Isotopic Labeling and Normal-Mode Analysis of a Model Green

May 18, 2002 - Unambiguous vibrational band assignments have been made to 4'-hydroxybenzylidene-2,3-dimethyl-imidazolinone (HBDI), a model ...
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6056

J. Phys. Chem. B 2002, 106, 6056-6066

Isotopic Labeling and Normal-Mode Analysis of a Model Green Fluorescent Protein Chromophore Xiang He, Alasdair F. Bell, and Peter J. Tonge* Department of Chemistry, SUNY at Stony Brook, Stony Brook, New York 11794-3400 ReceiVed: December 18, 2001; In Final Form: March 6, 2002

Unambiguous vibrational band assignments have been made to 4′-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI), a model compound of the green fluorescent protein (GFP) chromophore, with the use of isotopic labeling at positions C1, C3, N4, C5, and the bridging CR. Vibrational spectra were collected using Raman and IR spectroscopy and ab initio normal mode calculations were performed using density functional theory (DFT) and a 6-31G** basis set. Although several reports of calculations and measurements on GFP model compounds have recently appeared, we are able to definitively assign normal modes for the first time because of the use of isotopic labeling. Specifically, in the region between 1750 and 1550 cm-1, we have identified marker bands both in Raman and IR spectra for cationic, neutral, and anionic forms of the chromophore. The Raman bands at 1744 (cation), 1697 (neutral), and 1665 cm-1 (anion) are assigned to νCdO arising from the imidazolinone carbonyl group, whereas the bands at 1647 (cation), 1642 (neutral), and 1631 cm-1 (anion) are assigned to νCdC for the exocyclic CdC double bond. In addition, a band at 1567 (neutral) and 1556 cm-1 (anion) is assigned to a normal mode delocalized over the imidazolinone ring and exocyclic double bond. Importantly, a band at 1582 cm-1 in cationic HBDI also involves a contribution from N-H bending of the protonated imidazolinone N4-H and consequently is very sensitive to deuteration. Because the Raman spectra of neutral and anionic HBDI in H2O and D2O are virtually identical, the sensitivity of the 1582 cm-1 band in cationic HBDI to deuteration provides a means of identifying protonation of the imidazolinone ring in green fluorescent protein. These assignments are discussed with reference to the Raman spectra of GFPs obtained in an earlier study (Bell, A. F.; He, X.; Wachter, R. M.; Tonge, P. J. Biochemistry 2000, 39, 4423-4431) and are crucial for interpreting the vibrational spectra of GFPs.

Introduction The green fluorescent protein (GFP) from Aequorea Victoria has developed into an extremely valuable research tool in molecular and cell biology because of its unique protein scaffold enclosing the 4′-hydroxybenzylidene-imidazolinone fluorescent chromophore.1,2 The chromophore is generated autocatalytically via a posttranslational internal cyclization, dehydration, and oxidation of the Ser65-Tyr66-Gly67 tripeptide.1,3,4 Because chromophore generation occurs inside the folded protein without the need for any cofactors other than molecular oxygen, GFP can be genetically fused to a protein of interest, and the fluorescent signal from the GFP can then report on the expression and transport of the target protein within living cells. In addition, GFP constructs are also being used extensively to analyze protein-protein interactions using fluorescence resonance energy transfer. Finally, irradiation of GFP results in the formation of nonfluorescent dark states (photobleaching and photoconversion), making it possible that GFPs can be used as optical switches or to monitor protein movement within cells using fluorescence recovery after photobleaching.5 All of these applications require a complete understanding of the mechanism of fluorescence. Consequently, it is of great importance to understand the relationship between chromophore structure and fluorescence and the role of the protein matrix in modulating the spectral properties of the chromophore. In this regard, GFP is an excellent model system for understanding how proteins control the photochemistry of a bound chromophore. * To whom correspondence should be addressed. Phone: (631) 6327907. Fax: (631) 632-7960. E-mail: [email protected].

The 27 kDa GFP forms a β-can structure that protects the chromophore from solvent.6,7 The absorption and fluorescence spectra of GFP and model chromophores have been extensively studied.8-11 For wild-type GFP, two absorption bands at 398 and 478 nm are attributed to the neutral and anionic forms of the chromophore, respectively.12,13 This compares to a model compound, ethyl 4′-hydroxybenzylidene-2-methyl-imidazolinone-3-acetate (HBMIA), for which the neutral and anionic forms in aqueous solution absorb at 368 and 425 nm, respectively.13,14 These results demonstrate the importance of both the protonation state and protein environment of the chromophore in determining the absorption maximum. Excitation into the absorption bands of both neutral and anionic forms of the chromophore produces the characteristic green fluorescence of GFP (λmax ) 508 nm), and it is believed that the neutral form undergoes an excited-state proton transfer to an intermediate form that then produces the observed fluorescence.10 Considerable efforts have been made to produce new mutants with altered properties.8,15-18 Recently, a red fluorescent protein has been isolated and cloned from Discosoma st. which has 558 and 583 nm for absorption and emission, respectively.18,19 To design GFPs with novel spectroscopic properties, it is necessary to understand how the protein matrix influences light absorption and emission by the embedded chromophore. Moreover, because it has now been clearly demonstrated that light absorption alters the emission properties of individual GFPs, it is important to analyze the light-induced changes in chromophore structure and determine the spectroscopic properties of the different chromophore structures.10,20,21 Our principal

