UV Transition Moments of Tyrosine - The Journal of Physical

Article pubs.acs.org/JPCB

UV Transition Moments of Tyrosine Louise H. Fornander, Bobo Feng, Tamás Beke-Somfai, and Bengt Nordén* Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden S Supporting Information *

ABSTRACT: To assist polarized-light spectroscopy for protein-structure analysis, the UV spectrum of p-cresol, the chromophore of tyrosine, was studied with respect to transition moment directions and perturbation by solvent environment. From linear dichroism (LD) spectra of p-cresol aligned in stretched matrices of poly(vinyl alcohol) and polyethylene, the lowest π−π* transition (Lb) is found to have pure polarization over its entire absorption (250−300 nm) with a transition moment perpendicular to the symmetry axis (C1−C4), both in polar and nonpolar environments. For the second transition (La), polarized parallel with the symmetry axis, a certain admixture of intensity with orthogonal polarization is noticed, depending on the environment. While the Lb spectrum in cyclohexane shows a pronounced vibrational structure, it is blurred in methanol, which can be modeled as due to many microscopic polar environments. With the use of quantum mechanical (QM) calculations, the transition moments and solvent effects were analyzed with the B3LYP and ωB97X-D functionals in cyclohexane, water, and methanol using a combination of implicit and explicit solvent models. The blurred Lb band is explained by solvent hydrogen bonds, where both accepting and donating a hydrogen causes energy shifts. The inhomogeneous solvent-shift sensitivity in combination with robust polarization can be exploited for analyzing tyrosine orientation distributions in protein complexes using LD spectroscopy.

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INTRODUCTION A better understanding of the immensely diverse roles that proteins play in cellular operations, and also how they may function in new synthetic materials, beckon the development of new approaches that can report on the structural and dynamic properties of proteins in solution. Today, detailed information on the static structural properties of proteins is provided predominantly by X-ray crystallography1 and NMR spectroscopy.2,3 These methods, though, are severely limited in several important cases of structural biology, such as when studying the noncrystalline samples of fibril-forming proteins or of membrane proteins in a true membrane environment.4,5 Here other techniques, including optical spectroscopic methods, where information from electronic transition moments is exploited, become increasingly important in the study of protein structure and dynamics. The optical spectroscopic techniques range from routine use of UV absorption and emission measurements6 to more specialized applications such as Förster resonance energy transfer (FRET)7,8 and linear and circular dichroism (LD and CD).9,10 In order to understand and interpret the results of any absorption or fluorescence measurement, knowledge of the electric dipole transition moments of the molecules involved is essential. Structural information, such as distances, relative orientations, or changes in environmental conditions, can be obtained from FRET, LD, and CD once the spectroscopic results can be put into the appropriate context of the corresponding active transition moments.11−14 Accurate information about the directions of the transition moments and the purity of polarization (i.e., overlap between differently polarized transitions) are then particularly important to have. In addition, advances in © 2014 American Chemical Society

quantum mechanical (QM) computation have made theoretical approaches very useful in the molecular understanding of the effects the surrounding environment may have on electronic properties of a given chromophore.12,15−25 Linear dichroism (LD), circular dichroism (CD), and fluorescence anisotropy are commonly used spectroscopic techniques that utilize the chromophores within the protein for structural and kinetic determination.10 CD can be used to study secondary structures of proteins,26 and if coupled with time-resolved experiments, protein folding may be investigated as well.27,28 Fluorescence anisotropy can be used to assess binding constants and reaction kinetics.6 To efficiently extract as much information as possible, detailed knowledge about the transition moments of the chromophores within the protein is of particular interest. The electric dipole transition moment is defined as

μj0 = where |0> and |j> refer to the ground and excited states, respectively, and μ is the electric dipole operator, which is a summation over all the charges of the molecule (i.e., ∑qiri), where qi is the charge at position ri. μj0 is a vectorial property, whose direction is determined by the properties of the molecular wave functions of the |0> state and |j> state. The UV absorption of proteins is due to their peptide backbone whose π → π* transitions dominate in the far UV, together with three basic chromophores absorbing between 200 Received: July 1, 2014 Revised: July 12, 2014 Published: July 14, 2014 9247

