Partial Orientation of Cytochrome c in a Lyotropic Liquid Crystal

J. Phys. Chem. B 2000, 104, 10653-10658

10653

Partial Orientation of Cytochrome c in a Lyotropic Liquid Crystal: Residual H-H Dipolar Coupling Ivano Bertini,*,†,§ Federica Castellani,† Claudio Luchinat,†,‡ Giacomo Martini,§ Giacomo Parigi,†,‡ and Sandra Ristori§ CERM, UniVersity of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy, Department of Agricultural Biotechnology, UniVersity of Florence, P.le delle Cascine 28, 50144 Florence, Italy, and Department of Chemistry, UniVersity of Florence, Via G. Capponi 9, 50121 Florence, Italy ReceiVed: March 14, 2000; In Final Form: August 9, 2000

It is shown that dissolving a paramagnetic protein in a lyotropic liquid crystal solvent results in measurable splittings of several hyperfine-shifted proton lines, without excessive broadening. These splittings are due to residual dipolar coupling of neighboring geminal protons and are interpreted in terms of an orientation tensor, whose magnitude corresponds to about 1% orientation. The reasonable agreement obtained between observed splittings and their calculated absolute values shows that the simplified analysis used is essentially correct. Advantages of this strategy with respect to the milder orientation obtained by using phospholipid mixed micelles (usually called bicelles) were that the induced splittings were so large that they could be measured in homonuclear proton spectra of paramagnetic proteins, in particular for the heme signals in heme proteins, whose 13C enrichment may not always be trivial. The sizable broadening induced by the strong orientation effect is not a problem as long as well-resolved hyperfine-shifted signals are considered.

Introduction In partially oriented systems the anisotropic spin-spin interactions are not completely averaged by the rapid tumbling of molecules in solution. In the absence of quadrupolar nuclei, the principal anisotropic interactions involved are the dipolar interaction (either from nuclear spins or nuclear and electron spins) and the chemical shift anisotropy, the latter usually being small for protons. In oriented media, the proton-proton dipolar interaction gives rise to a splitting of signals into multiplets. The analysis of dipolar interactions has been used to determine the structure of small molecules in liquid crystals.1-5 When this kind of approach is extended to larger molecules, such as proteins, the knowledge of dipolar anisotropy can provide useful constraints for protein structure determination in high-resolution NMR.6-12 Self-orientation of paramagnetic molecules has also been exploited.13-17 For partially oriented paramagnetic molecules, the dipolar interaction between the unpaired electrons and protons also results in a variation of the hyperfine shift. This is due to the fact that in partially orienting media the paramagnetic dipolar shift is a weighted average of the dipolar shift for each orientation with respect to the external magnetic field. Dilute lyotropic phases have been considered as possible orienting media for the study of protein structure in solution,18,19 although mainly phospholipid mixed micelles have been used.6,9-12,18,20-24 These latter systems exhibit very small orien* To whom correspondence should be addressed. Phone: +39 055 4209272. Fax: +39 055 4209271. E-mail: [email protected]. † CERM. E-mail: [email protected] (F.C.). ‡ Department of Agricultural Biotechnology. E-mail: luchinat@cerm. unifi.it (C.L.); [email protected] (G.P.). § Department of Chemistry. E-mail: [email protected] (G.M.); [email protected] (S.R.).

tation effects; moreover, these systems being out of thermodynamic equilibrium, reproducibility problems may arise. Many conventional surfactants are known to give lyotropic phases in binary or ternary systems,25-27 and their phase diagrams have been well established by scattering, microscopy, and magnetic resonance techniques.28-35 By varying either the temperature and/or composition of the system, different phases characterized by order in 1, 2, or even 3 dimensions can be obtained. Usually, at low concentration and high temperature isotropic phases take place, made by micelles with random orientation. Only in a small range of concentration and temperature can a nematic phase occur. In this case, anisotropic micelles are able to orient themselves cooperatively, so that a 1-dimensional order is established. These kinds of phases have been classified as NC or ND, according to the micellar shape (rodlike and disklike, respectively), and as negative (-) or positive (+), according to the sign of the diamagnetic anisotropy. To establish the presence of a nematic phase, the splitting of the 2H signal of deuterated water in the NMR spectrum may be used.36-38 At higher concentration the most common structures are the hexagonal and lamellar phases, respectively, made by infinite cylinders and bilayers, depending on the type of surfactant. The high viscosity of these phases prevents their spontaneous alignment in the presence of an external magnetic field. In this paper we report the results obtained from NMR measurements on cytochrome c (cyt c), a low-spin Fe(III) protein, in solution with synthetic surfactant lyotropic aggregates, which are able to induce partial orientation of the protein. A paramagnetic protein has been chosen to obtain wellspread 1H signals, far away from the crowded diamagnetic region of the NMR spectrum. Thus, we were able to measure, accurately and unambiguously, both splitting into multiplets and variations in the peak position, despite a sizable line broadening.