10.1021/jp0145560 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/18/2002

Model Green Fluorescent Protein Chromophore approach to addressing these issues is to use vibrational spectroscopy, which is a powerful method for providing detailed information on the structure of chromophores bound to proteins.22-24 In particular, many studies have demonstrated the sensitivity of vibrational spectroscopy to important structural details such as the protonation state and the effect of light absorption on the structure of bound chromophores, for example, in systems such as photoactive yellow protein (PYP)25-28 and rhodopsin (reviewed in Mathies, 198729). It is worth noting that important information on both the isomerization and protonation states of the chromophore in the photobleached intermediate of PYP was acquired with the aid of isotopic labeling experiments and DFT calculations.28 The sensitivity of vibrational spectroscopy to protonation and isomerization is particularly important in the context of GFP, because these are two methods through which the protein matrix can modulate the photophysics of the chromophore. In addition, because vibrational frequencies are a function solely of the electronic ground state, combination with absorption spectroscopy enables interactions that alter the ground and excited-state structures to be differentiated.30 Several vibrational studies have already appeared on GFP and model compounds.14,21,31-34 Recently, we obtained Raman spectra on wild-type GFP and the S65T mutant together with data on a model chromophore (HBMIA).14 These data enabled us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It was found that the ground state structure of the anionic form of the chromophore is strongly dependent on the chromophore environment, whereas the neutral form is considerably less sensitive. In addition, a linear correlation between the absorption properties and the groundstate structure was demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm-1. Hellingwerf and co-workers have characterized the photoconversion of GFP with Fourier transform infrared (FTIR) spectroscopy.21 Their results showed that the chromophore in the protein changes its protonation states upon photoconversion. However, no evidence was found for a change in the protonation states of residues close to the chromophore or for the isomerization of the double bond which links the two rings of the chromophore upon absorption of light. Recently, Langhoff et al. reported GFP mode assignments by comparing Fourier transfer infrared (FTIR) difference spectra of GFP and PYP with ab initio calculations of the ground electronic state structures and vibrational spectra of their chromophores in selected protonation states.31 On the basis of their studies, the protonation state of the chromophore (neutral or anionic) was correlated with the protonation state of Glu222 which is believed to lie at the end of a proton shuttle connecting the chromophore and the remainder of the protein. The role of Glu222 in GFP photochemistry has recently been further refined on the basis of X-ray crystallographic studies.35 Schellenberg et al. used resonance Raman spectroscopy to examine the protonation states and excitation dynamics of GFP together with another model chromophore, 4′-hydroxybenzylidene-2,3-dimethyl-imidazolinone, (HBDI, Figure 1).32,33 With the aid of ab initio normal mode calculations they assigned several vibrational transitions. More importantly, it was suggested by their data that the protein influences the structural evolution of the excited states. Tozzini et al. have also calculated the ground-state geometries, electronic structures, and vibrational spectra of a model GFP chromophore using ab initio methods.34 Although the earlier experimental and theoretical studies on GFPs and model compounds have provided useful insights into the

J. Phys. Chem. B, Vol. 106, No. 23, 2002 6057

Figure 1. Structural formula of 4′-hydroxybenzylidene-2,3-dimethylimidazolinone, HBDI.

structure of GFP chromophores, more detailed and accurate vibrational band assignments are required to reach firm structural conclusions. A powerful method to achieve this goal is to use isotope labels together with normal mode calculations. In the current study, we have extended the previous work on the vibrational spectra of GFP and model compounds by introducing isotope labels at key positions in HBDI. The Raman and IR spectra of neutral, anionic, and cationic HBDI and its isotopically labeled analogues have been obtained, and the majority of the most prominent vibrational bands have been assigned with the aid of ab initio normal mode calculations. Using this information we are able to revisit the wild-type GFP vibrational data and improve our understanding of the role of protein interactions in determining the properties of GFP chromophores. Experimental Section Chemicals. 4-Hydroxybenzaldehyde was purchased from Aldrich. N-Acetylglycine and methylamine were purchased from Acros. 4-Hydroxybenzaldehyde-R-13C and acetic anhydride-1,1′13C were purchased from Isotec. Glycine-1-13C, glycine-2-13C, 2 and glycine-15N were purchased from Cambridge Isotope Labs. Synthesis. 4′-Hydroxybenzylidene-2,3-dimethyl-imidazolinone, HBDI. HBDI was prepared basically as described by Kojima et al. and recrystallized from ethanol/methylene chloride (10: 1).2 The only modification to this synthetic route was that 4-hydroxybenzaldehyde was used as the starting material instead of 4-acetoxybenzaldehyde because 4-hydroxybenzaldehyde was converted to 4-acetoxybenzaldehyde when the corresponding azlactone was formed. 1H NMR (250 MHz, methanol-d4): δ 2.38 (s, 3H, 3-CH3), 3.18 (s, 3H, 2-CH3), 6.83 (d, 2H, J ) 8.8 Hz), 7.01 (s, 1H), 7.98 (d, 2H, J ) 8.8 Hz). 13C NMR (250 MHz, DMSO-d6): δ 15.24 (C-CH3), 26.17 (N-CH3), 115.75 (C2′ and C6′ on phenyl ring), 125.40 (C1′ on phenyl ring), 125.48 (bridging C), 134.10 (C3′ and C5′ on phenyl ring), 136.31 (C5 on imidazolinone), 159.56 (C4′ on phenyl ring), 162.25 (C3 on imidazolinone), 169.85 (C1 on imidazolinone). Molecular weight: EI-MS M+ calculated for [C12H12N2O2] ) 216.0899, found ) 216.0896. 4′-Hydroxybenzylidene-2,3-dimethyl-imidazolinone-3-13C, HBDI-3-13C. HBDI labeled with 13C on the imidazolinone 3-C

6058 J. Phys. Chem. B, Vol. 106, No. 23, 2002 carbon (HBDI-3-13C) was prepared as described for HBDI except that N-(acetyl-1-13C)-glycine was used instead of Nacetylglycine. N-(Acetyl-1-13C)-glycine was prepared by Nacylating glycine with acetic anhydride-1,1′-13C2.36 1H NMR chemical shifts were identical to those given above for unlabeled HBDI. The 13C NMR spectrum was identical to that of the unlabeled HBDI, except that the peak at 162.25 ppm was greatly increased in intensity, consistent with 13C labeling of the C3 imidazolinone carbon. Molecular weight: EI-MS M+ calculated for [C1113C1H12N2O2] ) 217.0932, found ) 217.0940. 4′-Hydroxybenzylidene-2,3-dimethyl-imidazolinone-4-15N, HBDI-4-15N. HBDI labeled with 15N on the imidazolinone 4-N nitrogen (HBDI-4-15N) was prepared as described for HBDI except that N-acetylglycine-15N was used. N-Acetylglycine-15N was prepared by N-acylating glycine-15N with acetic anhydride.36 1H NMR chemical shifts were identical to those given above for unlabeled HBDI except that a singlet at 7.01 ppm, assigned to the proton attached to the bridging CR carbon, was split into a doublet (1JH-C ) C-N ) 4.8 Hz), and the singlet at 2.38 ppm, assigned to the methyl group attached to the imidazolinone C3 carbon, was split into doublet (1JH-C-CdN ) 2.8 Hz), consistent with 15N labeling at N4. The 13C NMR spectrum was identical to that of unlabeled HBDI. Molecular weight: EI-MS M+ calculated for [C12H12N115N1O2] ) 217.0869, found ) 217.0867. 4′-Hydroxybenzylidene-2,3-dimethyl-imidazolinone-5-13, HBDI13 5- C. HBDI labeled with 13C on the imidazolinone 5-C carbon (HBDI-5-13C) was prepared as described for HBDI except that N-acetylglycine-2-13C was used. N-Acetylglycine-2-13C was prepared by N-acylating glycine-2-13C with acetic anhydride.36 1H NMR chemical shifts were identical to those given above for unlabeled HBDI. The 13C NMR spectrum was identical to that of the unlabeled HBDI, except that the peak at 136.31 ppm was greatly increased in intensity, consistent with 13C labeling of the imidazolinone C5 carbon. Molecular weight: EI-MS M+ calculated for [C1113C1H12N2O2] ) 217.0934, found ) 217.0934. 4′-Hydroxybenzylidene-2,3-dimethyl-imidazolinone-1-13C, HBDI-1-13C. HBDI labeled with 13C on the imidazolinone 1-C carbon (HBDI-1-13C) was prepared as described for HBDI except that N-acetylglycine-1-13C was used. N-Acetylglycine1-13C was prepared by N-acylating glycine-1-13C with acetic anhydride.36 1H NMR chemical shifts were identical to those given above for unlabeled HBDI, except that the singlet at 7.01 ppm, assigned to the bridging CR carbon, was split into a doublet (1JH-C ) C-C ) 4.8 Hz), and the singlet at 3.18 ppm, assigned to the methyl group attached to N2 on the imidazolinone ring, was split into a doublet (1JH-C-C-C ) 2.5 Hz). The 13C NMR spectrum was identical to that of unlabeled HBDI, except that the peak at 169.85 ppm was greatly increased in intensity, consistent with 13C labeling of the imidazolinone C1 carbon. Molecular weight: ESI-MS M+ calculated for [C1113C1H12N2O2] ) 217.0932, found ) 217.0927. 4′-Hydroxybenzylidene-R-13C-2,3-dimethyl-imidazolinone, HB13 R- C-DI. HBDI labeled with 13C on the benzylidene carbon (HB-R-13C-DI) was prepared as described for HBDI except that 4-hydroxybenzaldehyde-R-13C was used instead of 4-hydroxybenzaldehyde. 1H NMR chemical shifts were identical to those given above for unlabeled HBDI, except that the singlet at 7.01 ppm, assigned to the bridging CR carbon, was split into a doublet (1JH-C ) 154 Hz). The 13C NMR spectrum was identical to that of the unlabeled HBDI, except that the peak at 125.48 ppm was greatly increased in intensity, consistent with 13C labeling of the bridging CR carbon. Molecular weight: EI-MS M+ calculated for [C1113C1H12N2O2] ) 217.0931, found ) 217.0932.