dx.doi.org/10.1021/jp5065352 | J. Phys. Chem. B 2014, 118, 9247−9257

The Journal of Physical Chemistry B

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calculations, we address the first two low-lying La and Lb π → π* states (Platt’s nomenclature37) of the spectrum (Figure 1). The Lb transition is characteristic for tyrosine; it has a significant oscillator strength with a clear polarization, in contrast to phenylalanine in which the Lb transition is weak with polarizations subject to vibronic borrowing. This difference is because the symmetry in the benzene ring is perturbed by the hydroxyl group in tyrosine, causing the Lb transition to become partly allowed. It has previously been noted that both the vibrational pattern of phenol38 as well as the electronic transitions of p-cresol39 change significantly depending on the solvent. Variations in hydrogen bonding for p-cresol have been examined theoretically using high-level multiconfigurational self-consistent field, and complete-active-space perturbation theory methods.15 Such studies have reported on structural and electronic properties of the chromophore of tyrosine in gas phase models or for models treating solvent effects by implicit methods.15,39−44 The calculations with implicit models have demonstrated that exclusion of explicit solvent molecules cannot reproduce experimentally observed spectral changes between solvents with different polarity.39 Therefore, to support the experimental observations, we employed QM calculations using combined explicit and implicit solvent models and performed similar analysis to resolve, again, the shift and the orientation of the transition moments in different polar conditions. Additionally, the effect exerted on the spectral properties of p-cresol when immobilizing the otherwise freely rotating hydroxyl group, for example through hydrogen bonding with neighboring residues in a protein, was also examined. We note a rather large blue shift when the hydroxyl group is turned out of plane from its original, in solution, planar position. This paper reports results obtained by a combination of experimental and computational means. By aligning p-cresol in two different stretched films with contrasting polar characteristics: poly(vinyl alcohol) (polar) and polyethylene (apolar), we can experimentally assess the directions of the transition moments and the polarization purity of these transitions. We show from the computations how the polarity of the solvent and, more importantly, the presence of hydrogen bonds, as well as the rotation of the hydroxyl group, all affect the La and Lb transition moments of p-cresol. This information is crucial for all interpretations of spectra that involve the electronic transition moments of tyrosine residues for structural analysis of proteins. On the basis of the data for p-cresol presented in this paper, in combination with a rather simplistic spectroscopic measurement, sometimes unique structural information about a protein system can be obtained. As an example, an LD experiment on an aligned protofilament may provide information about the average orientation of the tyrosine residues in the fiber which, when combined with knowledge about polarity dependence of the close environment, can allow the hydrogen-bonding affinity to be deduced and the orientation of specific tyrosine residues to be distinguished. As a demonstration, we address a protein−DNA filament, Rad51-DNA, in which different tyrosines may be shifted differently depending on environmental variations.

and 300 nm: phenol, indole, and benzene. These constitute the side chains of the amino acids tyrosine, tryptophan, and phenylalanine, respectively, and all three are aromatic chromophores containing a π-cloud that strongly influences the physical and chemical behavior of the molecules. For indole and phenol, respectively, a hydrogen is attached to a ring (aza) nitrogen and an exocyclic hydroxyl group is perturbing the πconfiguration somewhat. Moreover, these groups give the molecules ability to form hydrogen bonds in polar solvents, which can further affect the spectroscopic properties depending on the solvent. Indole, the chromophore in tryptophan, has a fairly large extinction coefficient and emission quantum yield, and its fluorescent properties are known to be highly dependent on the environment: it is therefore routinely used as an intrinsic marker in proteins by fluorescence spectroscopy.6 The great sensitivity in both position and magnitude of the emission of tryptophan to changes in the microenvironment has made the less-sensitive chromophores tyrosine and phenylalanine considered often less useful for these kinds of diagnostic applications.29 Nevertheless, utilizing the spectral features of tyrosine to this end is indeed feasible: phenol is more polar than indole and is therefore usually more exposed to solvent in a protein. Thus, such a tyrosine residue is expected to react more strongly upon environmental changes than tryptophan. Also, the shifts in the absorbance spectrum due to changes in the polarity of the environment are generally larger for tyrosine than for tryptophan.29 Thus, inclusion of tyrosine in the arsenal of chromophores used for characterization of the protein environment has clear advantages. In order to extract structural information from UV−vis spectra of proteins containing tyrosine residues, detailed information about the electronic transitions responsible for the light absorption is needed. We shall assume that the basic tyrosine chromophore, a phenol, with a methyl linker in the para position, can be represented by para-cresol (p-cresol, Figure 1). The electronic ground state of p-cresol has been

Figure 1. (A) Amino acid tyrosine, here highlighted in red, inside the recombination protein Rad51. (B) The basic chromophore of tyrosine is shown with its methyl linker as modeled by p-cresol. Approximate orientations of the electric dipole transition moments for the two first electronic transitions (La and Lb) are shown dotted in black, and the dihedral angle δ denotes the rotation of the hydroxyl group around the C−O bond. The La transition is basically parallel to the symmetry axis, along the z axis, and the Lb transition lies along the y axis perpendicular to La, in the plane of the molecule.

studied experimentally by IR and Raman spectroscopy,30−32 stimulated emission dip spectroscopy, and dispersed fluorescence spectroscopy,33,34 as well as in theoretical studies.35,36 In this paper, preparing for applications to protein structure analysis, we combine experimental and theoretical methods in order to get a detailed picture of the electronic spectrum of pcresol and its polarization properties in a solution environment. On the basis of polarized absorption measurements and QM

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EXPERIMENTAL SECTION Reagents and Solutions. para-Cresol (p-cresol or 4methylphenol), poly(vinyl alcohol) (PVA, MW 89000-98000, degree of hydrolysis 99+ %), and cyclohexane (99.5% 9248



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the experimentally obtained polarization spectra, A⊥ and A∥. Ay and Az are obtained from the Lambert−Beer equations Ay = εycl and Az = εzcl, and from the use of eqs 2−4: (concentration, c, and path length, l, are not taken into account).

anhydrous) were supplied from Sigma-Aldrich, and the polyethylene (PE) film was supplied from VWR International. Film Preparation and UV−vis. A PVA solution of 10 wt % concentration was prepared by dissolving PVA into Millipore-Q water at 80 °C during continuous stirring until a transparent viscous solution was obtained and thereafter letting it cool to room temperature. A small volume of p-cresol dissolved in ethanol was mixed with the PVA solution (1.25% p-cresol/ EtOH of the total volume), so that the final concentration of pcresol in the mixture was 1.17 mM. The solution was stirred vigorously, and typically 1 mL/sample was smeared out on a glass slate and dried overnight, forming a homogeneous dehydrated PVA film. The dried PVA films were assembled into a stretching device and heat was applied to avoid ruptures in the film while stretching. The PE film was soaked in a concentrated solution of p-cresol in chloroform, and the solvent was allowed to evaporate before the film was stretched. Films used as baselines were prepared in the same way as described but with no chromophore incorporated. For the isotropic UV− vis spectra, p-cresol was weighed accurately and dissolved in a proper amount of methanol or cyclohexane to a final concentration of 140 μM. All spectra were recorded on a Cary 4000 UV−vis spectrophotometer (Varian), and orthogonal polarization filters were used for measuring polarized absorption. Analysis of Dichroic Spectra. LD is defined as below:

LD = A − A⊥

Az = (3SyyAiso − LD)/(Syy − Szz)

(5)

Szz = 1/3LD(r∼ 290nm)

(6)

1/3LD(r∼ 230nm)

(7)

Syy =

Sxx is then calculated from Sxx = Szz − Syy. Computational Details. UV−vis spectra and excitation energies of p-cresol were calculated with Gaussian 09, using time-dependent density functional theory (TDDFT).45 Becke’s three parameter density functional46 with the Lee-Yang-Parr correlation functional47 (B3LYP) was used to obtain initial molecular structures. Further on, in order to consider dispersion correction and long-range corrections for electron−electron interactions, the ωB97X-D functional was also employed.48 Previous photochemical studies have shown that TDDFT calculations can accurately describe spectral properties of dyes with low computational costs using middle-sized basis sets.22,23 Accordingly, optimization of the molecular structure was done using the basis set 6-31+G(d,p), and photochemical properties were calculated using the 6-31++G(d,p) basis set. Solvent effects of cyclohexane, methanol, and water were accounted for by applying the integral equation formalism polarizable continuum solvent model (IEFPCM).49 In addition, one or two explicit methanol or water molecules were added to include effects of the hydrogen-bonding capability for the respective solvent. These explicit solvent molecules were oriented in such a way so that p-cresol could act either as a hydrogen-bond donor or hydrogen-bond acceptor (Figure 2). Spectroscopic results were averaged from calculations with different initial water arrangements, using two sets for models

(1)

LD A iso

(4)

In this paper, Szz and Syy were obtained by assigning the absorption at the highest LDr value in the 270−290 nm band to be purely z-polarized (Ay = 0), and the highest LDr value in the 210−230 nm region to be purely y polarized (Az = 0).

where LD∥ is the absorbance of the parallel polarized absorption relative to an orientation axis and LD⊥ is the perpendicularly polarized absorption.10 LD is thus a measure of the absorption of organized chromophores within a sample, where a positive LD stems from transition moments organized parallel to the orientation axis and vice versa. The reduced LD, LDr, is a measure that is independent of concentration and path length and is defined as LDr =

A y = (3SzzA iso − LD)/(Szz − Syy)

(2)

where Aiso is the absorbance of an unoriented sample, and based on the assumption that the orientation distribution around the orientation axis is equal, Aiso = 1/3(A∥ + 2A⊥). Similarly, the reduced molecular extinction coefficient, εr, is defined as εi εir = 1 (ε + εy + εz) (3) 3 x where the subscripts x, y, and z have the same coordinate system as the symmetry axes of the molecule. By the use of an orientation constant S, we can then rewrite LDr as LDr = εxr Sxx + εyr Syy + εzr Szz

(4)

Sii is given by Sii = 1/2(3 cos βi −1), where βi is the angle between the molecular axis and the stretching direction, thus if S = 1, the molecule is perfectly oriented. If the LDr is constant over the absorption band, the polarization is said to be “pure”, and no other polarizations contribute to the absorption band. Here we use the reasonable assumption that the out-of-plane polarization, εx, for the planar molecule p-cresol is negligible and that the polarizations are perpendicular to each other and along the symmetry axes z and y. We can thus resolve component spectra Ay and Az, which are linear combinations of 2

Figure 2. Optimized structures for p-cresol when acting as a (A) hydrogen donor or (B) hydrogen acceptor with methanol. The calculated hydrogen bond lengths are indicated. Several optimized conformations with different relative orientations are possible, nevertheless, these do not deviate significantly in their calculated energies or wavelengths. 9249



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of cyclohexane, are not apparent in the presence of methanol: this is due to hydrogen bonding between p-cresol and methanol, making the spectrum blurred as a result of the almost continuously varying environment.50 The absorption spectrum of p-cresol in methanol can be artificially reproduced by overlaying the absorption spectrum of cyclohexane and incrementally shifting it to model many microscopic polar environments (Figure S1 of the Supporting Information). This is also in harmony with the fact that several different organizations of the polar environment contribute to the final spectroscopic spectrum of p-cresol in methanol. We calculated the molar extinction coefficient for p-cresol for the two different solvents to εp‑cresol,MeOH(223 nm) = 6347 M−1 cm−1 and εp‑cresol,cyclohexane(221 nm) = 6548 M−1 cm−1. In order to resolve whether p-cresol acts as a hydrogen donor or a hydrogen acceptor in polar (hydroxylic) solutions, we assessed different possible organizations of hydrogen bond(s) with QM calculations. Four different arrangements were studied: the first being when the hydrogen on the p-cresol hydroxyl group acts as a donor to the electronegative oxygen atom in either water or methanol, the second arrangement when the oxygen on the hydroxyl group of p-cresol acts as a hydrogen acceptor, the third arrangement when p-cresol acts as both a donor and acceptor simultaneously, and last, the fourth arrangement when the solvent is added only implicitly to the molecule. When comparing the electronic transitions obtained from density functional calculations, the calculated wavelengths are shorter in absolute values than those experimentally obtained, which can be ascribed to a general overestimation of excitation energies in TDDFT calculations.12,23−25 However, since the errors are systematic, the calculated results are still valid for relative quantitative comparisons.11,15,18,22−24 Table 1 gives the calculated spectral shifts of the La and Lb transitions arising from the four previously mentioned arrangements of the hydrogen bonds when the molecule is in methanol or in water compared to when apolar cyclohexane is used as a solvent. The changes in wavelength for the Lb band were minimal when methanol or water was used as the implicit solvent. This inconsistency relative to our experimental results is in line with previous calculations, where only implicit models were employed.21 However, when adding an explicit solvent molecule (see Figure 2) in addition to the implicit solvent