10.1021/jp0009608 CCC: $19.00 © 2000 American Chemical Society Published on Web 10/24/2000

10654 J. Phys. Chem. B, Vol. 104, No. 45, 2000 In the analysis two main problems were faced: (i) the presence of a buffered protein could disturb the nematic phase formation and (ii) most of the protein signals were broadened beyond detection when the order induced on the guest molecules was too strong. To decrease the interactions between micellar aggregates and guest proteins, systems with electrical charge of the same sign have been chosen, as proved in other cases.39 Materials and Methods Cetyltrimethylammonium bromide (CTAB), myristyltrimethylammonium bromide (MTAB), KH2PO4 for buffer preparation, 1-pentanol, and the protein cyt c from bovine heart (MW ) 12 327) were purchased from Sigma and used without further purification. Cetyltrimethylammonium and myristyltrimethylammonium, usually in the form of bromide salts, are the most studied and best characterized cationic surfactants.40-43 A detailed phase diagram of CTAB has been published by Hertel and Hoffmann.44 The phosphate buffer was prepared as a solution of KH2PO4 (0.1 M) in D2O, and its pH was adjusted to 7.0 ( 0.1 with a KOH solution. The pH of each sample and its reference was measured with a Hanna pHep 2 pH-meter; the accuracy was (0.1 unit. pH variations in the range 7.0-8.4 were used to modulate the average surface charge of the guest protein. The lyotropic systems were prepared starting from the buffered 10-3 M protein solution and adding the required amount of surfactants by weight. The reference solutions were the buffered protein solutions without added surfactant or cosurfactant. The pH values of the lyotropic sample and of its reference solution were the same within the accuracy of the pH-meter used. Monodimensional 1H NMR spectra were recorded on Bruker Avance 400, Avance 500, Avance 600, and Avance 800 spectrometers operating at 400.13, 500.13, 600.13, and 800.13 MHz, respectively. Different field strengths were useful to test the reproducibility of the effect of the orienting media. For the cyt c reference solution the standard presaturation pulse sequence was used, whereas for the micellar solution the superWEFT45 (water-eliminated Fourier transform) pulse sequence was used with recycle delay ranging from 80 to 200 ms. This ensured an appreciable reduction of the intensity of HOD and hydrogenated surfactant signals. 2H NMR spectra were acquired using the lock channel, and for these experiments no proton decoupling was used. COSY46,47 and NOESY48 experiments were performed at 303 and 317 K, respectively, on the micellar sample. The COSY spectrum was recorded at 800 MHz with spectral windows of 80.11 ppm × 80.11 ppm for 4096 × 512 data points. A relaxation delay of 50 ms was used. The NOESY spectrum was recorded at 600 MHz with spectral windows of 100 ppm × 100 ppm for 4096 × 1024 data points. A relaxation delay of 176 ms was used. All NMR data were processed with standard Bruker software packages (XWINNMR). Results and Discussion CTAB was added to cyt c/phosphate buffer solution, pH 7, in the concentration range of 25-27% w/w. This was done to match the ND+ nematic phase of the CTAB/water binary system, around 298-313 K. In most of the samples only a partial formation of the nematic phase was observed, and accordingly, the deuterium NMR spectra showed the intense single peak of the isotropic phase and the weak doublet of the nematic part. Figure 1 shows the 2H (c) and 1H (b) NMR spectra of a sample

