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J. Phys. Chem. B 2001, 105, 2850-2857
Chromophore Protonation States and the Proton Shuttle Mechanism in Green Fluorescent Protein: Inferences Drawn from ab Initio Theoretical Studies of Chemical Structures and Vibrational Spectra† Hi-Young Yoo,‡,§ J. A. Boatz,§ Volkhard Helms,|,⊥ J. Andrew McCammon,⊥ and Peter W. Langhoff∇,*,§,#, Department of Chemistry, UniVersity of California, IrVine, California 92619-2025, Air Force Research Laboratory (AFRL/PRS), 10 East Saturn BouleVard, Edwards AFB, California 93524-7680, Max-Planck-Institute of Biophysics, Kennedyallee 70, 60596 Frankfurt, Germany, Department of Chemistry and Biochemistry and Department of Pharmacology, UniVersity of CaliforniasSan Diego, 9500 Gilman DriVe, La Jolla, California 92093-0365, Department of Chemistry, Indiana UniVersity, Bloomington, Indiana 47405-4001, and San Diego Supercomputer Center, UniVersity of CaliforniasSan Diego, 9500 Gilman DriVe, La Jolla, California 92093-0505 ReceiVed: October 6, 2000; In Final Form: January 9, 2001
Assignments are provided of prominent features in the recently measured Fourier transform infrared (FTIR) difference spectra of green fluorescent and photoactive yellow proteins (GFP, PYP) employing ab initio calculations of the ground electronic state structures and vibrational spectra of their chromophores in selected protonation states. Particular attention is addressed to inferring the protonation states of wild-type GFP chromophore and to reconciling the measured FTIR difference spectrum with a proposed proton shuttle mechanism in which protonated and deprotonated forms of the chromophore are paired with corresponding charge states of a Glu222 residue shuttle terminus. The calculated GFP IR difference spectrum obtained from the neutral-anionic pair of chromophores is found to be in general accord with the FTIR measurements on wild-type GFP in its protonated and deprotonated forms, whereas the spectrum obtained from the zwitterioniccationic pair of chromophores provides a less satisfactory simulation of the data. The apparent absence of a carbonyl band in the measured GFP FTIR difference spectrum, a feature expected upon protonation of the carboxylic Glu222 residue, is reconciled by the presence of a carbonyl mode in the imidazole ring of the neutral chromophore which partially obscures the anticipated R-COOH Glu222 feature in the calculated spectrum. By contrast, the corresponding carbonyl mode in the PYP chromophore is predicted to be significantly weaker and at lower frequency than in GFP, accounting in part for the presence of an unobscured prominent R-COOH Glu46 residue carbonyl mode in the measured PYP FTIR difference spectrum. Accordingly, the present ab initio theoretical study supports the predominance of neutral and anionic forms of wild-type GFP chromophore, and it argueably reconciles the available FTIR data with a probable Glu222 terminus of the proposed proton shuttle mechanism in the protein. Additional experimental studies of IR and Raman difference spectra in GFP and PYP, including particularly isotopic substitutions, are suggested to complement additional theoretical studies in progress.
1. Introduction Crystallographic,1-6
absorption/emission,7-18
optical and theoretical19-23 studies of wild-type and mutant green fluorescent proteins (GFP) have contributed to development and refinement of a comprehensive model of the structure and functioning of this important fluorescent marker.24-26 Wild-type GFP is widely regarded to be comprised of two forms of the protein which † Work supported in part by grants from the National Research Council, the U.S. Air Force Office of Scientific Research, and the National Science Foundation. * Please address all correspondence in connection with this manuscript to Professor Peter W. Langhoff, San Diego Supercomputer Center, University of CaliforniasSan Diego, 9500 Gilman Drive, La Jolla, CA 92093-0505. E-mail:
[email protected]. Fax: 858-534-5113. Phone: 858-822-3611. ‡ University of CaliforniasIrvine. § Air Force Research Laboratory. | Max-Planck-Institute of Biophysics. ⊥ Department of Chemistry and Biochemistry and Department of Pharmacology, University of CaliforniasSan Diego. # Indiana University. ∇ San Diego Supercomputer Center, University of CaliforniasSan Diego.
