Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2116-2120, March 1995 Biophysics

Stark-effect experiments on photochemical holes in chromoproteins: Protoporphyrin IX-substituted myoglobin (chromophore-protein interaction/order-disorder phenomena/spectral-structural correlations) J. GAFERT*, J. FRIEDRICH*, AND F. PARAKt *Physikalisches Institut und Bayreuther Institut fCir Makromolekulforschung, D-95440 Bayreuth, Germany; and tPhysik-Department, Technische Universitat Munchen, D-85748 Garching, Germany Communicated by Hans Frauenfelder, Los Alamos National Laboratory, Los Alamos, NM, November 15, 1994

ABSTRACT We performed comparative Stark-effect ex- pattern. From this specific pattern we will draw an important periments on spectral holes in a protein and a glass sample. conclusion as to the range of the interaction of the chro- The protein was protoporphyrin IX-substituted myoglobin in mophore with its environment: this range does not exceed the a glycerol/water solvent. The glass sample was a protopor- typical dimensions of the protein. The influence of the host phyrin IX-doped mixture of dimethylformamide/glycerol. As glass can be neglected. Hence, the chromophore is indeed a expected, in both cases the spectral holes varied linearly with probe of the protein. This is an important statement: it implies, the electric field. Yet, whereas in the protein the holes showed for instance, that inhomogeneous line broadening in chro- a clear splitting, they showed no splitting in the glass sample, moproteins is due to conformational substates; it confirms in irrespective of the chosen polarization of the laser. In both a direct way that the properties measured in optical experi- samples the hole broadened in the applied field. The magni- ments-for instance, the compressibility (28-30)-are really tude of the broadening was about the same in both cases. The properties of the protein. Stark-effect experiments on proteins following conclusions were drawn. The absence of a splitting have been performed so far on photosynthetic reaction centers in the glass signals an effective global inversion symmetry of (for a review, see ref. 31), which are characterized by strong the chromophore, despite its low symmetry group. The dipole charge transfer states. Experiments in solution with biophys- moment changes are random. In the protein the inversion ical relevance to signal transduction in visual pigments were symmetry is broken through the spatial correlation of the carried out by Mathies and Stryer (32) on retinal, but to our protein building blocks, leading to a molecular frame-fixed knowledge hole-burning Stark experiments on proteins have dipole moment difference and, hence, to the observed splitting. not been published. Despite these symmetry-breaking properties, the local struc- tural randomness is of the same magnitude in the glass and Basic Aspects of Hole-Burning Stark Spectroscopy in in the protein, as is obvious from the broadening. The distinct Proteins difference in the Stark pattern shows that the range of the relevant chromophore interactions is confined to typical To sketch the characteristic features of the Stark effect, we dimensions of the protein. start with the assumption that the molecular probe can be described approximately as a point dipole with dipole moment The characterization of the solid state of proteins is not ilg in the ground state and IUe in the excited state. An external straightforward. Proteins do not fit into the usual categories of field Eo induces a frequency shift Av of the transition under solid-state phases. They have many "in-between" properties. consideration: At sufficiently high temperatures, say above 200 K, they show a behavior in between those of liquids and crystals (1-10). At hAv = -fAi0E. - -f2 E.A .- E0, [1] sufficiently low temperatures their properties are in between those of crystals and glasses. For instance, their specific heat at low temperature is glass-like (11, 12), their dielectric as well where Ap,,, is the difference of the dipole moments between as ultra-sound absorption is glass-like (11), and many of their ground and excited states, optical as well as their Mossbauer properties are glass-like (2, /lo = Ile - lAg, 4, 5, 13-18). On the other hand, their x-ray diffraction pattern [2] is well resolved and crystal-like (19). Yet, even the x-ray and A& is the difference of the polarizability tensors in the diffraction reveals glass-like properties when the Debye- respective states. Waller factor is analyzed (20, 21): the mean-square displace- faccounts for the fact that in a host matrix the local field Ej.c ment (x2) averaged over the atoms of an amino acid residue may be quite different from the external field. In most cases varies along the backbone and, as the absolute temperature one uses the so-called Lorentz field correction, which is based goes to zero, shows large deviations from the usual zero-point on the assumption that the molecular probe sits in a spherical vibrational amplitudes. These findings show that there is an hole of a dielectric continuum which is polarized through the additional structural uncertainty in the positions of the protein external field. In this case building blocks which arises from a broad distribution of conformational substructures. +3 2 f= [3] In this paper we will show by using hole-burning Stark 3 ' spectroscopy (22-27) that, on the one hand, the randomness in the structure of a protein is comparable to the randomness of with e being the dielectric constant. The problems with the a glass. Yet, on the other hand, there is a high spatial point dipole approximation and the Lorentz correction were correlation in the protein which leads to a very specific Stark discussed by Hanson et al. (33). As to proteins, nothing is known about the quality of these approximations, but two The publication costs of this article were defrayed in part by page charge problems are immediately obvious: (i) the medium, as seen payment. This article must therefore be hereby marked "advertisement" in from the chromophore, is no longer homogeneous and (ii) the accordance with 18 U.S.C. §1734 solely to indicate this fact. continuum approximation for a protein seems to be question- 2116 Downloaded by guest on September 24, 2021 Biophysics: Gafert et aL Proc. Natl Acad Sci. USA 92 (1995) 2117 able. But for the qualitative features of our experiments this is (Fig. 1) dissolved in 0.1 M NaOH. In the reconstituted of no concern. iron-free Mb, the absorption ratio at 405 and 280 nm was 3.26. In