He et al.

Figure 2. Characteristic absorption spectra for the cationic (-; 1 M HCl), neutral (‚‚‚; acetate buffer, pH 5.5, 20 mM), and anionic (- -; 1 M NaOH) forms of HBDI.

UV-Visible Absorption, Fluorescence, Raman, and FTIR Spectroscopies. UV-visible absorption spectra were obtained on a Cary 100 Bio spectrophotometer (Varian). The Raman spectra were acquired using an instrument described previously.37 500 mW of near-IR excitation (752 nm) was provided by a model 890 Ti:sapphire laser (Coherent, Santa Clara, CA), pumped by an Innova 308C argon ion laser (Coherent). The FTIR spectra were obtained using a Bio-Rad FTS 40A spectrometer. Fluorescence spectra were obtained using a Spex Fluorolog-3 spectrofluorimeter. Neutral solutions of HBDI in H2O/D2O were prepared by dissolving compounds in 20 mM sodium acetate and adjusting the pH/D to 5.5 using dilute HCl/ DCl. Acidic and basic solutions of HBDI in H2O and D2O were prepared by dissolving HBDI in H2O/D2O and adjusting the pH/D to 1 or 14 using 12 M HCl/DCl or 10 M NaOH/15 M NaOD, respectively. For FTIR spectroscopy, solutions of HBDI were prepared in dimethyl sulfoxide (DMSO) and methanol-d4 at concentrations of 30 mM and 15 mM, respectively. Vibrational Calculations. The ab initio normal mode calculations on HBDI and its isotopically labeled analogues were performed on an Indigo II SGI using Gaussian 98.38 Results Absorption and Fluorescence Spectra of HBDI. The absorption spectra of HBDI shown in Figure 2 are almost identical to those of HBMIA.14 λmax for the neutral, cationic, and anionic forms of HBMIA appear at 368, 393, and 428 nm, respectively, compared to 368, 391, and 425 nm for HBDI. The slight difference between the absorption maxima of the two model chromophores in the cationic and anionic forms is presumably due to the acetate substituent in HBMIA. Titration of HBDI yields two macroscopic pKa’s of 1.4 and 8.0, which are similar to the previously reported values of 1.8 and 8.2 for HBMIA and which have been assigned to the ionization of the imidazolinone N3 nitrogen and of the phenolic hydroxyl group, respectively.14 The absorption spectrum of the GFP chromophore anion in vacuo was obtained recently by Andersen et al., and the absorption maximum was found at 479 nm, which is close to the value for the anion within the protein.39 The fluorescence spectra of neutral, anionic, and cationic HBDI are extremely weak (data not shown). For wild-type GFP, the quantum yield has been reported to be about 0.8, whereas in model compounds at RT, a value of less than 0.0001 has

Model Green Fluorescent Protein Chromophore

Figure 3. Raman spectra of HBDI and its isotopically labeled analogues in sodium acetate buffer (pH 5.5, 20 mM). The solvent background has been subtracted. The concentrations were 0.3 mM, and the spectra were accumulated for 10 min.

been observed.13 It is possible that the lack of fluorescence for the model compounds in solution results from excited-state deactivation via rotation around the exocyclic CdC double bond of the chromophore,40 although some recent studies have cast doubt on this mechanism.41 In contrast, it has been hypothesized that in the GFP the chromophore is constrained by the protein environment and is not able to freely isomerize.13,40,42,43 Vibrational Assignments for HBDI. To be able to assign which form of the chromophore predominates inside the protein, it is of great importance to assign the vibrational spectra for each protonation state of the model GFP chromophore. Our initial model studies have focused on ethyl 4′-hydroxybenzylidene-2-methyl-imidazolinone-3-acetate (HBMIA). Raman studies revealed that each of the different protonated forms of HBMIA gave a clear signature in the double bond stretching region between 1500 and 1700 cm-1 and that the ground state structure of the anionic form of the chromophore was strongly dependent on the chromophore environment whereas the neutral form seemed to be insensitive.14 We are now able to refine these observations using isotope labeling and ab initio calculations to provide unambiguous vibrational band assignments on the related chromophore HBDI. HBDI has been used for these studies as it can be produced in much higher yields than HBMIA, an important concern when generating isotope labels.2,44 The Raman spectra of HBDI and HBMIA are basically the same except for some minor differences in the region between 1000 and 1100 cm-1 which are ascribed to the change in substituent (methylene acetate to methyl) at the 2 position. Our results on HBDI will be discussed with reference to recent publications by Parson and co-workers,32,33 who used vibrational spectroscopy and computational methods for the mode assignments of HBDI, and Tozzini et al., who used DFT methods to

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Figure 4. IR spectra of HBDI and its isotopically labeled analogues in DMSO. The solvent background has been subtracted. The concentrations were 30 mM.