are related by deprotonation of the p-hydroxylbenzylideneimidazolidinone chromophore through a proton shuttle equilibrium which favors the neutral form in vivo, but which can be photoconverted into the anionic form by excited-state proton transfer.7-18 The shuttle mechanism is thought to be disrupted in selected mutant forms of GFP, favoring one or the other of the charge states of the chromophore in these cases, and accounting thereby for associated changes in optical absorption/ emission spectra and other attributes of the protein. This model of GFP provides a plausible basis for reconciling a large body of experimental observations,1-18 and it is supported in certain of its aspects by quantum-mechanical studies of the ground and excited electronic states of neutral, anionic, zwitterionic, and cationic forms of the chromophore.19-22 Moreover, recent electrostatic calculations of protonation and conformational substates in wild-type GFP are in accord with the essential features of the model and, in particular, largely support the notion that the carboxylic group of the Glu222 side-chain residue is the probable terminus of the proton shuttle connecting neutral and anionic forms of the protein-bound chromophore.23
10.1021/jp003683d CCC: $20.00 © 2001 American Chemical Society Published on Web 03/21/2001
Assignments in the FTIR spectra of GFP and PYP Fourier transform (FT) measurements of the infrared (IR) spectrum of wild-type GFP performed prior to and after ultraviolet photoconversion largely confirm a change in protonation state of the chromophore and consequent formation of a second (deprotonated) form,27 and recently reported preresonance-enhanced Raman spectra further support the preponderance of the neutral and anionic forms of the chromophore in wild-type GFP, with neither the cationic or zwitterionic forms judged to be present in significant concentration.28 The measured FTIR difference spectrum, however, has been interpreted as being at odds with protonation of the carboxylic group of the Glu222 residue, with the expected carbonyl band arising from the R-COOH moiety apparently absent in the reported data.27 Moreover, the Raman difference spectra (protein plus solvent minus solvent alone) of wild-type and S65T GFP observed in the double-bond stretching region (1500 to 1800 cm-1) have been interpreted by comparisons with modes localized in the 4-hydroxylbenzylidene ring of a relevant model compound without reference to the presence or absence of contributions from carbonyl or carboxylic vibrations,28 the Raman data consequently neither confirming or contesting protonation of the Glu222 residue in GFP. These circumstance are in contrast to the situation in photoactive yellow protein (PYP), where there is clear evidence of protonation of the Glu46 residue in the measured IR difference (pB - pG) spectrum,27,29 and very recent resonance-Raman spectra of the protonated cis form of wildtype PYP chromphore have correspondingly detected a weak chromophore carbonyl mode in the double-bond spectral region employing isotopic substitution.30 Inferences drawn from the FTIR experiments on GFP and PYP seemingly cast doubt on the role of Glu222 as the terminal site of the shuttle mechanism in GFP and, accordingly, to some degree on the integrity of the overall proton shuttle mechanism in this important protein more generally,24-26 whereas the recently reported Raman data for GFP do not provide unambiguous vibrational assignments of the observed spectral features in the absence of isotope labeling studies.28 Clarifying calculations of the structures and vibrational spectra of the suggested protonation forms of the GFP chromphore, of the complementary forms of Glu residue, and of relevant model compounds can provide timely aid in interpreting the FTIR and Raman measurements and in reconciling these data with the canonical model of the protonation states and proton shuttle mechanism in GFP.1-26 In this article, we report ab initio quantum-chemical studies in restricted Hartree-Fock (RHF) approximation of the structures and vibrational spectra of wild-type GFP chromophore in neutral, anionic, zwitterionic, and cationic forms, of the Glu residue in its neutral and anionic forms, and of model compounds (methyl ester and thioester of p-coumaric acid) found useful in interpretation of the FTIR measurements on GFP and PYP.27,29 The calculated IR difference spectrum constructed from the neutral and anionic forms of the GFP chromophore, and from the corresponding anionic and neutral forms of Glu residue, is found to be in better accord with the measured FTIR difference spectrum constructed from spectra taken prior to and after ultraviolet irradiation than is the spectrum obtained from the zwitterionic and cationic forms of chromophore. Accordingly, the present calculations support the preponderance of the neutral and anionic charge states of the chromophore in wildtype GFP, and the absence of significant concentration of zwitterionic and cationic forms of the GFP chromophore, in agreement with inferences drawn from the recent FTIR and