with one explicit molecule and four sets for models with two explicit molecules. For these calculations, the energetic differences within each set were minimal. To investigate the effect the orientation of the hydroxyl group of p-cresol has on the transition dipole moments, their energy levels, as well as their orientations, the dihedral angle δ was rotated in steps of 10 degrees, from 0° to 180°, while the rest of the molecule was optimized using ωB97X-D with the 6-31+G(d,p) basis set (see Figure 1). A TDDFT calculation was performed as described above for each 10° step at the ωB97X-D/6-31++G(d,p) level of theory.

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RESULTS Spectral Shift Depends on a Donating Hydrogen Bond. The isotropic UV−vis absorbance spectrum of p-cresol shows two distinct bands arising from the La and Lb transitions centered at 225 and 280 nm, respectively. In Figure 3, the

Figure 3. Absorbance spectra of p-cresol in cyclohexane (solid line) and methanol (dotted line). A red shift of the spectrum in methanol by approximately 3 nm relative to that in cyclohexane is observed.

absorbance spectra of p-cresol in cyclohexane and methanol are shown. A red shift of the Lb transition of approximately 3 nm can be clearly distinguished when the apolar solvent cyclohexane is exchanged for the polar methanol. Also, the highly structured vibrational peaks, which are distinct in the presence

Table 1. Calculated Energies and Lb, La Transition Wavelengths for p-cresol in Various Solventsa solvent

model

ΔE (kcal/mol) ωB97X-Dd

cyclohexane MeOH

H20

implicitb Dc Ac D+Ac implicit D A D+A implicit

−2.5 0.0 −2.5 0.0 -

Lb (nm)

B3LYPe −2.7 0.0 −2.3 0.0 -

ωB97X-D 244.5 247.3 241.8 245.0 244.1 247.3 241.6 244.4 244.1

shift (nm)

B3LYP 254.2 258.0 250.9 254.9 253.6 257.5 250.6 254.2 253.6

ωB97X-D +0.0 +3.1 −2.7 +0.5 −0.4 +2.8 −2.9 −0.1 −0.4

La (nm)

B3LYP +0.0 +3.8 −3.3 +0.7 −0.6 +3.3 −3.6 +0.0 −0.6

ωB97X-D 210.5 213.0 209.7 212.4 210.2 212.5 209.4 211.4 210.1

shift (nm)

B3LYP 221.0 226.0 219.5 224.5 220.2 224.1 219.2 225.4 220.3

ωB97X-D +0.0 +2.5 −0.8 +2.0 −0.3 +2.0 −1.1 +0.9 −0.4

B3LYP +0.0 +5.0 −1.6 +3.5 −0.8 +3.1 −1.8 +4.4 −0.8

ΔE is the difference in total energy between hydrogen bond donating and hydrogen bond accepting configurations. The wavelength shifts are calculated with implicit cyclohexane calculations as reference. For the explicit solvent calculations, the results of different initial solvent arrangements were averaged, for which deviations were less than 1 nm. For more details, see Computational Details. bImplicit solvent calculation using the IEFPCM model. cp-cresol acts as hydrogen bond donor (D), acceptor (A), or both donor and acceptor (D+A) using IEFPCM and one or two explicit solvent molecules (see Figure 2). dEnergies and photochemical properties obtained at the ωB97X-D/6-31++G(d,p)//ωB97X-D/631+G(d,p) level of theory. eEnergies and photochemical properties obtained at the B3LYP/6-31++G(d,p)//B3LYP/6-31+G(d,p) level of theory. a

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Figure 4. Polarized absorption spectra of p-cresol in (A) PVA and (B) PE. The solid line (A∥) corresponds to the polarization parallel to the orientation of the stretched film, thus along the long axis (z axis) of the molecule. The dashed line (A⊥) shows the polarization perpendicular to the stretching orientation of the molecule (y axis). In (C and D), the calculated long- and short-axis polarizations for PVA and PE are shown, respectively. The Lb transition (around 280 nm) is purely polarized for both PVA and PE, this since the perpendicular transition (Ay, solid line in C and D) contributes almost solely to the 280 nm absorption. The La (around 225 nm) transition stems mostly from a parallel contribution (Az, dashed line in C and D); however, a slight perpendicular contribution can also be detected at 215−220 nm.