study the vibrational properties of 4′-hydroxybenzilidene imidazolinone (HBI).34 Neutral HBDI. The Raman and IR spectra of neutral HBDI and its isotopically labeled analogues are shown in Figures 3 and 4, respectively. The Raman data were obtained in sodium acetate buffer at pH 5.5, whereas the IR data were obtained in DMSO, because of the higher concentration requirements for the IR experiments and the relatively low solubility of neutral HBDI in water. The observed transitions together with their assignments are listed in Tables 1 (Raman) and 2 (IR). In addition, Table 3 contains the corresponding Raman band assignments for neutral HBDI in D2O (spectra not shown). The highest frequency band below 1800 cm-1 in unlabeled HBDI is the weak 1697 cm-1 Raman band and the strong 1699 cm-1 IR band. This band appeared at 1677 cm-1 in the IR spectrum of HBDI reported by Esposito et al.,32 likely reflecting the use of KBr pellet over solution, and was assigned to the imidazolinone CdO stretching vibration. This assignment can now be verified on the basis of isotope labeling. Specifically, in the IR spectrum of neutral HBDI-1-13C, the 1697 cm-1 band is shifted 30 cm-1 to 1667 cm-1. In the Raman spectrum, the weak band at 1697 cm-1 disappears, with the new band presumably shifting under stronger bands at lower wavenumber. In addition, the position of the 1697 cm-1 band is unaffected in any of the other labeled compounds confirming its assignment. The 1642 cm-1 Raman band and 1641 cm-1 IR band for unlabeled HBDI are assigned by isotope labeling to the CdC stretching mode associated with the exocyclic CdC double bond. We separately 13C-labeled the two carbons in the exocyclic CdC double bond (CR and C5) and obtained their vibrational spectra. The 1641 cm-1 band observed in the IR spectrum of unlabeled HBDI shifts to 1622 and 1625 cm-1 in HBDI-5-13C and HB-R-13C-DI, respectively. In addition, the

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He et al.

TABLE 1: Positions of Raman Bands for the Neutral, Anionic, and Cationic Forms of HBDI and Its Isotopically Labeled Analogues in H2O between 1000 and 1800 cm-1 HBDI

HB-R-13C-DI

HBDI-1-13C

HBDI-3-13C

HBDI-4-15N

HBDI-5-13C

description

a 1642 1603 a 1567 1449 1317 1234 1178 1036

1697 1624 1603 1588 1559 1450 1314 1231 1178 1034

a 1660 1600 a 1565 1447 1315 1233 1176 1028

a 1642 1603 a 1567 1450 1320 1235 1178 1038

Neutral 1697 1643 1606 1591 1561 1452 1316 1233 1178 1031

1665 1631 1579 1556 1533 1503 1439 1371 1310 1246 1171 1144 1037

a 1615 1579 1549 1534 1500 1436 1367 1308 1242 1169 1140 1033

a 1643 1579 1553 1534 1501 1438 1370 1306 1244 1169 1138 1027

a 1630 1581 1557 1533 1502 1438 1372 1310 1246 1172 1145 1037

Anionic a 1630 1578 1554 1533 1502 1438 1369 1306 1245 1171 1142 1033

a 1613 1581 1554 1534 1497 1436 1356 1306 1240 1169 1131 1033

CdO str CdCstr phenol imidazolinone + CdC str phenol phenol phenol C-H def

1744 1647 1595 1582 1378 1338 1289 1234 1181 1151 1020 1009

1744 1633 1596 1582 1381 1338 1289 1235 1183 1155 1022 1011

1692 1645 1600 1584 1381 1345 1289 1237 1183 1150 1023 997

1744 1650 1600 1586 1381 1341 1289 1237 1183 1155 1026 1012

Cationic 1746 1650 1600 1580 1381 1341 1290 1236 1183 1151 1022 1008

1743 1628 1595 1582 1377 1333 1290 1236 1182 1144 1024 1009

CdO str CdCstr phenol N-H bend phenol phenol phenol phenol phenol C-H def

a

1697 1622 1602 a 1562 1450 1312 1229 1178 1033

CdO str CdCstr phenol phenol imidazolinone + CdC str C-Hdef phenol phenol Phenol C-H def imidazolinone C-C str + CdO bend

phenol phenol C-Hdef imidazolinone imidazolinone C-C str + CdO bend

phenol imidazolinone C-C str + CdO bend

Not observed; str: stretching; def: deformation.

TABLE 2: Positions of IR Bands for the Neutral, Anionic, and Cationic Forms of HBDI and Its Isotopically Labeled Analogues between 1500 and 1800 cm-1 HBDI

HB-R-13C-DI

HBDI-1-13C

HBDI-3-13C

HBDI-4-15N

HBDI-5-13C

description

1697 1622 1602 1582 1558 1513

CdO str CdCstr phenol phenol imidazolinone + CdC str phenol

1699 1641 1602 1583 1562 1514

1697 1625 1602 1585 1557 1514

1667 1633 1602 1583 1561 1514

Neutral (in DMSO) 1699 1697 1641 1641 1602 1602 1583 1582 1562 1555 1514 1514

1669 1630 1583 1557 1499

1665 1619 1581 1548 1498

1645 1622 1579 1552 1499

Anionic (in 1 M NaOD) 1670 1670 1629 1629 1581 1579 1557 1554 1500 1498

1663 1615 1581 1554 1495

CdO str CdCstr phenol imidazolinone + C-C str phenol

1748 1648 1599 1538 1518

1746 1630 1595 1539 1517

1702 1643 1598 1538 1517

Cationic (in 1 M DCl) 1747 1748 1648 1649 1599 1597 1538 1536 1517 1519

1748 1627 1596 1539 1516

CdO str CdCstr phenol phenol phenol

str: stretching; def: deformation.

1642 cm-1 band observed in the Raman spectrum of unlabeled HBDI is not observed in the Raman spectra of the two labeled compounds because it has presumably shifted underneath the high wavenumber wing of the strong 1603 cm-1 transition.

The next Raman band at 1603 cm-1 and the two bands at 1602 and 1583 cm-1 in the IR spectrum are unaffected by isotope labeling. Because all of the bonds in the imidazolinone ring and the bridging carbon have been labeled, this is consist-

Model Green Fluorescent Protein Chromophore

J. Phys. Chem. B, Vol. 106, No. 23, 2002 6061

TABLE 3: Positions of Raman Bands for the Neutral, Anionic, and Cationic Forms of HBDI and Its Isotopically Labeled Analogues in D2O between 1000 and 1800 cm-1 HBDI