J. Phys. Chem. B, Vol. 105, No. 14, 2001 2851 Raman data.27,28 It is argued on the basis of the present calculations that carbonyl (R-COOH) and corresponding carboxylic (R-COO-) vibrational modes, anticipated on the basis of the shuttle model upon protonation/deprotonation of the Glu222 residue, may, in fact, be present in the measured GFP FTIR difference spectrum27 but that their assignments are complicated by the presence of related carboxylic and carbonyl modes arising from R-CdO-/R-CdO moieties present in the imidazole ring of the GFP chromophore which partially obscure the former features in the measured spectrum. Similar modes are identified on the basis of the calculations performed on the methyl ester and thioester of p-coumaric acid in the measured difference spectrum (before and after alkalization) of the former compound in solution27 and in the measured FTIR difference spectrum (before and after photoconversion) of PYP.29 In the latter case, the carbonyl mode in the PYP chromophore is found to be significantly weaker and at lower frequency than in the GFP chromophore, a difference attributed to the presence of the heavy adjacent sulfur atom in the PYP chromophore, accounting in part for the unobscured appearance of a prominent R-COOH Glu46 residue mode in the measured PYP FTIR difference spectrum. In view of the significant implications of the absence of protonation of the Glu222 residue for the integrity of the proton shuttle mechanism in wild-type GFP, attention is focused here largely on contributions from this particular residue and from the aforementioned relevant chromophore vibrations to the IR difference spectrum. More detailed interpretive descriptions and assignments of the reported FTIR and Raman measurements in GFP and PYP, which include contributions to the spectra from protonation/deprotonation of other residues, incorporation of the perturbing effects of the protein environment on chromophore modes, and higher-level quantum calculations of the chromophore structures and vibrational states, are reported separately elsewhere.31 2. Calculations Good quality 6-31G(d) basis sets,32 which include d-type polarization functions on all atoms except hydrogen, are employed in RHF calculations of the equilibrium structures and vibrational spectra of the GFP chromophore in its four protonation states. Additionally, related calculations of Glu residue in its anionic (R-COO-) and neutral (R-COOH) forms, and of the methyl ester and thioester of p-coumaric acid chromophores, are also performed in these basis sets. In Figure 1 are shown the structures of the (trans) methyl ester of p-coumaric acid (panel A) and of the neutral (panel B) and zwitterionic (panel C) forms of wild-type GFP chromophore obtained from the present RHF calculations. As indicated in the figure, the anionic forms of p-coumaric acid and GFP are obtained by removal of the hydroxyl proton from the phenol ring of the neutral forms shown (panels A and B), whereas the cationic form of GFP chromophore is obtained by addition of a proton to the negatively charged oxygen of the zwitterionic form (panel C). The calculated structures of the four charged forms of the GFP chromophore are found to give true (local) minima in the total electronic energy surfaces and, correspondingly, to provide all real positive vibrational frequencies in RHF approximation. Similarly, the methyl ester and thioester of p-coumaric acid are found to exhibit stable neutral and anionic structures in RHF approximation in the 6-31G(d) basis sets employed. The neutral and anionic structures of GFP are found to be largely but not exactly planar (panel B), whereas the cationic and zwitterionic forms exhibit some further degree of nonplanarity (panel C).
2852 J. Phys. Chem. B, Vol. 105, No. 14, 2001
Yoo et al. provide an undercorrection for localized stretching modes, which are generally found to be too stiff in RHF approximation relative to experimental values, and an overcorrection for other (bending, twisting, ...) modes. Moreover, such linear scaling leaves the calculated vibrational (IR and Raman) intensities uncorrected for the effects of electron correlation. An alternative procedure which involves adjustments of the calculated Hessian force constant matrix to provide agreement with measured frequencies can also provide adjustments of the calculated intensitites, although this semiempirical method is not adopted here. Rather, the effects of electron correlation on the calculated vibrational frequencies and intensities are estimated directly by performing higher-level ab initio calculations described in sections 3 and 4 below. The calculated frequencies and IR intensities are employed in construction of continuous IR spectra by representing each of the individual calculated lines as Gaussian profiles having spectral widths chosen to simulate the line widths observed in the FTIR measurements. This procedure is largely subjective but serves only to aid comparison between theory and experiment by including the effects of environmental line broadening and experimental line width limiting factors which contribute to the measured profiles in the absence of specific calculations of these effects. The integrated intensities and frequencies of the resulting theoretical line profiles are not subjective and are determined entirely by the calculations performed. 3. Results
Figure 1. Calculated RHF structures of the neutral methyl ester of p-coumaric acid in trans form (panel A), of the neutral form of wildtype GFP chromophore (panel B), and of the zwitterionic form of the GFP chromophore (panel C), all obtained employing 6-31G(d) Gaussian basis sets. The former compound in its thioester form is employed as an appropriate model chromophore for photoactive yellow protein, as described in the text. The anionic forms of the methyl ester of p-coumaric acid and of the GFP chromophore are obtained by removing the hydroxyl proton from the phenol ring of the indicated neutral forms (panels A and B), whereas the cationic form of the GFP chromophore is obtained by adding a proton to the negatively charged oxygen in the indicated zwitterionic form (panel C).