Bertini et al. containing 27% w/w CTAB, at 302 K. The 1H NMR spectra contained only the narrow peaks of the isotropic phase, shifted by a few tens of parts per billion with respect to the reference solution (a). The assignment of the downfield and upfield paramagnetic signals is also reported in the same figure.49 Comparable results were obtained for the shorter-chain surfactant MTAB. By increasing the pH to 8.4, an entirely nematic sample was obtained; its 1H NMR spectrum, shown in Figure 2, contained few and very broad signals. A complete orientation of the sample was reached after the system was kept overnight in the magnetic field of the spectrometer (11.7 T) at 302 K. The deuterium spectrum consisted of a doublet (Figure 2c), and this confirmed the presence of a well-established nematic phase. On the other hand, the broadening of the 1H NMR peaks with respect to the sample at pH 7 indicated that the total positive charge on the protein was lowered and that a stronger interaction with the positive CTAB micelles occurred. By increasing the temperature, a nematic-isotropic transition took place. In the intermediate range 306-309 K both phases were present to a comparable extent. The addition of an alcohol as cosurfactant can stabilize the nematic phase. Moreover, as reported in the literature,27 for certain alcohol/surfactant molar ratios, a change in the shape of the micelles is induced and a different orientation in the external magnetic field can be obtained. This effect can be explained by considering that molecules with different shapes and polarities are inserted into the micellar solution of the original surfactant. We thus investigated the systems with CTAB/C5H11OH ) 1.25 w/w at various surfactant contents, where discoid micelles and a ND- phase are present.50 In the system with [CTAB] ) 10%, a well-defined, stable, and reproducible nematic phase was obtained, in the range of temperature between 295 and 311 K. Figure 3 shows the 1H spectrum of cyt c in CTAB/pentanol mixed micelles and in its reference solution, recorded at 800 MHz and 303 K. This sample was entirely nematic and well aligned, as shown by the 2H spectrum, also reported in the figure. Several reasonably well-resolved multiplets can be observed in this spectrum, which are due to the residual dipolar interactions between protons and their nearest neighbors. In particular, there are triplets with relative intensities 1:2:1 due to the interaction among the three protons of the methyl groups, and doublets with 1:1 intensities due to the interaction between the two geminal protons of the methylenes. This effect is expected and general, but is particularly evident in paramagnetic proteins, such as cyt c, that possess several CH3 and CH2 groups sufficiently close to the paramagnetic center to yield signals well separated from the diamagnetic region of the spectrum. For these signals it is possible to establish an unambiguous correspondence between each multiplet and the peaks of cyt c in the buffer solution. For instance, the two heme methyls (8-CH3 and 3-CH3, which have shifts at 34.59 and 31.78 ppm, respectively) are split into triplets, the latter not completely resolved, and the propionate 7R′-CH (at 18.56 ppm) and histidine 18β-CH (at 14.15 ppm) are split into doublets due to coupling with their geminal partners. A comparison between the spectra in CTAB micelles (Figure 2) and in CTAB/pentanol micelles (Figure 3) shows that different orienting media are able to induce splittings of different extents in the same signal (see, for example, the above-quoted 3-CH3 and the signal around -24 ppm, due to -CH3 of the methionine 80). Figure 4 shows an 800 MHz COSY spectrum of cyt c in CTAB/pentanol mixed micelles. Cross-peaks mainly due to dipolar couplings between protons are present. The two insets

Orientation of Cytochrome c in a Liquid Crystal

J. Phys. Chem. B, Vol. 104, No. 45, 2000 10655

Figure 1. 1H NMR spectra (500 MHz) at T ) 302 K of 0.5 mM cyt c: (a) in 100 mM phosphate/D2O buffer solution (pH 7.0), (b) in the same buffer solution containing 27% w/w CTAB. Several assignments are shown. (c) 2H NMR spectrum at T ) 302 K, corresponding to system b.

in this figure are portions of a NOESY spectrum of the same system, recorded at 600 MHz. The better resolution permits a better appreciation of the correlations between peaks inside a multiplet. The separation among the components in a multiplet depends on the orientation of each individual group with respect to the external magnetic field; in turn, this arises from the orientation of the protein induced by the lyotropic system in which it is dissolved. Thus, the splittings in rodlike CTAB micelles, which are known to orient themselves parallel to the magnetic field,44 are expected to be different from those in the disklike CTAB/ pentanol micelles, which, on the contrary, are preferentially oriented perpendicularly to the magnetic field.50 Therefore, the observed differences are in line with expectations. In Table 1 the variation of shifts and splittings of cyt c paramagnetic signals in the ternary system CTAB/pentanol/D2O are reported at two different magnetic fields (9.4 and 18.8 T). For signals split into multiplets, the values of shift variations have been calculated as the difference between the multiplet center and the peak of the reference sample. As expected, shift variations are field dependent, so that their values are constant at different fields when expressed in parts per million; on the contrary, splittings do not depend on the external field strength, and they are constant when expressed in hertz. The theoretical interpretation of the proton paramagnetic shifts in Table 1 is not straightforward, as they can be affected by a number of factors, including slight conformational changes induced in the protein by the interaction with the liquid crystal phase. It must also be taken into account that the 1H chemical

shift anisotropy could play a role, even if scaled with respect to the oriented protein fraction. Values reported in the literature for this effect are scarce and often very scattered, due to the many experimental difficulties in obtaining reliable results.51-53 So, in our case, it was not possible to estimate a priori the anisotropy value for each proton. The observed splittings, on the contrary, are expected to be less sensitive to conformational changes, and could be satisfactorily interpreted in terms of partial orientation. The splitting due to the dipolar coupling between two protons in partially oriented systems, obtained by calculating the energy difference between two states differing by ∆MI ) (1, is provided by6