HB-R-13C-DI

HBDI-1-13C

HBDI-3-13C

HBDI-4-15N

HBDI-5-13C

description

a 1625 1602 a 1562 1235 1178 1038

CdO str CdCstr phenol phenol imidazolinone + CdC str C-O str phenol C-H def imidazolinone C-C str + CdO bend

a 1643 1603 a 1568 1236 1179 1039

1692 a 1603 a 1560 1237 1181 1037

a 1641 1606 a 1567 1220 1181 1030

1692 1642 1607 1579 1570 1223 1181 1039

Neutral 1677 1643 1603 a 1560 1237 1181 1034

1627 1579 1552 1531 1506 1438 1369 1313 1242 1166 1139 1036

1618 1579 1551 1532 1501 1438 1369 1311 1243 1167 1141 1033

1646 1579 1555 a 1501 1438 1370 1312 1244 1168 1138 1024

1629 1579 1557 1533 1503 1438 1371 1313 1245 1169 1142 1035

Anionic 1628 1576 1551 1533 1502 1438 1369 1313 1244 1168 1140 1035

1611 1579 1553 1532 1498 1436 1357 1313 1240 1168 1132 1031

CdCstr phenol imidazolinone + CdC str phenol phenol phenol C-H def

1749 1650 1598 1572 1438 1380 1290 1239 1184 1155

1747 1630 1594 1568 1438 1379 1284 1237 1183 1154

1704 1644 1598 1571 1438 1379 1285 1238 1183 1152

1749 1646 1597 1572 1438 1380 1290 1239 1184 1155

Cationic 1747 1650 1598 1563 1438 1379 1289 1237 1183 1153

1749 1626 1595 1569 1437 1373 1290 1235 1183 1143

CdO str CdCstr phenol imidazolinone + CdC str phenol C-H def phenol phenol phenol phenol C-H def

a

phenol phenol phenol C-H def phenol imidazolinone C-C str + CdO bend

Not observed; str: stretching; def: deformation.

ent with their assignments as phenol modes. The earlier study by Esposito et al. designated these bands as phenol 1 and phenol 2 modes.32 However, it should be noted that lack of sensitivity to isotopic labeling may reflect the fact that these modes are highly delocalized rather than localized to the phenol ring. There is some doubt concerning the previous assignment of the 1567 cm-1 Raman band as the “CdN stretch” mode.32 We would expect changes in the frequency of the 1567 cm-1 band in both HBDI-3-13C and HBDI-4-15N, if this band results predominantly from a CdN stretching vibration. However, this band is insensitive to labeling the C3 carbon. Although the 1567 cm-1 band is also insensitive to labeling the C1 imidazolinone carbon, this band shifts about 5-8 cm-1 to lower energy in the other labeled analogues (4-15N, 5-13C, and R-13C). Similar changes were also observed in D2O buffer (Table 3), suggesting that the 1567 cm-1 band does not solely arise from the CdN bond but rather that it is localized over most of the imidazolinone ring and the exocyclic CdC double bond. Ab initio calculations, which in general agree very well with experimental observations for the neutral chromophore, predict that this is a highly delocalized mode over both rings and the exocyclic double bond. However, because of the lack of appropriate isotopic labels, we cannot experimentally confirm the involvement of the phenol ring in this mode. The assignment of the 1449 cm-1 Raman band to C-N stretch is doubtful because it is unaffected in any of the labeled analogues. Instead, the 1449 cm-1 band probably involves the C-C-H in-plane deformation mode of the phenol ring. Finally, the position of the IR band at 1514 cm-1 (Table 2) is unaffected in all of the isotopically labeled neutral analogues. This is

consistent with the previous assignment of this band to a mode involving the phenol ring (phenol 3 mode32). In the spectral region below 1430 cm-1, it has been suggested that most of the vibrational modes arise from motions of the phenol group.32 This would certainly be consistent with our isotope labeling studies which for the most part reveal only very small wavenumber shifts. However, the lack of isotope shifts could also represent the fact that the normal modes are highly delocalized over the two rings, so that a change in mass of any individual atom has only a relatively small effect on the band position. This would be in agreement with our normal mode calculations which predict that many of the normal modes in this region involve both rings and that labeling at a single position will only have a small effect. Because we are unable to distinguish between these possibilities without isotopic labels in the phenol ring, the isotopically insensitive bands at 1317, 1234, and 1178 cm-1 are by default assigned to the phenol ring. The one exception is the band at 1036 cm-1 which decreases 8 cm-1 upon 1-13C labeling and is assigned to an imidazolinone C-C stretching mode coupled to CdO bending.45 Effect of Deuteration. Because there is an exchangeable hydroxyl proton in the neutral form of HBDI, it might be expected that some changes would occur in the vibrational spectra upon deuteration. To test this, we obtained Raman spectra in D2O as shown in Figure 5. Comparison with the spectra in H2O and D2O reveals only minor differences reflecting little participation of the O-H group in normal modes below 1800 cm-1. Anionic HBDI. In the anionic form of HBDI, the phenolic hydroxyl group is deprotonated. The absorption spectra (Figure 2) of HBDI reveal that the anionic form of HBDI in H2O has

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Figure 5. Raman spectra of HBDI in (A) sodium acetate buffer (pD 5.5, 20 mM), (B) 1 M DCl, and (C) 1 M NaOD. The solvent background has been subtracted. The concentrations were 0.3, 5, and 10 mM, respectively, and the spectra were accumulated for 10 min.

Figure 7. IR spectra of HBDI and its isotopically labeled analogues in aqueous 1 M NaOD in D2O. The solvent background has been subtracted. The concentrations were 10 mM.

Figure 6. Raman spectra of HBDI and its isotopically labeled analogues in aqueous 1 M NaOH solution. The solvent background has been subtracted. The concentrations were 10 mM, and the spectra were accumulated for 10 min.

an absorption maximum at 425 nm, which is 57 nm higher than the neutral form of HBDI. The vibrational spectrum of anionic HBDI displays key differences from that of neutral HBDI reflecting significant structural changes upon deprotonation. Figures 6 and 7 contain the Raman and IR spectra, respectively, of anionic HBDI in H2O and D2O, respectively, and the major bands are assigned in Tables 1 (Raman H2O), 2 (IR D2O), and 3 (Raman D2O; spectra not shown).

The CdO stretching band is observed in the IR spectrum of HBDI at 1669 cm-1 and appears as a weak shoulder in the Raman spectrum at around 1665 cm-1. Upon labeling the carbonyl carbon (1-13C), this band decreases 24 cm-1 to 1645 cm-1 confirming the assignment to CdO stretch. Similarly, the 1631 cm-1 Raman band (1630 cm-1 IR) can be assigned confidently to the CdC stretch mode because it is shifted to 1613 cm-1 (1615 cm-1 IR) and 1615 cm-1 (1619 cm-1 IR) in HBDI-5-13C and HB-R-13C-DI, respectively. Finally, the effect of 1-13C labeling on the exocyclic double bond is difficult to determine in the Raman spectra because the CdC band is obscured by the CdO stretching band which has shifted and increased in intensity upon labeling (1643 cm-1 H2O, 1646 cm-1 D2O). However, the better resolution of the IR data (Figure 7) reveals a shoulder at around 1622 cm-1 on the low wavenumber side of the CdO stretching band (1645 cm-1), suggesting that the CdC stretching band is sensitive to 1-13C labeling. The 1579 cm-1 Raman band (1583 cm-1 IR) is assigned to the phenol 1 mode because it is unaffected by isotope labeling. The 1556 cm-1 peak is the most intense band in the unlabeled anionic form of HBDI and is sensitive to labeling. This band is shifted 7 cm-1 in HB-R-13C-DI and by about 2-3 cm-1 in HBDI-4-13C, HBDI-5-13C, and HBDI-1-13C. Consequently, we assign this band to a normal mode that has some contribution from the -CdC-CdN- portion of the imidazolinone ring. Finally, the 1533 and 1503 cm-1 bands are assigned to phenol modes. In general, the bands in the lower wavenumber region in the anionic form are more sensitive to labeling than for the neutral form. The bands at 1439, 1310, and 1171 cm-1 in unlabeled HBDI are unaffected or exhibit only small changes in position in the labeled analogues and are probably due to contributions from the unlabeled phenolic ring or highly delocalized modes.