model, a red shift of approximately 3 nm is produced when pcresol donates a hydrogen to the solvent and a blue-shift of approximately equal size when the donation pattern is reversed (see Table 1). The directions of the shifts remain the same for water and methanol, using either B3LYP or ωB97X-D functionals, and also the size of the shift is in the same range. These values are in very close agreement with the experimental observations detailed above. Our results imply, therefore, that for p-cresol the wavelength shift is not caused by the bulk polarity of the solvent dielectric but rather mainly by the local capability of the solvent to form hydrogen bonds where p-cresol is the donor. The higher probability of p-cresol acting as a donor in hydrogen bonds rather than as an acceptor is also shown by the ∼2.3−2.7 kcal/mol lower relative energies predicted for the hydrogen-donating models in both methanol and water (Table 1). This is also supported by previous calculations on gas phase phenol−water complexes.15 Band Polarizations of La and Lb. The La transition moment lies parallel with the principal symmetry axis, the z axis (Figure 1), while the Lb transition, which is also in the plane of the molecule, lies perpendicular to La, along the y axis. In order to completely utilize the La and Lb transitions in spectroscopic analyses, the exact orientations of La and Lb have to be determined. We have examined the directions of the transition moments by polarized absorption and by QM calculations, and we have also investigated if the hydrogen bonding that arises in polar solvents affects the directions and magnitudes of the transitions. By orienting the molecule in stretched polymer films with different polarity, the directions of the two transitions could be

identified. The method of using a stretched polyethylene matrix was applied to para-dimethoxybenzene and other, bigger aromatic molecules by Thulstrup and co-workers, who were first to put such measurements on a correct mathematical footing.51−54 Poly(vinyl alcohol) (PVA) was used as a polar film, since in addition to being an alcohol (corresponding to ethanol), it also accommodates some water molecules that may also form hydrogen bonds with p-cresol. As a corresponding apolar model medium, a polyethylene (PE) film was used. Figure 4 (panels A and B) show the raw data for the polarized absorption spectra of p-cresol oriented in stretched PVA and PE, respectively, normalized with respect to the parallel contribution of the La transition, A∥. The internal difference between the parallel and perpendicular polarized absorption spectra depends on the directions of the transition moments within the molecule and on how well the molecules are oriented in the film. Even without further analysis of the raw data (Figure 4, panels A and B), one can see that the molecules have a better orientation in the PVA film compared to the PE film, considering the orthogonal polarizations give larger differences here. Notably, this difference is more significant for the La transition (225 nm) than for the Lb transition (280 nm). For the La transition, a clear horizontal character can be distinguished, while a perpendicular contribution is dominating the Lb transition; however, this perpendicular Lb contribution is not as obvious. To be able to differentiate the vertical and horizontal characters of the La and Lb transitions, the long- and short-axis polarized component absorption spectra were calculated (Figure 4, panels C and D) as well as the LDr (Figure S2 of the Supporting Information), as described in 9251



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Experimental. The LDr (Figure S2 of the Supporting Information) for PE and PVA have a more or less constant value between 260 and 290 nm, demonstrating that the Lb transition is pure in this spectral region. The LDr of p-cresol in both the PVA and the PE films shows fluctuations in the region 210−240 nm, indicating an admixture of different polarizations, possibly due to overlap with or perturbation-induced mixing with higher transitions. The orientation parameters, Sii, for the molecule within the two films were calculated, and, as expected, the orientation of the molecules was significantly better in the PVA film compared to the PE film (Szz = 0.337 for PVA, Szz = 0.069 for PE) (see Experimental and Table S1 of the Supporting Information). With the orientation parameters of the molecule known, in combination with the preliminary assumption that the transitions are pure at 230 and 290 nm, the long- and shortaxis polarizations could be calculated (Figure 4, panels C and D). In Figure 4 (panels C and D), we can clearly see that the Lb transition is indeed purely polarized (along the y-axis) in both PVA and PE, and that there is basically no contribution from the z axis absorption in this region. For the somewhat stronger La transition, most of the absorption intensity stems from a zpolarized contribution, but there is some influence from the y axis as well at 215 nm. This feature seems somewhat more prominent in the apolar PE film than in the polar PVA film. A second absorption peak, slightly blue-shifted relative to the main La peak, can also be detected in the isotropic absorption in apolar cyclohexane solvent but not for the polar methanol (Figure 3). In accordance with the nondegenerate perturbation theory, weaker transitions are more conspicuously affected by stronger, close-lying transitions, although the total intensity changes are the same.55 Changes in both the direction of polarization and the absorption intensity of the weaker, perturbed transition are based on the relative directions of the two initial transitions and the inverted value of the energy difference between them. Also, the shift in the absorbance spectrum of the perturbed transition is related to the inverted energy difference between the two interacting transitions.55 The stronger transition from which La may borrow intensity could be any of several higher transitions that lie below 200 nm, a spectral region in which we cannot make accurate measurements with the present experimental settings. The reason why we see a second perpendicular contribution to the La peak in apolar solutions but not in polar solutions will be discussed later. Our TDDFT calculations show that the transition dipole moment of the Lb absorption band is nearly perpendicular to the long axis of the p-cresol molecule. However, when using the ωB97X-D functional, the direction of the Lb transition moment in cyclohexane deviated by approximately 10° from perfect perpendicularity to La. The deviation was influenced mostly by the rotation of the hydroxyl group and very little by the orientation of the methyl group, which has its optimum conformation when one of the methyl hydrogens lies in the plane of the aromatic ring on the same side as the hydroxyl group.35 However, when the hydroxyl group is turned 180°, the Lb transition is again shifted from the optimal perpendicular orientation but, as expected, by −10° instead. Since the energy barrier from hydroxyl (and methyl) rotation is very low compared to kT at room temperature, free rotation of the hydroxyl group is expected in solution. With this averaging, symmetry dictates that the Lb transition is observed as lying perfectly along the y axis. Still, however, the spectral rotational