Most noticeable in this connection is the difference in the conformation of the hydroxyl tail on the imidizole ring, which is significantly out of plane in the zwitterionic-cationic forms and largely in plane in the anionic-neutral forms of the chromophore. Of course, the present calculations do not include the interactions of the chromophores with the surrounding protein environment, and so possible steric effects consequent of these interactions not included in the development can cause the chromophore geometries to differ in the protein from those obtained in vacuo, issues which are treated in detail elsewhere.31 The bond distances and angles obtained from the calculations on GFP are in good accord with previously reported RHF results for the neutral and anionic forms of a GFP chromophore which did not include the hydroxyl tail on the imidazolidinone ring of Figure 1B.20,22 The RHF prediction for the structure of the neutral GFP chromophore is in good quantitative accord with available crystallographic data1-5 and with the recently reported structure of a compound that is highly similar to the neutral GFP chromophore.6 The vibrational frequencies obtained from the RHF calculations have been scaled by the factor 0.89 commonly employed to correct uniformly for the absence of electron correlation in the Hartree-Fock approximation.33 This ad hoc procedure can
In Figure 2 are shown calculated IR absorption spectra in RHF approximation for the anionic and neutral trans forms of p-coumaric acid methyl ester (Figure 1A) and the associated difference (anionic-neutral) spectrum in comparison with an experimental difference spectrum obtained from FTIR measurements performed prior to and after alkalization of this compound in solution.27 Note that the spectrum for the neutral form (pCA-ME Neutral) has been plotted upside down to aid in identification of the negative-intensity peaks in the difference spectrum (A-N). The RHF results of Figure 2 are obtained from the calculated frequencies and intensities employing Gaussian full line widths at half-maximum of 35 cm-1. Use of other line widths in the range 10-50 cm-1 provides spectra (not shown) which are also generally satisfactory for purposes of comparison with experiment but which are narrower or broader than the measured line widths. As indicated in the figure caption, the amplitude of the measured profile, reported in arbitrary units, has been scaled to accommodate comparison with the calculated IR intensity profile in km/mol/cm-1. The calculated bands are evidently in one-to-one accord with those in the measurement, as indicated by the correspondence numbers provided in the spectrum, although there are differences in detail in the positions and intensities of the calculated RHF and measured features. The vibrational characters of the prominent features in the calculated spectrum, as determined by inspection of the associated normal-mode displacement vectors, provide assignments of the features in the measured spectrum and aid in assignments of corresponding vibrational modes in the GFP and PYP chromophores, as indicated in section 4 below. Figure 3 depicts the vibrational frequencies and intensities obtained from the RHF calculations on p-coumaric methyl ester, shown as stick heights and in the form of a cumulative intensity distribution, as well as corresponding values obtained from density functional (DFT) and second-order Møller-Plesset perturbtion theory (MP-2). These results provide an estimate
Assignments in the FTIR spectra of GFP and PYP
Figure 2. Calculated RHF IR absoption profiles for the neutral and anionic forms of the methyl ester of p-coumaric acid (Figure 1A) and the corresponding difference (anionic-neutral) spectrum, as indicated, in comparison with experimental values.27 Gaussian line shapes having full line widths at half-maximum of 35 cm-1 are employed in conjunction with the calculated frequencies (scaled by 0.89) and intensities to facilitate comparison with experiment. The calculated IR intensity profiles are in units of km/mol/cm-1, whereas the amplitude of the experimental profile, reported in arbitrary units,27 has been scaled to accommodate comparison with the calculated values. The numbers shown provide assigned correspondences between prominent positiveintensity (circles) and negative-intensity (squares) features in the calculated and measured spectra. Note that the spectrum for the neutral form has been plotted upside down to facilitate identification of the numbered features.
of the effects of electron correlation, absent in the RHF spectra reported in Figure 2, on the vibrational frequencies and IR intensities in p-coumaric acid. The DFT calculations, which employ the familiar B3LYP energy functional, and the MP-2 results are obtained in the 6-31G(d) basis sets at the appropriate optimized chemical stuctures employing standard options in the GAMESS and Gaussian 98 code suites.34,35 Representation of the IR absorption intensities in the form of both stick heights and cumulative distributions provides detailed values for the individual features, as well as convenient overviews of each of the calculated spectra. Evidently, there is some sensitivity of the results to the level of electron correlation included in the calculation, the nature and consequences of which are discussed in section 4 below. In Figure 4 are shown the calculated Glu residue IR difference (neutral-anionic) spectrum, the corresponding GFP chromophore IR difference spectrum for the anionic and neutral forms of the choromophore (Figure 1B), and the sum of the two difference spectra in comparison with the measured FTIR difference spectrum.27 Figure 5 depicts corresponding values obtained employing the zwitterionic and cationic forms of the GFP chromophore (Figure 1C). The contribution from the Glu222
J. Phys. Chem. B, Vol. 105, No. 14, 2001 2853
Figure 3. Vibrational frequencies and IR intensities for the neutral form of p-coumaric acid calculated in restricted Hartree-Fock approximation (bottom panel), B3LYP density-function theory (center panel), and second-order Møller-Plesset perturbation theory (top panel) employing the basis sets indicated in Figure 1 and in the text. The calculated frequencies have been scaled by the indicated factors commonly employed to improve agreeement betweeen theory and measured values,33 whereas the intensities are reported as calculated in the indicated units. Also shown are cumulative intensity distributions to provide an overview of the spectra and to aid in identifying similarities and differences in the calculated values.