all our experiments the observed spectral features are (For details of the sample preparation, see ref. 37.) The final linear in Eo, hence, the quadratic term in Eq. 1 can safely be sample was obtained by mixing 2.4 ml of water-free glycerol neglected. and 1 ml of water containing 290 mg of Mb. Since protopor- Next, we have to include the influence of the matrix in more phyrin IX is practically insoluble in glycerol/water, this final detail. This influence is twofold. As mentioned above, the solution did not contain any significant amount of free chro- matrix modifies the external field via the f factor (Eq. 3) but mophores. For comparison, protoporphyrin IX was also dis- can generate a field of its own as well. The matrix field, in turn, solved in a glass matrix. We chose a mixture of dimethylform- can induce a dipole moment pi (i stands for induced) in the amide and glycerol with a volume ratio of 1:3. chromophore. This induced dipole moment may contribute Spectroscopic Measurements. Holes were burnt in proto- significantly to the observed Stark pattern because the matrix porphyrin IX-substituted Mb at three different frequencies in fields can be extremely high (34, 35). Note that ,iL contains the the inhomogeneous band located at 15,818, 16,021, and 16,195 polarizability tensor a, although the effect is still linear in cm-1 as indicated by arrows in Fig. 4. In the glass sample we the external field Eo. If the matrix were a perfectly ordered performed hole burning at one frequency only, 15,977 cm-1 crystal, it would change the molecular parameter A,I.( by an (Fig. 2). The field Eo under burning conditions was -5.835 additional contribution Ap,i with a constant magnitude and kV-cm-'. The holes were burnt at 1.7 K to relative depths of spatial direction. Both contributions would add up, but it about 30%. They were saturated to some extent, yet they could would not be possible to separate them experimentally. In case still be well fitted to Lorentzians. The hole-burning laser the matrix is disordered, A,ui attains a random character, which system was a ring dye laser pumped by a 6-W argon ion laser. gives rise to a broadening linear in E.. Burning times were on the order of 15 s at radiation power Let us describe the general situation of an asymmetric levels of 50 ,W. The bandwidth of the laser was on the order chromophore in a random matrix in more detail. AIpO is a of a few megahertz. The holes were detected by scanning the vector fixed in the frame of the chromophore. As a conse- laser over the burnt-in hole and measuring the transmission. quence there is a fixed angle y between A1.o and the transition The sample was kept in cuvettes with a thickness of 1 mm. dipole moment ILeg. Hence, the direction of AIpO with respect The optical density varied between 0.09 and 0.7, depending on to Eo can be selected by choosing the polarization of the laser sample and spectral range. The cuvette was put between two in a proper way. As long as Ai0o is selected so that it has its plane electrodes. The voltage across the electrodes was varied major component parallel to Eo, a spectral hole would split in between -3.5 and 3.5 kV. The distance between the electrodes a symmetric fashion since, in a random matrix, there is with was 6.5 mm. equal probability a component which is antiparallel to the Polarization Selection and Fitting Procedure. An important field. From the splitting the quantity fASzo in Eq. 1 can be aspect in a hole-burning Stark experiment concerns the laser determined. On the other hand, if the polarization of the laser polarization. In a weakly bleached sample, the hole is strongly is chosen so that Aix, is mainly oriented orthogonal to the anisotropic. The distribution of selected molecules is -cos2 E, external field, only a broadening results. where E is the angle between the polarization direction of the In addition to the characteristic features resulting from Ag.o, laser field and the transition dipole moment Pieg. As a conse- there is AliL, which is random and, hence, can have any quence, the Almo distribution is also strongly anisotropic, orientation in the frame of the chromophore. Moreover, also because the angle y between ILeg and Ai.0 is fixed. By choosing its magnitude is subject to a variation. For the distribution of the polarization of the burning laser properly, the Aim. distri- the magnitude Ali we chose a Maxwell-Boltzmann-like dis- bution can be optically selected so that its main component tribution (25, 36), with the restriction that this distribution is falls either parallel or perpendicular to the external field Eo. confined to two dimensions, taking into account the disk-like We chose the laser field parallel as well as perpendicular to Eo, polarizability tensor of a porphyrin molecule (26): because it takes at least two different polarization directions to get unambiguous results from the fit. In addition, both patterns 2 2_j have to be fitted with the same parameters simultaneously. For P(Agi) = (r)PA2ti expi - ( I) [4] the whole fit procedure we used the formulas as given by Schatz and Maier (36). For the protein sample, the resulting where or is the respective width. It is a parameter characteristic electric-field features of the hole were distinctly different: the for the randomness of the guest-host interaction. If we take parallel orientation led to a splitting and a broadening of the into account the influence from a random matrix on the hole, whereas the orthogonal orientation led to a broadening effective dipole moment, Eq. 1 changes to only. hAv = -fE(A\u cos 0 + Aiii cos Oj), [5] /CH2 with 0 and Oi being the angles between the external field E., HC CH3 and the respective dipole moment differences A,IO and A,ui. The Stark spectra are obtained from a convolution of the -CH burnt-in hole with the homogeneous lineshape function shifted in the electric field according to Eq. 5. The convolution is then CH2 averaged over all possible orientations of the chromophore with respect to the laser field and over the distribution as given in Eq. 4 (36). Materials and Methods CH3 Sample Preparation, The natural heme group does not HO-C-CH2CH2 CH 2CH 2-C-OH allow for narrow-bandwidth hole burning because of its ultra- II II short lifetime. Hence, horse myoglobin (Mb) (Sigma) was 0 0 deprived of the heme group by a butanone extraction at pH 2.3. The apoprotein was reconstituted with protoporphyrin IX FIG. 1. Protoporphyrin IX. Downloaded by guest on September 24, 2021 2118 Biophysics: Gafert et al. Proc. Natl Acad ScL USA 92 (1995)