dependence on the hydroxyl group orientation for the Lb transition moment could be important in context of tyrosine residues in proteins, where the hydroxyls often are immobilized through hydrogen bonding in folded structures, resulting in nonplanar orientations of the hydroxyl group.13 Angle of the Hydroxyl Group Affects the Transitions. In order to further investigate how the orientation of the hydroxyl group affects the La and Lb transitions, we performed a series of calculations where the hydroxyl group, which without any external influences lies in the plane of the aromatic ring, was moved in small steps completing a half-circle. When pcresol is in a polar solvent or forms the residue on tyrosine within a protein, the hydroxyl group of p-cresol can form hydrogen bonds with the surrounding solvent or polar side chains in its vicinity. With dependence on where these side chains are located, the hydroxyl group may be forced to rotate a little, an action that possibly affects the transition moments. The conformation for p-cresol with the lowest energy is its syn-conformation, meaning that one of the hydrogens in the methyl group is in the plane of the aromatic ring, and that this particular hydrogen is on the same side as the hydroxyl group.35 When started from this position, the hydroxyl group was moved in steps of 10° until 180° was reached. Energies and corresponding wavelengths for the Lb transition are shown in Figure 5. Note that the rotation of the hydroxyl group from 0° to 90° has a slight energy cost in the ground state, ∼3 kcal/mol. As the relative increase in energy at the corresponding excited state is larger, ∼5.5 kcal/mol, a clear wavelength shift of approximately −12 nm can be seen. At the same time, the oscillator strength of the transition is significantly decreased at 90°. While the hydroxyl group lies preferably in the plane of the aromatic molecule, the low rotational barrier indicates that in a protein environment, a hydrogen bond formation could easily move the hydroxyl group out of plane. This is important since it may cause a blue shift in the absorption spectrum from such a rotated tyrosine residue, or, if the angle is big enough, making the oscillator strength low or even disappear.

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DISCUSSION We have studied the aromatic chromophore of the amino acid tyrosine, modeled by p-cresol, with the aim of providing a data set useful for future protein analyses. When determining protein structures as well as molecular binding events in solution, techniques such as CD, LD, and fluorescence anisotropy may be utilized, requiring detailed knowledge about the corresponding electronic transitions of the species involved: the directions and magnitudes of the transition moments, as well as how these are affected by the solvent or other local environments. We have studied the first two lowlying transitions, La and Lb, which involve the electronic excitations from bonding π orbitals to antibonding π* orbitals. Here we have assessed their exact orientations both experimentally and by TDDFT calculations. Also, we have addressed in detail how asymmetry in the molecule caused by rotation of the hydroxyl group, likely to occur in protein structures, affects both the energies and the directions of the La and Lb transitions. Polar Solvent Causes a Red Shift in the UV−vis Absorbance Spectra. We have verified that when an apolar solvent (cyclohexane) is exchanged for a polar one (methanol), a red shift emerges in the absorbance spectrum of p-cresol. In the polar methanol solution, the excited states of the La and Lb transitions are stabilized, resulting in a decrease of the 9252



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ring system. Our QM calculations have in addition shown that at the molecular level, the main contribution to this spectral shift does not lie in the continuous bulk polarity variations of the solvents but rather in their capacity to form discrete hydrogen bonds, where p-cresol acts as a hydrogen donor. This will be discussed further below. In contrast to our experimental and theoretical studies, another study on the solvent effect of tyrosine performed by ab initio calculations resulted in a blue shift in solvent compared to vacuum, the authors claim their conflicting result be a consequence of not including hydration effects.36 Like Chignell and Gratzer,39 we notice a slight increase in intensity (3% at 220−223 nm) when the apolar solvent was changed to a polar solvent. p-Cresol Acts as a Hydrogen Donor. We shall discuss the nature of the hydrogen bonding58 of p-cresol. Besides the spectral shift, another obvious effect in the absorbance spectrum when exchanging an apolar solvent to a polar one is that the vibronic structure, which is clearly detected in the Lb transition of cyclohexane, disappears. The reason for this spectral smoothening is that in a polar medium multiple transient conformations of p-cresol bound to solvent molecules will coexist (see simulation in Figure S1 of the Supporting Information). These conformations have transitions with slight energetic variations between them, and in a polar solution these different transitions are averaged together resulting in a smooth but broadened absorbance spectrum. Thus, for cyclohexane, the number of conformations is highly restricted, while in a polar medium there is a loss of fine structure by inhomogeneous broadening due to hydrogen bonding effects.50 Scheiner et al.15 have analyzed potential hydrogen bonds that can be formed between a polar solvent molecule and the aromatic group of various amino acids, including tyrosine (modeled by phenol).15 They found that the strongest of all hydrogen bond conformations formed between phenol and a polar solvent molecule are the conventional hydrogen bonds (e.g., OH−O). A detailed comparison of various possible orientations of chloroform, fluoroform, and water as solvent molecules around p-cresol and phenol performed by Shirhatti et al.59 and Sheiner et al.15 indicate that CH---Y, or OHφ, type hydrogen bonds also can be formed when a proton donor interacts with the π-cloud of the aromatic ring system. However, these bonds are much weaker than the conventional hydrogen bonds, and therefore they do not contribute significantly to spectral changes.15 Consequently, we neglected potential effects of these bonds and focused on the spectral effects of the conventional hydrogen bonds in different solvents. Our TDDFT calculations demonstrate a red shift of 3 nm for Lb, and a somewhat lower ∼2.5 nm red-shift for La, when p-cresol acts as a donor (see Table 1), which corresponds very well to our experimental data (Figure 1). When p-cresol is set to act as a hydrogen acceptor instead, there is still a shift of 3 nm but now to the blue. Note that through its hydroxyl group, p-cresol possesses the bivalent possibility of acting both as a hydrogen donor and a hydrogen acceptor,56 and we therefore investigated this scenario as well. We found that when p-cresol acts simultaneously as a donor and acceptor, the shift is close to zero for Lb (Table 1). In agreement with our calculations, previous ab initio calculations have demonstrated that p-cresol forms a more stable hydrogen bond with water when it acts as a donor as compared to when it acts as an acceptor.15 This indicates that in proteins, tyrosine residues will predominantly form hydrogen bonds where they act as hydrogen bond donors.