Model Green Fluorescent Protein Chromophore

Figure 8. Raman spectra of HBDI and its isotopically labeled analogues in aqueous 1 M HCl solution. The solvent background has been subtracted. The concentrations were 5 mM, and the spectra were accumulated for 10 min.

However, the bands at 1371, 1246, 1144, and 1037 cm-1 are sensitive to isotope labeling. The band at 1371 cm-1 is shifted by 15 cm-1 to lower wavenumber in 5-13C labeled HBDI but does not shift in any of the other labeled spectra. This observation is difficult to explain in terms of simple stretching motions because each of the atoms attached to C5 has also been labeled without affecting the position of the 1371 cm-1 band. The band at 1246 cm-1 exhibits small shifts of 6 and 4 cm-1 on labeling the C5 and CR positions, respectively, indicating a contribution from the stretching of the bridging double bond to this mode. The 1144 cm-1 band experiences shifts on labeling C5, C1, and CR of 13, 6, and 4 cm-1, respectively, indicating contributions from the CR-C5-C1 group to this particular mode. Finally, the band at 1037 cm-1 is shifted by 10 cm-1 to 1027 cm-1 in 1-13C labeled HBDI and is assigned to a C-C stretching mode with some contribution from carbonyl bending.45 Effect of Deuteration. As expected, because there are no exchangeable protons in the anionic form of HBDI, the vibrational band positions are not significantly altered in D2O compared to H2O. Cationic HBDI. For cationic HBDI, both the imidazolinone ring N4 nitrogen and the phenolic hydroxyl group are protonated. The cationic form of HBDI in 1 M HCl has an absorption maximum at 391 nm that is about 23 nm higher than the neutral form (Figure 2). The Raman spectrum of cationic HBDI has key differences from both the neutral and anionic forms highlighting the importance of protonation on the chromophore’s structure. The 1744 cm-1 band observed in the Raman spectrum of cationic HBDI (1748 cm-1 IR) shifts to 1692 cm-1 in HBDI1-13C, consistent with the assignment of this band to a pure CdO stretching mode (Figures 8 and 9). Similarly, the 1647

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Figure 9. IR spectra of HBDI and its isotopically labeled analogues in aqueous 1 M DCl solution in D2O. The solvent background has been subtracted. The concentrations were 5 mM.

cm-1 Raman band (1648 cm-1 IR) observed in cationic HBDI shifts to 1628 and 1633 cm-1 in HBDI-5-13C and HB-R-13CDI, respectively, consistent with assignment to the CdC stretching mode of the exocyclic double bond. The Raman spectrum of cationic HBDI is dominated by two intense bands at 1595 and 1582 cm-1. Although the 1595 cm-1 band can be assigned to a phenol ring mode (phenol 1), based on its insensitivity to labeling, the 1582 cm-1 Raman band, which is not observed in the IR spectrum, decreases in intensity and shifts to 1572 cm-1 in D2O (Figure 5 and Table 3). The sensitivity of the 1582 cm-1 Raman band to deuteration on the imidazolinone N4 nitrogen, suggests that this band has a strong contribution from N-H bending. Deuteration alters the sensitivity of this band to isotopic labeling of the imidazolinone ring. For example, in H2O, the 1582 cm-1 band is insensitive to 4-15N labeling, indicating that CdN stretch is not involved in this mode. However, in D2O, the 1572 cm-1 band decreases 9 cm-1 upon 4-15N labeling. Consequently, deuteration alters the normal mode composition of this band by uncoupling the N-H bending coordinate. Because of the lack of any significant change on isotopic labeling, the bands observed in cationic HBDI at 1378, 1338, 1289, 1234, 1181, and 1020 cm-1 are all assigned predominantly to the phenol ring or to highly delocalized modes. However, a band at 1151 cm-1 decreases by 7 cm-1 in 5-13C labeled HBDI and a band at 1009 cm-1 band shifts to 997 cm-1 in HBDI-113C. The 1151 cm-1 band is reminiscent of the 1370 cm-1 band in anionic HBDI in that it is only sensitive to C5 labeling. The 1009 cm-1 band is similar to the 1037 cm-1 band in anionic HBDI because it decreases by 10 cm-1 in HBDI-1-13C and is again assigned to a single bond stretching mode with some contribution from carbonyl bending. The Raman and IR band positions and their assignments are summarized in Tables 1-3.

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TABLE 4: Positions of Calculated Raman Bands for the Neutral and Anionic Forms of HBDI and Some of Its Isotopically Labeled Analogues between 1500 and 1800 cm-1 HBDI HB-R-13C-DI HBDI-1-13C HBDI-3-13C HBDI-4-15N mode 1802 1704 1663 1631 1618 1558