vb = 16,021 cm-1

= 0.65 0

10 * 0.60 0o

0.55 15,600 16,000 o V V, cm-1

-5 0 5 Frequency, GHz Frequency, GHz

FIG. 4. Deformation of a spectral hole in an external field 0, FIG. 2. Deformation of the shape of a spectral hole in an external E.. electric field E.. 0 indicates the hole without external field, 11 indicates without field; 11, laser polarization parallel to E.; I, laser polarization perpendicular to Eo. The sample was protoporphyrin IX in myoglobin. the hole with the polarization of the laser parallel to E., and I with Temperature was 1.7 K. was 11.67 kV-cm-1. (Inset) Inhomogeneous the polarization perpendicular to E.. The sample was protoporphyrin E. band with the positions indicated where Stark experiments were IX in a dimethylformamide/glycerol glass. Temperature was 1.7 K. E. performed. was 11.67 kV-cm-'. (Inset) Inhomogeneous absorption band with the burn frequency indicated. a.u., Arbitrary units. faE./h, as a function of Eo. In addition, the angle y between Results the transition dipole moment IUeg and A,IO is shown as a function of the field. -y did not show any field dependence, as Our goal was to find out whether the ordered structure of the expected. Again, the data shown refer to the band center. protein leads to characteristic features in the Stark pattern of Our observations can be summarized as follows: (i) all the chromophore as compared with a random environment. spectral changes are linear in the external field EO; (ii) in the Fig. 2 shows a hole burnt into the glass system as it deformed glass, we have broadening only; (iii) in the protein, we have a under an external field E.. The field was varied from -5.835 splitting and a broadening as well; (iv) broadening in the glass to +5.835 kV*cm-l. The polarization of the burning laser was and that in the protein are of comparable magnitudes; (v) parallel (11) or perpendicular (I) to Eo. There was no essential although the magnitude of the broadening is different for the difference: the hole broadened in a symmetric fashion in both three frequencies measured in the protein, the qualitative bases. Fig. 2 Inset shows the inhomogeneous spectrum and the aspects of the Stark spectra do not depend on frequency. spectral position where the hole-burning experiment was carried out. Discussion Fig. 3 shows the broadening of the hole in the glass as a function of Eo. The respective dependence is perfectly linear. The characteristic features of a Stark spectrum-namely, the The linearity indicates a first-order Stark effect. splitting and the broadening-depend on two parameters of Fig. 4 shows the deformation of a hole under an electric field the model: Al.i, the molecular frame-fixed change of the in the protein system. This figure should be compared with Fig. dipole moment, and a-, the mean value of the matrix-induced 2. There is a striking difference: when the laser polarization dipole moments. was chosen parallel to Eo, the hole showed a clear splitting in The Glass Sample. There are two interesting and surprising addition to a On the other broadening. hand, when the laser observations. In the glass there is no splitting but a broadening polarization was perpendicular to E., there was a broadening only, irrespective of the polarization, and the broadening is only. This is quite in contrast to the glass sample, where no splitting was found. Fig. 4 Inset shows the inhomogeneous spectrum with the positions indicated where Stark experiments were performed. The experiment shown is for the band center. Fig. 5 shows the splitting, fAl0oEo/h, and the broadening,