residue to the IR absorption difference spectra shown in both cases is obtained by performing RHF calculations on its neutral (R-COOH) and anionic (R-COO-) forms employing a methyl terminal group (RdCH3). The localized nature of the COOH and COO- group vibrations, which are found to be simple Cd O stretching and COO- asymmetric stretching motions, respectively, ensures that the calculated results are largely insensitive to the nature of the terminating group R. As in the case of Figure 2, the calculated GFP IR difference spectrum of Figure 4 is seen to be in general but not precise accord with the measured FTIR spectrum. Specifically, although there is a general correspondence between the calculated and observed features, as indicated by the assignment numbers in the spectrum, the individual positions and intensities of the bands obtained from the RHF calculations are not in precise accord with the measured difference spectrum, particularly in the double-bond spectral region above 1500 cm-1. The calculated zwitterionic-cationic difference spectrum of Figure 5 is seen to be in less satisfactory accord with the measured spectrum and, in particular, does not compare well with the experimental data in the 1550-1750 cm-1 interval. Moreover, there are more significant differences in the predicted and observed intensity distributions in Figure 5 than there are in Figure 4 over the entire spectral region shown, making the peak correspondences indicated in the former largely tenative. Of course, in the absence of isotopic substitution studies the correspondences between the
2854 J. Phys. Chem. B, Vol. 105, No. 14, 2001
Figure 4. As in Figure 2, for the RHF IR absoption difference profiles of Glu residue (neutral-anionic), of the anionic-neutral forms of wildtype GFP chromophore, and the sum of the two profiles, as indicated, in comparison with experimental values.27 The structures R-COOH and R-COO- (RdCH3) are employed to represent the neutral and anionic forms of the Glu residue, whereas the neutral and deprotonated forms of Figure 1B are employed to represent the GFP chromophore. Gaussian full line widths at half-maximum of 15 cm-1 are employed in constructing the profiles shown from the calculated RHF frequencies (scaled by 0.89) and intensities. The assigned correspondences between calculated and observed features are suggestive rather than definitive in the absence of isotopic substitution studies.
calculated and experimental features indicated in Figure 4 are also suggestive rather than definitive. The 15 cm-1 line widths employed for the Glu residue and GFP chromophores in Figures 4 and 5 are significantly smaller than those (35 cm-1) required to fit the solution-phase FTIR spectrum of trans-p-coumaric acid methyl ester reported in Figure 2. In Figure 6, the calculated Glu (anionic-neutral) IR difference spectrum is combined with the difference spectrum obtained for the neutral and anionic forms of the methyl thioester of p-coumaric acid in its trans configuration, which provides an appropriate model chromophore for wild-type PYP in its pB and pG forms, respectively.29,30 As indicated in the figure, full line widths at half-maximum of 25 cm-1 are employed for all the spectra shown. The experimental difference spectrum in this case, obtained from measurements performed before and after photoconversion of the protein, evidently includes a missing data interval (≈1700-1500 cm-1), precluding comparison with the prominent features at 1470 and 1440 cm-1 in the calulated spectrum. There is, however, good correspondence between the calculated and measured four prominent (negative intensity) features in the 1400-1000 cm-1 interval and between the calculated and observed higher-frequency feature in the spectrum. As in the case of Figures 4 and 5, the correspondences betweeen theory and experiment indicated in Figure 6 are tentative in the absence of isotopic substitution studies.
Yoo et al.
Figure 5. As in Figure 4, for the zwitterionic-cationic forms of the GFP chromophore, employing the values and procedures indicated there and in the text.
4. Discussion The methyl ester of p-coumaric acid (Figure 1A) exhibits some of the structural features of the GFP and PYP chromophores, so comparisons between the theoretical and experimental IR difference spectra depicted in Figure 2 provide an illustration of the strengths and weaknesses of the RHF approximation to calculations of IR absorption profiles relevant to these two proteins. Of particular interest in this connection is the highest-frequency negative-intensity band (square 1) at 1760 cm-1 in the calculated neutral spectrum of Figure 2, which clearly corresponds to the feature at 1710 cm-1 in the measured spectrum. This feature, which is found upon examination of the associated normal-mode displacement vector to be a localized carbonyl stretching mode (R-CdO) in the neutral form of the compound, is evidently predicted to appear ≈50 cm-1 above the corresponding measured feature at 1710 cm-1, despite the use of a 0.89 scaling factor for the calculated RHF frequencies in Figure 2. Similarly, the highest-frequency positive-intensity band (circle 1) in the calculated spectrum at 1705 cm-1, which is found to be the corresponding softened R-Cd O- stretching mode in the anionic form of the compound, also appears at (≈25 cm-1) higher frequency than the corresponding feature at 1680 cm-1 in the measured spectrum. By contrast, the calculated lowest frequency positive-intensity mode (circle 8) at 1110 cm-1, which is comprised largely of in-plane hydrogen bending motions in the phenol ring of the anionic form of the compound, appears significantly lower in the spectrum than does the corresponding measured feature at 1175 cm-1. Accordingly, it is clear that the use of a single scaling factor (0.89) is inadequate to bring the calculated RHF vibrational frequencies into coincidence with the measured values, resulting in an undercorrection of the calculated highfrequency stretching modes and an overcorrection for other
Assignments in the FTIR spectra of GFP and PYP
Figure 6. As in Figure 4, for the RHF IR absorption difference spectra of Glu residue (A-N), wild-type PYP (N-A), and the sum of the two profiles, as indicated, in comparison with experimental values.27 The methyl thioester form of the compound of Figure 1A in its neutral and anionic forms is employed to represent the PYP chromophore in its pB and pG states, respectively. Gaussian full line widths at halfmaximum of 25 cm-1 are employed for all calculated spectra shown.