1801 1685 1661 1630 1611 1557

1742 1690 1644 1633 1598 1552 1491

1742 1686 1639 1630 1582 1551 1489

Neutral 1758 1701 1662 1631 1618 1558 Anionic 1718 1690 1643 1633 1598 1551 1491

1802 1699 1660 1629 1591 1558

1802 1703 1662 1630 1608 1558

1 2 3 4 5 6

1742 1690 1638 1624 1576 1551 1492

1742 1690 1641 1631 1590 1552 1490

1 2 3 4 5 6 7

Ab Initio Normal Mode Calculations. Our principal interest is in understanding the normal mode composition of the bands involving the CdC, CdO, and CdN stretching vibrations, because these modes are most likely to be sensitive to local structural interactions within the protein and also reflect the electronic structure of the chromophore. To supplement our experimental observations on the effects of isotopic labeling, ab initio normal mode calculations were performed on HBDI and its isotopically labeled analogues using density functional theory (DFT) and a 6-31G** basis set. Table 4 shows a comparison of the calculated frequencies of the Raman bands for labeled and unlabeled neutral and anionic HBDI. The principal bands that change upon labeling are obtained from the calculations in the double bond stretching region (18001500 cm-1). For neutral HBDI, there are six modes in this frequency range. The highest energy mode, mode 1, is the CdO stretch mode with weak coupling to CdC stretch. The calculations predict a 44 cm-1 decrease in the calculated frequency of mode 1 upon 1-13C labeling. Experimentally, a somewhat smaller decrease is observed in the IR spectrum of neutral HBDI of 32 cm-1 (Figure 4). The calculations predict that mode 2 involves a major contribution from stretching motions of the exocyclic CdC bond coupled to the phenol ring and predict a 19 cm-1 decrease in this band upon R-13C labeling. Experimentally, the CdC stretching mode is sensitive to both R-13C (-18 cm-1) and 5-13C (-20 cm-1) labeling. The only other mode that is predicted to be sensitive to labeling in the imidazolinone ring or exocyclic CdC bond is mode 5, which was previously assigned to the CdN stretching vibration.32 Although our calculations predict that mode 5 is sensitive to labeling at the C3, N4 and R positions, in contrast, experimentally, this mode is insensitive to labeling at C3 and is assigned to a delocalized imidazolinone mode involving the exocyclic double bond. As noted above, the calculations predict that mode 5 is delocalized over both rings; however, we cannot confirm the involvement of the phenol ring in this mode experimentally. In agreement with previous studies, modes 3, 4, and 6 are predicted to involve mainly the phenol ring.32 Although the calculated isotopic shifts for neutral HBDI generally agree well with the experimentally observed values, our calculations are less successful at predicting the isotopic shifts for anionic HBDI. This may partly reflect the importance of counterions present in solution with the charged HBDI molecules, and preliminary calculations (data not shown) indicate that the normal mode composition is very sensitive to the presence of solvating counterions. For example, preliminary calculations on the anion with the inclusion of a suitable counterion demonstrates that the gas phase calculations over-

Figure 10. Two resonance structures for the anionic form of HBDI.

estimate the shortening of the phenolic C-O bond upon deprotonation. This has a severe effect on the compatibility of the calculations with relation to solution phase experimental data. Calculations are currently in progress to address this issue. Unfortunately, we were unable to find a global energy minimum for the cationic form of the chromophore. All our calculations on the planar structures of the cation produced imaginary frequencies in the vibrational calculations which may indicate that in vacuo this form adopts a nonplanar conformation. Discussion Importantly, in this study, we are able to provide the first definitive assignments of the vibrational spectra of a model GFP chromophore by using isotope labeling. This is valuable for establishing the nature of the changes in the vibrational spectrum because of the protein environment. In turn, these changes can be related to the structure of the chromophore within the protein and linked to the absorption and emission spectra observed for GFPs. Specifically, in the region between 1750 and 1450 cm-1, we identify marker bands in both the Raman and IR spectra for cationic, neutral, and anionic forms of the chromophore. Furthermore, we can confidently assign these bands to specific vibrational modes. The Raman bands at 1744, 1697, and 1665 cm-1 for cationic, neutral, and anionic HBDI, respectively, are assigned to a CdO stretching mode arising from the imidazolinone carbonyl group. This band is much stronger in the IR spectra, consistent with the prediction that the highest-frequency mode is strongly IR-active and weakly Raman-active. The decrease in νCdO upon deprotonation of neutral HBDI reflects a weakening of the Cd O bond in the anionic form probably because of larger contributions from resonance structures such as II in Figure 10. Conversely, the increase in νCdO for cationic HBDI indicates a strengthening of the C-O bond upon imidazolinone ring protonation. The bands at 1647, 1642, and 1631 cm-1 for cationic, neutral, and anionic HBDI, respectively, are assigned to the CdC stretching mode associated with the exocyclic double bond. This band is both strongly IR and Raman active. In an analogous fashion to the CdO bond, deprotonation of the phenol reduces the bond order of the exocyclic double bond as shown by the 11 cm-1 reduction in νCdC for the anion compared to neutral HBDI. The experimental data suggest that the 1602 and 1583 cm-1 IR bands (1603 cm-1 in Raman) in neutral HBDI are phenol ring modes beause they are unaffected in the labeled compounds. Interestingly, the 1603 cm-1 Raman band is shifted to 1579 cm-1, and its intensity is greatly decreased in the anionic form of HBDI. This suggests that the negative charge in the anionic chromophore is delocalized over the phenol ring, thereby reducing its’ aromaticity and causing a dramatic decrease in this band. The assignment of the 1567 cm-1 band in neutral HBDI to the CdN stretch is questionable because it is not confirmed by our isotopic labeling results.32 Although our normal mode

Model Green Fluorescent Protein Chromophore calculations are consistent with this band having a contribution from CdN stretch (Table 4 mode 5), it is more appropriate to describe this as a delocalized mode involving both rings and the exocyclic double bond. Our experimental data indicate that this mode is delocalized over the imidazolinone ring and the exocyclic double bond for both neutral and anionic HBDI. The corresponding Raman band in cationic HBDI at 1582 cm-1 is very sensitive to deuteration, decreasing in intensity and shifting to 1572 cm-1 in D2O. Consequently, the corresponding normal mode has a considerable contribution from N-H bending. The sensitivity of this band to deuteration at N4 will provide an important marker for imidazolinone ring protonation in our hunt for cationic and/or zwitterionic chromophores within the protein matrix. GFP Raman Spectra Revisited. The isotope labeling studies detailed above provide an excellent opportunity to revisit the Raman band assignments for the chromophores within folded GFPs. Previously, we obtained Raman spectra of S65T GFP at pH 5 and 8 where the neutral and anionic forms of the chromophore predominate. For the neutral form of S65T GFP, the principal Raman bands in the 1500-1700 cm-1 region are observed at 1662, 1646, 1599, 1560, and 1541(sh) cm-1.14 The 1662 cm-1 band can been assigned to the protein’s amide I mode, whereas the 1541 cm-1 is due to a small amount of anionic chromophore present at pH 5 (pKa 6). The three remaining bands in the protein spectrum at 1646, 1599, and 1560 cm-1 correspond to the three principal bands for the unlabeled neutral HBDI model, at 1642, 1603, and 1567 cm-1. Consequently, the 1646 cm-1 band observed in the S65T GFP spectrum can be assigned to the exocyclic CdC stretching mode, whereas the 1603 cm-1 band is associated with the phenol ring (phenol 1 mode). Finally, the 1560 cm-1 Raman band can be assigned to a normal mode delocalized over the imidazolinone ring and exocyclic double bond. This band is the most intense in the resonance Raman spectrum of wtGFP.33 The similarity in band position for neutral HBDI and neutral S65T GFP indicates that the protein matrix has little effect on the neutral form of the chromophore compared to interactions present in aqueous solution. Consequently, the 26 nm red shift in λmax for the neutral chromophore in the protein compared to HBDI in solution must arise from interactions between the protein and the chromophore’s excited state. In the anionic form of S65T GFP, the principal Raman bands in the 1500-1700 cm-1 region are observed at 1664, 1618, 1537, and 1495 cm-1.14 Again, the 1664 cm-1 band can be assigned to the protein amide I mode. The 1618 cm-1 band likely corresponds to the band observed at 1631 cm-1 in anionic HBDI and is assigned to a mode that involves the exocyclic CdC double bond. Both model and protein CdC stretch modes fit on the linear correlation between ground-state structure and absorption maxima reported previously.14 The 1495 cm-1 band corresponds to the 1503 cm-1 phenol mode observed for anionic HBDI. Finally, the 1537 cm-1 band is assigned to the delocalized imidazolinone/exocyclic CdC mode observed at 1556 cm-1 in anionic HBDI. The assignment of the latter band is based primarily upon the expectation that this band will have substantial intensity in the Raman spectra. Consequently, all three protein chromophore bands are shifted to lower wavenumber compared to the corresponding bands in anionic HBDI, indicating that interactions with the protein shift the phenol toward a quininoid structure and weaken the exocyclic CdC bond. These changes are consistent with an increased contribution from a quininoid-like resonance structure such as II in Figure 10.