0

1 1 35 O 0 - ~~~~~~. * * 30 L K S 20 125 ?r 0 4 8 12 E., kVcm-1 kV E., cm-1 FIG. 5. Splitting (fAA0E./h) and broadening (foEo/h) of a spec- tral hole as a function of the external field. The sample was proto- FIG. 3. Broadening of a spectral hole in the glass sample as a porphyrin IX in myoglobin. Temperature was 1.7 K. Burn frequency function of the external field Ee. The plotted quantity is foaE0/h (see was 16,021 cm-'. Also shown is the calculated angle -y between Al,o Eqs. 1 and 4). and the transition dipole moment Ileg. Downloaded by guest on September 24, 2021 Biophysics: Gafert et al. Proc. NatL Acad Sc. USA 92 (1995) 2119 comparable to the respective one in the protein. From the not those between chromophore and host glass. In other absence of any measurable splitting we have to conclude that words, the chromophore is indeed a probe for the structure Ago, the molecular frame-fixed change of the dipole moment, and for the dynamics of the protein. is very small compared with ur. Since we are dealing with Broadening and structural disorder. Inhomogeneous broad- allowed transitions, the symmetry of the excited state is ening always reflects structural disorder. Stark broadening is different from that of the ground state. Hence, the straight- inhomogeneous. Hence, we can take the width oa as a measure forward conclusion to satisfy the observation Ag. = 0 would of structural randomness. Unfortunately, our experiments do be to assume that the dipole moment in the ground state as not directly yield this quantity. Instead, we get the scaled well as in the excited state is close to zero. In this case, the quantity f.o, where f corrects for the local field. f may be molecular IT electron system must have approximate inversion different for the protein and the glass, but for a rough estimate symmetry. That is, the asymmetric substituents-namely, the we can safely assume thatf is of the same order of magnitude propionic acid groups and the methyl as well as the vinyl in both samples. Then we can directly compare the broadening groups-are of a negligible influence on the 7relectron system. in both samples, and we have the astonishing result that it is of (Although this argument seems to be straightforward, we have the same order of magnitude. In other words, the fluctuation to keep in mind that in the protein, things seem to be of the induced dipole moment in the spatially highly correlated different.) Then, if the symmetry argument above is right, we protein is comparable to that in the totally random glass phase. have to conclude that the total contribution to the Stark It is only the average value of the dipole moments which is broadening comes from the matrix. The matrix destroys the close to zero in the glass. We stress that these results from the symmetry of the Xf system by inducing a dipole moment. Since Stark experiment fit beautifully to the observations from the solvent cage around the dye probe in the glass varies in a pressure broadening experiments (30), where it was also completely statistical fashion, the induced dipole moment is concluded that the scales of randomness in a protein and a random, giving rise only to a broadening. glass are comparable, despite the high spatial correlation ofthe The Protein Sample. Splitting and spatial correlation. Con- protein building blocks. trary to the glass, we observe a splitting of the hole in the Summary and Conclusion. We have presented hole-burning protein; hence, we have to assume that Al0o 0. How can this Stark experiments on protoporphyrin IX-substituted Mb and assumption be reconciled with what we concluded from the on a protoporphyrin IX-doped glass. We found a clear splitting experiment in the glass-namely, a nearly inversion symmetry of the hole in the protein, depending on the laser polarization, for the IT electron system of the chromophore? but not in the glass. The Stark broadening was of the same Our argument is based on the strong spatial correlation of order of magnitude in the glass and protein sample, respec- chromophore and protein environment. In other words, it is tively. based on the long-range structural order of