Matrix Elements (in Debye) M ) ∑µ|〈p|xµ|h〉|2 and Energy Differences (in eV) for the cis and trans States in the Dressed Model system
∆E
transition
M
Acis
2.20 π2π* 2.14 π1π* 2.01 π′π* 1.04 π′′π* 2.32 π2π* 2.51 π1π* 1.63 π′π*
(HOMO-7)-(LUMO+1) (HOMO-6)-(LUMO+1) (HOMO-4)-(LUMO+1) (HOMO)-(LUMO+1) (HOMO-5)-(LUMO+3) (HOMO-6)-(LUMO+3) (HOMO-2)-(LUMO+3)
2.11 1.87 0.98 1.23 2.10 1.00 0.36
Atrans
TABLE 4: First-Order Perturbation Theory Shifts (in eV) for the Most Relevant Particle-Hole States in the cis and trans Configurations for the Dressed Chromophore Subject to the External Field system
transition
∆Ep
∆Eh
shifta
Acis
π1π* π2π* π′π* π1π* π2π* π′π*
1.95 2.17 0.24 1.87 2.98 -0.1
1.92 1.92 1.92 1.97 1.97 1.97
0.03 0.25 -1.68 -0.10 1.01 -2.07
Atrans
a
Refers to the shift due to the perturbation.
The Kohn-Sham eigenstates of the system in the external field can be obtained on this basis by perturbation theory. To keep our analysis simple, we approximate the external electrostatic field b with its value between the six-membered rings of the chromophore and those of Thr203. A direct inspection shows that this is a good approximation, as both the module and direction of the field are approximately constant in the region, b ) (0.06, -0.49, 0.36) a.u. To first order, the Kohn-Sham eigenvalues are shifted by (0) E(1) i ) Ei +
∑R R ∫ d3r|φi(r)|2rR
where R ) x, y, z are the Cartesian directions, and φi(r) is the wavefunction of the ith orbital. The resulting shifts are reported in Table 4. In the cis case, the dipole diagonal matrix elements of the two π states that are excited to the antibonding π* state are almost collinear, and are thereby shifted by approximately the same energy. This symmetry is lost in the trans case, where one element is shifted more that 1 eV higher than the other; the transition splits into two bands that form the most intense peaks in the complete spectrum. 5. Comparison with Experimental Data We compare the calculated spectra with the spectra obtained for a solution of E2GFP before and after photoconversion using light at 3.5 eV as discussed in ref 16. Since the experimental sample is buffered close to the isoelectric point, both protonation states are expected to be present in solution. We thus fit the experimental data minimizing
∫dE|SExp(E) - ∑ f Ri S Ri (E)|2 iR
with respect to the weights f Ri where i is either the neutral or the anionic state, and R is either cis or trans. As far as the fluorescent sample (prior to photoconversion) is concerned, the best fit is obtained with no contribution from the trans states, in accordance with structural information on the most stable state. The obtained weight for the neutral state is f cis A ) 88%, in qualitative accordance with the fact that the isoelectric point
Figure 5. Comparison of the measured absorption spectra (black solid lines, from ref 16), with the spectra calculated including the effects of the nearby residues and the electrostatic contributions of the remainder of the protein (see text, red dashed lines). Panel a shows spectra for the bright sample; the weights of the different configurations obtained cis by fitting the experimental data are f cis A ) 0.88 and f B ) 0.12. The trans dark sample is in panel b, where f cis ) 0.48, f ) 0.46, f cis A A B ) 0.04, trans and f B ) 0.02.
of the solution is pKa ) 7.2, and the solution is buffered at pH ) 7.36 Both the position and the relative strength of the main peaks are well reproduced by the fit (see Figure 5). As we turn to the absorption of the nonfluorescent state, a fraction of molecules switches to the trans state, and we estimate trans f cis ) 46%. Best values of the weights for the A ) 48% and f A trans ) 2%. It should be anionic state are f cis B ) 4% and f B stressed, however, that due to the strong similarity of the spectra of the anionic cis and trans states (see Figure 4, dashed red curves), only their sum is statistically relevant for the fit. The relative strengths of the two main peaks are well reproduced, and an increased absorption around 3.5 eV, characteristic of the dark state, is obtained. Also, the positions of those peaks are reproduced within the accuracy given by the limited time-evolution of our calculation (∆E ) 0.2 eV). It should be remarked that the energy of some of the main peaks is slightly red-shifted. This is most likely due to the well-known underestimation of the excitation energy within the LDA. 6. Conclusions In conclusion, we have shown how particular care must be taken in comparing calculated absorption properties of systems in complex environments. In assessing the role of the environment, one should keep in mind that its effects are manifold: it induces structural modifications of the gas-phase chromophore, it changes the electronic structure by modifying the electronic states, and it makes local-field modifications of the external electromagnetic field. The effects of the protein matrix in E2GFP seems to be different from that in other proteins of the same family, and these differences could be relevant to account for the strange photophysical properties of this particular system. In wild-type GFP, this role can only be inferred from the fact that the vacuum chromophore has optical properties strikingly similar to those of the chromophore in the protein matrix. This is also the case of the blue fluorescent protein, where ab initio calculations4 have revealed a delicate cancellation of the shielding of the applied electromagnetic field, and only the structural effects seem to be relevant. Differently from those cases, and because the particular environment of E2GFP makes the electronic excitations delo-
10812 J. Phys. Chem. B, Vol. 111, No. 36, 2007 calized on the environment, the optical properties of this system are sensitive to the electrostatic coupling to the rest of the protein. Only when this coupling is included in the calculation can the experimental data be reproduced, and in such a case, the theoretical and experimental data agree to a very satisfactory accuracy. Apart from these general considerations, we can also focus on the implications for the physics of E2GFP. Although our results do not further clarify the origin of fluorescence quenching, where environment excitations and the structural transformations in the excited-state are crucial, we have shown that previous experimental data on this system are consistent with the cis-trans photoisomerization mechanism. With the available experimental data, it is difficult to assess whether this process is the only one involved in the photoconversion from bright to dark state. In particular, ref 16 does not contain information on the fluorescence intensity of the sample corresponding to the absorption spectrum in Figure 5. Without such information, it is not possible to know whether the residual 48% of molecules that we assign to the cis state are still bright or whether some other mechanism quenches their fluorescence. Like Webber et al. claim,35 however, the significance of these results for E2GFPs is that the protein is evidently acting as more than a cage restricting torsional motion of the chromophore. Interactions between the protein and the chromophore must be considered to understand the ability to switch between bright and dark states. Acknowledgment. We thank G. Chirico for stimulating discussions, and R. Nifosı` for kindly providing the structural data for the E2GFP mutant. Computational resources of CILEA have made this work possible. We acknowledge the financial support of the 2003 FIRB program of the Italian Ministry of Scientific Research. References and Notes (1) Phillips, G. N. J. Curr. Opin. Struct. Biol. 1997, 7, 821-827. (2) Tsien, R. Y. Annu. ReV. Biochem. 1998, 67 (1), 509-544. (3) Crameri, A.; Whitehorn, E. A.; Tate, E.; Willem, P. C. Nat. Biotechnol. 1996, 14, 315-319. (4) Lo´pez, X.; Marques, M. A. L.; Castro, A.; Rubio, A. J. Am. Chem. Soc. 2005, 127 (35), 12329-12337. (5) Shimomura, O. FEBS Lett. 1979, 104, 220-222. (6) Cody, C. W.; Prasher, D. C.; Westler, W. M.; Prendergast, F. G.; Ward, W. W. Biochemistry 1993, 32, 1212-1218.
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