∆ν )

µ0S hγI2 16π

3

3

r

[D (3 cos ϑ - 1) + 23D 2

ax

rh

]

sin2 ϑ cos 2φ

(1) where S is the generalized order parameter for internal motion of the internuclear vector, Dax and Drh are the axial and rhombic components of a molecular alignment D tensor, r is the protonproton distance, µ0 is the magnetic permeability of a vacuum, γI is the proton magnetogyric ratio, and ϑ and φ are the polar coordinates describing the orientation of the proton-proton direction in the D frame. The S parameter was assumed to be equal to 1. Therefore, the CH2 signals are split into two lines, at -∆ν/2 and +∆ν/2 with respect to the average signals, whereas the CH3 signals are split into three lines, at -∆ν, 0, and +∆ν. As far as the methyl protons are concerned, it must be taken into account

10656 J. Phys. Chem. B, Vol. 104, No. 45, 2000

Bertini et al.

Figure 2. 1H NMR spectra (500 MHz) at T ) 302 K of 0.5 mM cyt c: (a) in 100 mM phosphate/D2O buffer solution (pH 8.4), (b) in the same buffer solution containing 27% w/w CTAB. (c) 2H NMR spectrum at T ) 302 K, corresponding to system b.

Figure 3. 1H NMR spectra (800 MHz) at T ) 303 K of 1.2 mM cyt c: (a) in 100 mM phosphate/D2O buffer solution (pH 7.0), (b) in the same buffer solution containing 10% w/w CTAB and 8% w/w 1-pentanol. (c) 2H NMR spectrum at T ) 303 K, corresponding to system b.

that all protons are rotating freely around the methyl axis and their positions must be averaged correspondingly. The values for the splittings calculated in this way are equal to those calculated for two protons along the ternary symmetry axis of

the methyl (with S ) 1), at the same distance of the protons in the methyl group, scaled by a factor of 3/2. This factor must be introduced into eq 1 to take into account the fact that methyl protons constitute a magnetically equivalent group.

Orientation of Cytochrome c in a Liquid Crystal

J. Phys. Chem. B, Vol. 104, No. 45, 2000 10657

Figure 4. Low-field region of the 800 MHz COSY spectrum of cyt c (1.2 mM) in the system CTAB/1-pentanol (10%/8% w/w) at 303 K. The two insets are portions of the NOESY spectrum of the same system, recorded at 600 MHz.

TABLE 1: Shift Variations (∆δ, ppm), Experimental Absolute Values and Calculated Values of the Splittings (|∆ν|, Hz) of the Hyperfine-Shifted Signals of Cytochrome c in the Ternary System CTAB (10% w/w)/1-pentanol (8% w/w)/D2O at 303 K for Two Different Values of the Magnetic Field (800 and 400 MHz) shifta 1-CH3, heme 3-CH3, heme 5-CH3, heme 8-CH3, heme prop 7R′, heme QT2, heme His 18, Hδ(2) His 18, 2Hβ Met 80, Hβ1(2) Met 80, CH3 Met 80, Hγ1(2) Leu 68, CH3δ1 Gly 29, HR2 Pro 30, Hδ1(2)

∆δ (ppm) 400 MHzb 800 MHzc

(ppm)

-0.15 +0.16 -0.08 -0.15 -0.08 g -0.50 -0.02 -0.21 +0.82 -1.01 g +0.37 +0.58

7.10 31.78 10.15 34.59 18.57 -2.33 23.57 14.16 12.16 -23.81 -27.65 -2.66 -4.15 -6.21

-0.19 +0.16 -0.09 -0.17 -0.08 +0.14 -0.51 -0.01 -0.22 +0.83 -1.06 +0.05 +0.38 +0.56

|∆ν| (Hz) 400 MHzd 800 MHzd

mean of the calcd valuese ∆ν (Hz)

std devf ∆ν (Hz)

g 160 g 264 328 g

144 165 258 260 332 104

+132 +138 +263 +243 +160 +211

30 38 26 37 62 22

280 216 80 112 g

270 220 70 h 80

-239 -210 +59 +96 +58

34 53 24 63 40

164

120

+146

24

At 800 MHz. Accuracy (0.03 ppm. Accuracy (0.01 ppm. Accuracy ranges between (5 and (20 Hz. The fit was performed on a family of 35 members of the solution structure of horse heart cyt c. f Of the calculated values across the family. g Assignment of these peaks was uncertain. h This peak was too broad to evaluate the splitting. a