calculated modes. Despite these quantitative discrepancies, which follow one predictable pattern for pure localized stretching modes and another for other vibrational modes, the results of Figure 2 provide confidence in the RHF approximation to correctly predict the appropriate numbers of vibrational features and their correct relative positions and intensities. Of course, it should be noted that the measured spectrum reported in Figure 2 refers to the neutral and anionic forms of p-coumaric acid methyl ester in solution,27 whereas the calculations do not include shifts in vibrational frequencies and intensities due to the perturbing effects of solvent-solute interactions. The calculated vibrational frequencies and IR intensities for the neutral form of p-coumaric acid methyl ester depicted in Figure 3 show clearly the effects of electron correlation on the spectrum. Specifically, the high-frequency R-CdO neutral stretch and the other two high-frequency vibrations obtained from the DFT-B3LYP and MP-2 calculations are seen to be softer by up to ≈60 cm-1 than the corresponding RHF values. As a consequence, the predictions of the correlated calculations are in better accord with the three negative-intensity highfrequency features in the measured spectrum (Figure 2, squares 1, 2 and 3) than is the RHF spectrum. The frequency shifts and changes in intensity due to correlation are somewhat smaller for the vibrations in the 1200-1500 cm-1 interval, although there are clearly differences in detail throughout this interval as well, and the intense closely spaced low-frequency features in the three spectra are clearly sensitive to the computational approximation employed. Moreover, the correlated results of Figure 3 provide intensities that are generally smaller than the RHF predictions, giving rise to uniformly weaker cumulative
J. Phys. Chem. B, Vol. 105, No. 14, 2001 2855 spectral intensity distributions at all frequencies in the DFT and MP-2 spectra relative to the RHF results. Remarks similar to the foregoing apply to DFT and MP-2 calculations of the IR spectrum of the anionic form of p-coumaric acid methyl ester (not shown). Despite the aforementioned differences, the three vibrational spectra reported in Figure 3 are seen to be generally similar in appearance, largely because approximately the same number of strong features are present in each case and their predicted frequencies and intensities are in the same general order. It is only upon examination of an individual mode in the spectrum that the differences in the three predictions of Figure 3 become more apparent, in which case it is generally important to take into account the role of electron correlation in predictions of both vibrational frequency and IR intensity. However, since attention focuses largely on the higher-frequency mid-IR spectral interval, and particularly on R-CdO and R-CdO- modes which are seen from the results of Figures 2 and 3 to be shifted in predictable and well-understood manner by the effects of electron correlation, RHF calculations are judged satisfactory for discussion of the Glu residue and GFP and PYP chromophore carbonyl and carboxylic modes of primary interest here. The calculated RHF IR difference spectra for Glu residue and GFP chromophore depicted in Figure 4 are seen to exhibit features common to the spectrum of Figure 2 for p-coumaric acid methyl ester and to give a difference spectrum that is in general accord with the measured spectrum. Of course, quantitative agreement between theory and experiment is not expected, since the measured spectrum is constructed from the difference of IR spectra for two forms of the protein, each of which is comprised of the chromophore bands and of a great many individual amide bands which reflect their secondary structures.36 Apparently, the calculated and measured spectra are in somewhat better agreement in the 1000-1500 cm-1 interval than in the 1500-1800 cm-1 interval, although differences in detail in positions and intensities between the two results are evident throughout the entire mid-frequency IR range shown. Specifically, the anionic band predicted at 1425 cm-1 (circle 7) is evidently more intense than the corresponding feature in the measurements, and the strong anionic bands at 1345, 1320, and 1150 cm-1 (circles 8, 9, and 12) in the measured spectrum are only qualitatively reproduced by the calculations. Of particular interest in the calculated difference spectrum are the Glu residue and GFP chromophore modes in the double-bond stretching region, which include carbonyl stretching modes (RCOOH/R-CdO) in their neutral forms (circle 1/square 1) and corresponding softened R-COO-/R-CdO- stretching modes in their anionic forms (circle 2/square 2). These four features (squares 1 and 2 and circles 1 and 2) have counterparts in the measured spectrum, although the calculated position of the neutral Glu carbonyl (R-COOH) mode (circle 1) is significantly higher in the spectrum than is the corresponding experimental feature, which apparently appears as a weak shoulder in the measured data, and unambiguous correspondences between the other three calculated and experimental features cannot be drawn on basis of the results Figure 4 alone. It is important to note in the foregoing connections that the measured GFP IR difference spectrum in D2O (not shown) includes a more prominent shoulder feature corresponding to that of Figure 4,27 and that more refined density-functional and configuration-interaction calculations provide neutral Glu residue carbonyl (R-COOH) modes in better agreement with the experimental shoulder feature,31 similar to the results reported in Figure 3 for p-coumaric acid. Moreover, the significant amount of negative intensity in the experimental spectrum in