Figure 5. Calculated energy for the Lb transition of p-cresol as a function of angle δ of the hydroxyl group. (A) Wavelength shift of Lb relative to the lowest energy conformation (δ = 0), (B) energy difference between ground state (S0) and the first electronically excited state (S1), (C) increase in conformational energy relative the lowest energy conformation, and (D) oscillator strength of Lb. The results were obtained at the ωB97X-D/6-31++G(d,p) level in cyclohexane.

excitation energy.21,56 This phenomenon is clearly detected in our absorbance measurements: when cyclohexane is exchanged for methanol, a red shift of 3 nm occurs (Figure 3), consistent with previous studies of p-cresol39 and benzene.57 These spectral features of benzene are similar to that of its derivatives (e.g., phenol and p-cresol), but one has to keep in mind that these derivatives may have new characteristics that reflect a perturbation by their respective substituents to the benzene 9253



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Direction of the La and Lb Transitions. We have resolved the directions of the two low-lying transitions, La and Lb, to lie effectively perpendicular to each other: Lb perpendicular to the symmetry axis, C1−C4, while La lies essentially parallel with it. However, when p-cresol is in an apolar solvent, both our isotropic and polarized absorption spectra display a second, smaller absorption peak in addition to La. The extra peak is approximately 10 nm to the blue side of the UV peak of the La transition. Interestingly, this extra peak could not be detected when the molecule is in a polar solvent. According to our measured polarized absorption, this transition has a perpendicular character, as opposed to La, which lies parallel to C1−C4 in the symmetry plane. The reason for this second peak is probably due to the fact that a weaker transition (La) can borrow intensity from a stronger near-lying transition as mentioned earlier. There are two strong electronic transitions predicted just below 200 nm, Ba and Bb, also stemming from electronic excitation from bonding π orbitals to antibonding π* orbitals. The transitions of the more high-lying degenerate Ba and Bb states are electric dipole-allowed, resulting in a large oscillator strength, almost two magnitudes larger than La and Lb.21 Our TDDFT calculations have shown that both Ba and Bb are mixed excitations, where electrons from the HOMO and HOMO-1 are excited to higher orbitals. Note that Lb consists mainly of excitations from the HOMO-1 molecular orbital and La mainly from HOMO. Therefore, the directions of Ba and Bb involve both perpendicular and parallel characters (data not shown). Ba and Bb of phenol have been shown to undergo shifts in bulk solvent to the blue and to the red, respectively.21 A redshifted Bb will come in closer contact with the La transition, producing perturbation of La on Bb. This may be the main reason for the perpendicular influence detected for La (Figure 4.). Applicability to Protein Structure Analysis through Site-Specific Linear Dichroism. The data we present show that the spectral shifts of the tyrosine chromophore, p-cresol, due to environmental changes are mainly a result of its hydrogen bond forming capacity. The nature and magnitude of wavelength shifts are determined by the nature of the hydrogen bonds, donor or acceptor, and whether such hydrogen bonds enforce a rotation of the hydroxyl group out of plane. To demonstrate how one may exploit this information to obtain further structural details on tyrosine residues with key importance, we select a study where structural and mechanistic properties of a filament-forming protein, Rad51, were investigated with polarized light spectroscopy (i.e., LD).13 With the use of LD, oriented systems can be studied (e.g., amyloid fibrils related to Alzheimer’s or other disease60 or longer protein−DNA filaments, such as those formed by the recombination proteins RecA61 or Rad5114,15). The technique provides average angles of optically active residues relative to the filament axis, which in turn can give information about the structure of the protein filament. The additional information obtained in this paper on p-cresol can be combined with the previous LD data to better understand contributions from the local environment of the tyrosine residues studied. Using Site-Specific LD (SSLD), Reymer et al.13 have determined the LD signal contribution for each tyrosine residue in the Rad51 filament by mutating one residue at the time and incorporating this unique angular information together with previously developed fragments of protein structure based on crystallography and NMR14,62−64 to reach a three-dimensional model of the Rad51-DNA fiber.13