b

c

d

In principle, the splittings are expected to be both positive and negative, depending on the orientation of the vector connecting the dipole-dipole-coupled protons with respect to the external magnetic field. From the fitting of these absolute values, measured at 800 MHz, we obtained Dax, Drh, and three Eulerian angles, defining the orientation of the D tensor with respect to the molecular frame. The latter is taken with the x axis along the metal-pyrrole II direction and the z axis perpendicular to the heme plane, as usually done for heme

e

proteins.54,55 The coordinates of the atoms were taken from a family of 35 NMR solution structures of horse heart cyt c. The latter is the closest analogue to the bovine protein investigated here. The absolute experimental ∆ν values have been fitted by using eq 1, and the values of the fitting parameters Dax ) -0.011 and Drh ) -0.002 and R ) -0.055, β ) -0.495, and γ ) -0.196 for the three Eulerian angles have been found. The values of the splittings have then been calculated with the correct sign, and their mean value has been reported in Table 1 together

10658 J. Phys. Chem. B, Vol. 104, No. 45, 2000 with their standard deviation, to be compared with the absolute experimental values. The Dax value indicated that a protein fraction of about 1% is oriented. If the sign of the D components was reversed, an identical fitting with opposite calculated signs of ∆ν was obtained. The overall agreement between experimental and calculated values of the splitting is satisfactory, with two exceptions. In particular, the thioether 2-methyl group (QT2) and the propionate 7R′ proton exhibit a large difference between observed and fitted values. It is possible that these groups occupy a slightly different position in the bovine protein with respect to that shown by the NMR solution structure family of horse heart cyt c, which has been used to perform the present calculations. Indeed, these splittings are expected to be very sensitive to small angular variation, making them very good candidates to provide structural constraints for the refinement of structural features close to the metal center in paramagnetic metalloprotein. Conclusion The ternary system CTAB (10% w/w)/1-pentanol (8% w/w)/ D2O gives rise, around room temperature, to a stable nematic phase able to reproducibly induce a partial orientation in the protein cyt c. The effect of the partial orientation in the 1H NMR spectrum is a sizable splitting of many hyperfine-shifted 1H signals, which arises from dipolar couplings of neighboring geminal protons. A fit of the absolute values of these splittings to an orientation tensor gave reasonable agreement, although other effects may be introduced as responsible for the differences between experimental and calculated values. The present data demonstrate the potential use of liquid crystal solvents as a device to induce large orientation on protein molecules and sizable chemical shift variations and splittings, which can then be exploited to obtain structural information. Acknowledgment. For financial support, we thank MURST ex 40%, Italy, the European Union, TMR-LSF Contract ERB FMGE CT950033, and CNR, Progetto Finalizzato Biotecnologie, Italy. References and Notes (1) Saupe, A.; Englert, G. Phys. ReV. Lett. 1963, 11, 462. (2) Lawson, K. D.; Flautt, T. J. J. Am. Chem. Soc. 1967, 89, 5489. (3) Emsley, J. W.; Lindon, J. C. NMR Spectroscopy using liquid crystal solVents; Pergamon: New York, 1975. (4) Lee, Y.; Reeves, L. W. Can. J. Chem. 1975, 53, 162. (5) Veracini, C. A.; De Mummis, A.; Chidichimo, G.; Longeri, M.; Bertini, V. J. Chem. Soc., Perkin Trans. 1979, 2, 572. (6) Tjandra, N.; Bax, A. Science 1997, 278, 1111. (7) Clore, G. M.; Gronenborn, A. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5891. (8) Hansen, M. R.; Rance, M.; Pardi, A. J. Am. Chem. Soc. 1998, 120, 11210. (9) Ottiger, M.; Bax, A. J. Biomol. NMR 1998, 12, 361. (10) Wang, H.; Eberstadt, M.; Olejniczak, E. T.; Meadows, R. P.; Fesik, S. W. J. Biomol. NMR 1998, 12, 443. (11) Koenig, B. W.; Jin-Shan, H.; Ottiger, M.; Bose, S.; Hendler, R. W.; Bax, A. J. Am. Chem. Soc. 1999, 121, 1385. (12) Ottiger, M.; Bax, A. J. Biomol. NMR 1999, 13, 187. (13) Tolman, J. R.; Flanagan, J. M.; Kennedy, M. A.; Prestegard, J. H. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9279.

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