2856 J. Phys. Chem. B, Vol. 105, No. 14, 2001 the 1625-1700 cm-1 interval strongly implies the presence of a Glu or other residue carboxylic contribution (square 2) in this spectral interval. This negative intensity is not accounted for by the calculated GFP chromophore contributions, and the discrepancy between theory and experiment depicted in Figure 4 would be larger in the 1625-1700 cm-1 interval if it were not assumed that anionic Glu222 contributes to the difference spectrum. Finally, the proximity of the calculated positiveintensity Glu residue carbonyl (R-COOH) mode at 1820 cm-1 and the corresponding negative-intensity neutral GFP chromophore carbonyl (R-CdO) mode at 1775 cm-1 suggests the possiblity of more complete cancellation of these contributions to the IR difference spectrum when both electron correlation and the perturbing effects of the protein environment are included in the theoretical development.31 The calculated zwitterionic-cationic IR difference spectrum shown in Figure 5 appears to include some of the features of the measured spectrum, which have accordingly been assigned correspondence numbers but generally provides a less satisfactory representation of the data than does the anion-neutral spectrum of Figure 4. Specifically, the predicted frequency of the cation chromophore carbonyl stretch at ≈1830 cm-1 (square 1) is at higher frequency than the neutral Glu R-COOH vibration at 1815 cm-1, a situation that is reversed relative to the anionicneutral and apparent experimental results of Figure 4; the zwitterionic chromophore shows an intense carbonyl mode at ≈1715 cm-1 (circle 2) which has no apparent counterpart in the measured spectrum; there are no predicted (positiveintensity) zwitterionic features of sufficient intensity to account for those appearing in the 1550-1650 cm-1 interval in the measured spectrum; and the predicted intensities in the 10001500 cm-1 interval are very weak relative to the strong line predicted at 1520 cm-1 (circle 6) and in very poor accord with the measured intensities in this interval. It should be noted in the foregoing connections that scaling the intensity of the measured spectrum to provide better apparent agreement with the calculations in the 1000-1500 cm-1 interval does not improve agreement in the 1550-1650 cm-1 interval and gives rise to significant discrepancies between the calculated zwitterionic carbonyl (circle 2) and ring-bridging asymmetric C-CdC stretching (circle 6) modes and the measured spectrum. The very strong intensity of the latter vibration is a consequence of the negative and positive charges residing on the phenol and imidizole rings, respectively, in the zwitterionic form of the GFP chromophore, resulting a very large dipole moment derivative in this particular normal mode. It seems clear that the measured spectrum cannot be accommodated over the entire spectral interval by the calculated zwitterionic-cationic IR difference spectrum employing any adjustment of the experimental intensity values. In view of these observations, it can be expected on basis of the calculations that the anionic-neutral forms of the GFP chromophore play a more significant role in wild-type GFP than the zwitterionic-cationic forms under normal conditions, although the latter forms of chromophore may nevertheless be important in the function of GFP, particularly in singlemolecule situations, issues that have been addressed separately elsewhere.19-22 It is interesting to note, moreover, that the Glu and cation chromophore carbonyl modes approximately cancel in the calculated zwitterionic-cationic spectrum of Figure 5, as in the case of the anionic-neutral spectrum of Figure 4, suggesting that such cancellation between chromophore and residue carbonyl modes can be expected to be more rather than less likely a priori. As in the case of Figure 4, the correspondences indicated in Figure 5 between calculated and measured features are largely tenative in the absence of definitive isotopic substitution studies.
Yoo et al. The sign convention employed in the PYP spectra shown in Figure 6 has been reversed relative to that of Figure 4, with the neutral Glu residue features in the difference spectra now appearing as negative-intensity peaks. Despite a missing data interval in Figure 6, the calculations and measurements appear to be in generally good agreement, particularly in the 10001400 cm-1 spectral interval. Of course, it must be assumed that the two strong anionic PYP modes predicted at 1440 and 1480 cm-1 by the calculations have experimental counterparts in the missing data interval. It is seen that the neutral Glu residue carbonyl feature at 1815 cm-1 (square 1) in the calculated spectrum appears at 1740 cm-1 in the measured spectrum, whereas the corresponding neutral PYP (R-CdO) feature (circle 1) appears at 1740 cm-1 in the calculated spectrum. This latter prediction is ≈35 cm-1 lower in frequency than the neutral GFP chromophore carbonyl mode, which appears at 1775 cm-1 in the calculated spectrum of Figure 4, and is well separated from the neutral Glu residue carbonyl feature (square 1) at 1815 cm-1. Moreover, the calculated intensity of the neutral PYP R-CdO feature is significantly weaker than the corresponding neutral GFP chromophore carbonyl mode, largely as a consequence of the presence of the heavy sulfur atom adjacent to the carbonyl group in the methyl thioester of p-coumaric acid which affects both the frequency and intensity of this mode. These predictions suggest that cancellation of intensity between Glu residue and chromophore carbonyl modes is less likely in PYP than in GFP, in accord with the measured FTIR data of Figures 4 and 6.27,29 5. Concluding Remarks The present investigation has focused attention on selected contributions from the p-hydroxylbenzylideneimidazolidinone chromphore and from