the protein. Unlike We conclude that (i) in the glass the relevant symmetry of in the glass, the immediate environment around the dye is the protoporphyrin IX must have an approximate inversion cen- same in all protein molecules. In this environment there are ter; (ii) the splitting in the protein sample is induced through polar and even charged groups. They create an electric field at the high spatial correlation of chromophore and protein the chromophore. Because of the strict spatial correlation, this building blocks; and (iii) the specific Stark pattern in the field has a definite direction in a chromophore fixed frame. It protein sample shows that the range of the relevant chro- induces a well-defined dipole moment. Similar observations mophore-protein interaction does not exceed typical dimen- have been made for a chromophore in a lipid bilayer (35). sions of the Mb protein. Hence, there is a A,uo vector which has a fixed direction in the molecular frame. (Although, here AI,o is a quantity which is We gratefully acknowledge enlightening discussions with Lothar induced by the pocket field of the protein, it acts like a Kador (University of Bayreuth) and G. Ulrich Nienhaus (University molecular quantity. Hence, we use the respective notation.) It of Illinois, Urbana). We acknowledge support from the Deutsche is this molecular frame-fixed quantity A1io which leads to the Forschungsgemeinschaft (SFB 213, Graduiertenkolleg Nichtlineare observed splitting: AILO has a definite orientation (angle y) Dynamik und Spektroskopie) and from the Fonds der Chemischen with respect to the transition dipole moment. Through the Industrie. laser field an oriented ensemble of polarized anisotropically 1. Frauenfelder, H., Parak, F. & Young, R. D. (1988) Annu. Rev. protein molecules is selected. This selected ensemble gives rise Biophys. Biophys. Chem. 17, 451-479. to an anisotropic A/,o distribution (in the laboratory frame). 2. Parak, F. & Knapp, E. W. (1984) Proc. Natl. Acad. Sci. USA 81, If this anisotropic distribution has its major component par- 7088-7092. allel to the external field, a splitting results. In the opposite 3. Frauenfelder, H. (1984) Helv. Phys. Acta 57, 165-187. case there is only a broadening. Along these lines of reasoning 4. Parak, F., Hartmann, H. & Nienhaus, G. (1987) in Protein it is clear that the splitting signals structural order and spatial Structure: Molecular and Electronic Reactivity, eds. Austin, R., correlation in the protein as opposed to the glass. Buhks, E., Chane, B., DeVault, D., Dutton, P. L., Frauenfelder, There is another important aspect to be stressed. In the glass H. & Goldanskii, V. I. (Springer, New York), pp. 65-84. as well as in the protein sample the interaction which domi- 5. Parak, F., Heidemeier, J. & Knapp, E. W. (1988) in Biological and Artificial Intelligence Systems, eds. Clementi, E. & Chin, S. nates the Stark-effect features is the same: namely, the dipole- (ESCOM, Leiden), pp. 23-47. induced dipole interaction, which falls off as R6. We can draw 6. Iben, E. T., Braunstein, D., Doster, W., Frauenfelder, H., Hong, a definite conclusion on the range of this interaction. The M. K., Johnson, J. B., Luck, S., Ormos, P., Schulte, A., Steinbach, experiments clearly demonstrate that it dies out on a length P., Xie, A. H. & Young, R. D. (1989) Phys. Rev. Lett. 62, scale which cannot significantly exceed the typical dimension 1916-1919. of the protein. For Mb this scale is on the order of 15 A. If the 7. Frauenfelder, H., Sligar, S. G. & Wolynes, P. (1991) Science 254, interaction range exceeded significantly the dimension of the 1598-1603. protein, we would not observe the dramatic difference in the 8. Nadler, W. & Stein, D. L. (1991) Proc. Natl. Acad. Sci. USA 88, Stark pattern of the holes. The chromophore would feel the 6750-6754. 9. Doster, W., Cusack, S. & Petry, W. (1989) Nature (London) 337, influence of the host glass and, consequently, the spectral hole 754-756. would not show such a pronounced splitting but would broaden 10. Rasmussen, B. F., Stock, A. M., Ringe, D. & Petsko, G. A. (1992) only. 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