J. Phys. Chem. B, Vol. 106, No. 23, 2002 6065 Summary In summary we have explicitly assigned several of the key vibrational bands observed in the Raman and IR spectra of cationic, neutral, and anionic HBDI, a model of the GFP chromophore. Importantly, these assignments provide a firm foundation for assigning the vibrational spectra of the chromophore in GFP and in understanding the role of the protein matrix in controlling the photophysics of the chromophore. Although there is no evidence to date for the presence of cationic or zwitterionic chromophore in nonilluminated GFPs, the sensitivity of an intense Raman band in cationic HBDI to deuteration will be an important method for determining if imidazolinone ring protonation occurs upon photoexcitation of GFP. Acknowledgment. This work was supported by grants from NIH (AI44639 and GM63121) and NSF (MCB960254). In addition, this material is based upon work supported in part by the U. S. Army Research Office under Grant DAAG55-97-10083. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society for partial support of this research. A.F.B. is an American Heart Association postdoctoral fellow. The NMR facility at SUNY at Stony Brook is supported by grants from NSF (CHE9413510) and from NIH (1S10RR554701). References and Notes (1) Tsien, R. Y. Annu. ReV. Biochem. 1998, 67, 509. (2) Kojima, S.; Ohkawa, H.; Hirano, T.; Maki, S.; Niwa, H.; Ohashi, M.; Inouye, S.; Tsuji, F. I. Tetrahedron Lett. 1998, 39, 5239. (3) Shimomura, O. Febs Lett. 1979, 104, 220. (4) Cody, C. W.; Prasher, D. C.; Westler, W. M.; Prendergast, F. G.; Ward, W. W. Biochemistry 1993, 32, 1212. (5) Lippincott-Schwartz, J.; Snapp, E.; Kenworthy, A. Nat. ReV. Mol. Cell. Biol. 2001, 2, 444. (6) Ormo, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J. Science 1996, 273, 1392. (7) Yang, F.; Moss, L. G.; Phillips, G. N. Nat. Biotechnol. 1996, 14, 1246. (8) Heim, R.; Prasher, D. C.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12501. (9) Cubitt, A. B.; Heim, R.; Adams, S. R.; Boyd, A. E.; Gross, L. A.; Tsien, R. Y. Trends Biochem. Sci. 1995, 20, 448. (10) Chattoraj, M.; King, B. A.; Bublitz, G. U.; Boxer, S. G. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8362. (11) Yokoe, H.; Meyer, T. Nat. Biotechnol. 1996, 14, 1252. (12) Ward, W. W.; Prentice, H. J.; Roth, A. F.; Cody, C. W.; Reeves, S. C. Photochem. Photobiol. 1982, 35, 803. (13) Niwa, H.; Inouye, S.; Hirano, T.; Matsuno, T.; Kojima, S.; Kubota, M.; Ohashi, M.; Tsuji, F. I. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13617. (14) Bell, A. F.; He, X.; Wachter, R. M.; Tonge, P. J. Biochemistry 2000, 39, 4423. (15) Heim, R.; Cubitt, A. B.; Tsien, R. Y. Nature 1995, 373, 663. (16) Wachter, R. M.; King, B. A.; Heim, R.; Kallio, K.; Tsien, R. Y.; Boxer, S. G.; Remington, S. J. Biochemistry 1997, 36, 9759. (17) Wachter, R. M.; Elsliger, M. A.; Kallio, K.; Hanson, G. T.; Remington, S. J. Structure 1998, 6, 1267. (18) Matz, M. V.; Fradkov, A. F.; Labas, Y. A.; Savitsky, A. P.; Zaraisky, A. G.; Markelov, M. L.; Lukyanov, S. A. Nat. Biotechnol. 1999, 17, 969. (19) Yarbrough, D.; Wachter, R. M.; Kallio, K.; Matz, M. V.; Remington, S. J. Proc. Natl. Acad. Sci. U. S.A. 2001, 98, 462. (20) Dickson, R. M.; Cubitt, A. B.; Tsien, R. Y.; Moerner, W. E. Nature 1997, 388, 355. (21) van Thor, J. J.; Pierik, A. J.; Nugteren-Roodzant, I.; Xie, A. H.; Hellingwerf, K. J. Biochemistry 1998, 37, 16915. (22) Carey, P. R. Biochemical applications of Raman and resonance Raman spectroscopies; Academic Press: New York, 1982. (23) Callender, R.; Deng, H. Annu. ReV. Biophys. Biomol. Struct. 1994, 23, 215. (24) Carey, P. R.; Tonge, P. J. Acc. Chem. Res. 1995, 28, 8. (25) Kim, M.; Mathies, R. A.; Hoff, W. D.; Hellingwerf, K. J. Biochemistry 1995, 34, 12669.

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He et al. (38) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.7; Gaussian, Inc.: Pittsburgh, PA, 1998. (39) Nielsen, S. B.; Lapierre, A.; Andersen, J. U.; Pedersen, U. V.; Tomita, S.; Anderson, L. H. Phy. ReV. Lett. 2001, 87, 2281021. (40) Chen, M. C.; Lambert, C. R.; Urgitis, J. D.; Zimmer, M. Chem. Phys. 2001, 270, 157. (41) Litvinenko, K. L.; Webber, N. M.; Meech, S. R. Chem. Phys. Lett. 2001, 346, 47. (42) Phillips, G. N., Jr. Curr. Opin. Struct. Biol. 1997, 7, 821. (43) Weber, W.; Helms, V.; McCammon, J. A.; Langhoff, P. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6177. (44) Buck, J. S.; Ide, W. S. Org. Synth. Coll. Vol. 1943, 2, 55. (45) Pinchas, S.; Laulicht, I. Infrared Spectra of Labeled Compounds; Academic Press: New York, 1971.