the Glu222 residue to the anionic-neutral IR difference spectrum in wild-type GFP. This focus is motivated by the apparent absence of a discernible feature in the 1700-1780 cm-1 interval in the measured FTIR difference spectrum, cited as evidence of the corresponding absence of a (R-COOH) carbonyl stretching frequency in this interval and of protonation of the Glu222 residue, which would have significant implications for the integrity of the proton shuttle mechanism in this important fluorescent protein. The calculations reported in Figure 4 suggest that the expected neutral Glu residue carbonyl band may, in fact, be present in the measured spectrum but that its appearance is distorted by compensating contributions from the carbonyl (R-CdO) mode in the imidazole ring of the neutral GFP chromophore. Additionally, the measured FTIR difference spectrum shows intensity in the 1625-1700 cm-1 interval which would be inexplicable in the absence of contributions from a Glu residue carboxylic (RCOO-) or similar mode, strongly suggesting that the protonation state of Glu222 is different in the protonated and deprotonated forms of wild-type GFP. These assertions are indirectly supported by the presence of a mode in the calculated IR spectrum of the methyl thioester of p-coumaric acid of Figure 6 which corresponds to the GFP chromophore R-CdO mode, but which is significantly weaker and lower in frequency in this model of the PYP chromophore than it is the GFP chromophore. Consequently, the present calculations suggest the carbonyl mode in the FTIR spectrum arising from the Glu46 residue in PYP is less likely to be obscured by the corresponding PYP chromophore mode than is the Glu222 mode in GFP, in accordance with the measured spectra. Although the effects of electron correlation can be expected to give rise to changes from the RHF vibrational frequencies and IR intensities upon which the foregoing assertions are based, as illustrated by the calcula-
Assignments in the FTIR spectra of GFP and PYP tions reported in Figure 3 and by related results reported elsewhere,31 these changes appear to improve the agreement between theory and experiment in the high-frequency interval and so do not alter the foregoing conclusions. Accordingly, the present calculations suggest it is premature to conclude that the measured FTIR difference spectrum in wild-type GFP contradicts the role of the Glu222 residue as the probable terminus of the proton shuttle mechanism in this protein.1-26 It should be emphasized in the foregoing connection that the calculated difference spectra reported here refer to changes only in the in vacuo vibrational modes of the GFP and PYP chromophores and in Glu residue, whereas the experimental spectra refer to changes in vibrational modes of the overall proteins. The perturbing effects of protein solvation on the chromophores and residue vibrational modes, in particular, can affect the appearances of the corresponding features in the IR spectra. Significant differences, for example, are evident in the Glu46 and Glu222 residue protein environments, the former residue experiencing weak hydrophobic interactions37 but the latter residue subjected to stronger hydrogen bonding with a nearby crystal water molecule.4 These qualitative observations, which suggest a much broader and more perturbed carbonyl mode in Glu222 than in Glu46, provide additional support for the foregoing assertions that the Glu222 vibrational bands in GFP are expected to be more difficult to detect than are the corresponding Glu46 modes in PYP. To clarify these issues quantitatively, additional studies are in progress which include the effects on chromophore vibrational frequencies and intensities of electrostatic perturbations from charged and polarizable groups in the protein, and conformational changes in other residues and their possible protonation upon change in chromophore charge state. It is at this level of sophistication that quantum calculations of the required vibrational states which include electron corrrelation and can be expected to provide more accurate frequencies and intensities are also warrented.31 It would be highly desireable if corresponding refined experimental studies, including particularly appropriate isotopic substitutions, were performed of the IR and Raman difference spectra of GFP and PYP in the mid-infrared spectral interval as complements to the theoretical studies in progress. Acknowledgment. This work was supported in part by grants from the National Research Council, the U.S. Air Force Office of Scientific Research, and the National Science Foundation. We thank Professor Peter R. Taylor (SDSC/UCSD) for his hospitality to one of us (P.W.L.) during portions of the course of the investigation, and Professor K. J. Hellingwerf for clarifying correspondence in connection with the GFP FTIR measurements reported in reference 27. References and Notes (1) Ormo¨, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J. Science 1996, 273, 1392-1395. (2) Yang, F.; Moss, L. G.; Phillips, G. N., Jr. Nature Biotechnol. 1996, 14, 1246-1251. (3) Brejc, K.; Sixma, T. K.; Kitts, P. A.; Kain, S. R.; Tsien, R. Y.; Ormo¨, M.; Remington, R. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 23062311. (4) Wachter, R. M.; King, B. A.; Heim, R.; Kallio, K.; Tsien, R. Y.; Boxer, S. G.; Remington, S. J. Biochemistry 1997, 36, 9759-9765. (5) Palm, G. J.; Zdanov, A.; Gaitanaris, G. A.; Stauber, R.; Pavlakis, G. N.; Wlodawer, A. Nat. Struct. Biol. 1998, 4, 261-365. (6) Kurimoto, M.; Subramony, P.; Gurney, R. W.; Lovell, S.; Chmielewski, J.; Kahr, B. J. Am. Chem. Soc. 1999, 121, 6952-6953. (7) Chattoraj, M.; King, B. A.; Bublitz, G. U.; Boxer, S. G. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8362-8367.
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