The La signal appears more distinct than the Lb signal in the SSLD differential (Figure 3 in ref 13), and we therefore choose to use the La transition to test our polarization and shift results for p-cresol to address the structure of Rad51-DNA. Out of the tyrosines site-selected for SSLD in Rad51, two demonstrate clear red-shifts: Tyr54 and Tyr216, 232.1 and 232.8 nm, respectively, while Tyr205 and Tyr228 are more blue-shifted, 227.6 and 228 nm, respectively.13 Tyr54 is positioned close to glutamine and arginine residues that create a polar pocket. This polar pocket may attract and localize water molecules, thus a more prominent hydrogen bond with Tyr54 is formed, which could explain the detected red shift. The same reasoning is valid for Tyr216, where in the crystal structure a strong hydrogen bond can be seen (2.4 Å), which is most likely also present in the filament in solution phase. Here Lys156 and Ser214 form the polar pocket. These examples clearly correspond to the QM models where explicit water molecules were hydrogen-bonded to p-cresol, resulting in a red-shifted peak (Table 1.). In contrast, the blue-shifted residue Tyr205 is located at the open surface of the protein, hence, directly exposed to water solvent. This corresponds to our QM models where only implicit solvent effects were considered resulting in a slight blue shift (Table 1). When being exposed to a polar solvent, hydrogen bonds are formed and broken constantly, most likely leading to that the chromophore spends less total time hydrogen bonded, in contrast to the small polar pockets where the same water molecules reside longer. The reason for the blue absorption of Tyr228 is not as clear and could be explained by three different effects, nevertheless all of these would result in a blue-shifted La peak: (1) there is no hydrogen bond formed with the residue, or (2) a hydrogen bond is formed with the closest water molecule, which would result in a rotation of the hydroxyl group to δ ∼ 130°, according to the crystal structure, or (3) the tyrosine residue could also act as both a donor and an acceptor simultaneously, forming two hydrogen bonds with His244 and a water molecule. The most intriguing residue for this protein filament is Tyr232, which is not resolved in the crystal structures but has been suggested to have key mechanistic importance in the overall function of this repairing enzyme.13 It was earlier proposed to stack between the bases of the DNA, as it is in a region which is speculated to act as a flexible loop capable of coordinating the incoming DNA double helix by favorable interactions. Indeed, LD experiments have shown that in the protein−DNA complex this region is not a random coil, rather folded, as Tyr232 gives a clear LD signal.13 However, the SSLD shows that its peak is red-shifted compared to Tyr205 or Tyr228, and thus both our p-cresol cyclohexane experiments, as well as the QM models, disprove that the Tyr232 is fully intercalated between two base pairs. Since the spectral shift of Tyr232 is consistent with tyrosine acting as a donor in a hydrogen bond, and the SSLD angle indicates alignment with the DNA base pairs, the most probable scenario is that Tyr232 interacts by stacking with the DNA-pairs, but it is not intercalated between the bases. Tyr 232 rather forms a hydrogen bond with a polar group, most likely with the sugar−phosphate backbone of DNA. The dual presence of both types of favorable interactions (i.e., π-stacking with the base pairs in combination with the capacity of forming hydrogen bonds) seems to be crucial for the correct function of Rad51: when Tyr232 is mutated to tryptophan, an aromatic residue capable of the same stacking interaction but not capable of 9254



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and PE (Figure S2), and table of the corresponding calculated Sii for p-cresol in these two environments (Table S1). This material is available free of charge via the Internet at http:// pubs.acs.org.

hydrogen bond formation at the same position as a tyrosine, the DNA binding affinity of the filament is strongly reduced.65 The above example clearly demonstrates that the characteristic spectral shifts observed in the absorption peaks of the various tyrosines relative to each other can provide substantially more information when data on the p-cresol presented here is utilized. The number of probable and speculative structural scenarios can be reduced significantly. Several UV−vis techniques may benefit from the information presented for tyrosine, including isotropic absorbance, CD, fluorescence anisotropy, and LD.

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Corresponding Author

*E-mail: [email protected]. Tel: +46 317723041. Fax: +46 317723858. Notes

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The authors declare no competing financial interest.

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CONCLUSIONS In this study, we have addressed the UV spectrum of p-cresol, the chromophore in tyrosine, by combining spectroscopic and computational techniques. Using LD spectroscopy, we have demonstrated that the Lb transition moment (280 nm) is of pure polarization, while the La transition (225 nm) has a certain perpendicular contribution from Ba and Bb. We have also investigated how La and Lb are affected by using solvents with different polarity and hydrogen-bonding capacity. A spectral red shift of 3 nm is produced when cyclohexane is exchanged to methanol. The QM calculations, employing a combination of implicit and explicit solvent models, showed that this shift is not due to the more polar bulk environment but rather due to the fact that the hydroxyl group of p-cresol can be hydrogenbonded as a hydrogen donor in methanol. We have further analyzed how the directions of the transition moments and the wavelengths of the absorption peaks are affected when the hydroxyl group is rotated out-of-plane, a possible scenario when tyrosine is sitting in a specific position in a protein. Accordingly, a significant blue shift can be expected if the hydrogen group is turned out of plane by more than 40−50°. Our findings of solvent-shift sensitivity, but overall robustness of transition moment directions, for the UV transitions of tyrosine should be important for protein structure analysis. As an example, we demonstrate for the Rad51 protein how information can be gained from LD data about the local environment of different tyrosine residues. We will in the future further investigate the potentials of a more general methodology for structural analysis of proteins or protofilaments (including prions) by site-specific mutation of tyrosine residues, whose possible hydrogen bonding and/or out-of-plane rotation of hydroxyl group may be first identified by UV−vis absorbance measurements. Thereafter, the directions in which the respective tyrosine residues are pointing can be deduced from LD. This, we propose, could be a fast and cheap new approach to obtain structural information for complex protein systems, such as filament or fibril-forming proteins, which have key roles in important biological processes. Spectroscopic studies on proteins have usually underutilized the information obtained from transition dipole moments. Our data will help to cover this gap. We also envisage the application of our results for characterizing complex systems such as membrane proteins buried in lipid membranes and amyloid fibrils.

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AUTHOR INFORMATION

REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

Reconstruction of an absorbance spectrum of p-cresol in methanol is performed based on a set of infinitesimally shifted absorbance spectra from cyclohexane (Figure S1), figure comparing the reduced LD (LDr) spectra of p-cresol in PVA 9255



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UV Transition Moments of Tyrosine - The Journal of Physical

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