- Email: [email protected]
Collective vibrational effects in hydrogen bonded liquid amides and proteins studied by isotopic substitution
Journal of Molecular Structure 552 (2000) 71±80
www.elsevier.nl/locate/molstruc
Collective vibrational effects in hydrogen bonded liquid amides and proteins studied by isotopic substitution O.F. Nielsen a,*, C. Johansson a, D.H. Christensen a, S. Hvidt b, J. Flink c, S. Hùime Hansen c, F. Poulsen d a
Chemical Institute, University of Copenhagen, 5-Universitetsparken, DK-2100 Copenhagen, Denmark b Department of Chemistry, Roskilde University, DK-4000 Roskilde, Denmark c Novo Nordisk A/S, Novo AlleÂ, DK-2880 Bagsvñrd, Denmark d Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-1353 Copenhagen, Denmark Received 6 September 1999; received in revised form 1 November 1999; accepted 1 November 1999
Abstract Raman spectroscopy is used to study the fast dynamics of simple liquid amides and proteins. Raman spectra in the visible region of liquid amides are obtained with a triple additive scanning monochromator, whereas FT-Raman technique is used in the near-IR region in order to avoid ¯uorescence from impurities in the proteins. Raman spectra are shown in the amide-I region of HCONHCH3 (N-methylformamide with all isotopes in their natural abundance), H 13CONHCH3, HC 18ONHCH3, human growth hormone, frog tropomyosin and chymotrypsin inhibitor 2 including C-13 and N-15 enriched samples of the latter. Resonance energy transfer (RET) between amide molecules gives rise to a non-coincidence effect of the anisotropic and the isotropic components of the amide-I band. This effect in¯uences the band position in mixtures of liquid amide isotopomers. A further spectral feature caused by collective vibrational modes in the hydrogen bonded liquid amides is named coalescence of bands in mixtures of isotopomers (CBMI). The result of this effect is that only one band is found in mixtures of isotopomers where bands at different frequencies are observed for each of the isotopomers. A similar effect may account for the observation of protein amide-I bands with frequencies dependent only on the secondary structure of the protein and not on the amino acid residues. RET and CBMI are due to a collectivity of vibrational modes in different amide molecules. This collectivity may be related to a cooperativity of hydrogen bonds. A low-frequency band around 100 cm 21 is observed in hydrogen bonded liquid amides and proteins. Isotopic substitution shows that the mode corresponding to this band involves displacements of atoms in hydrogen bonds. This mode may drive a breaking of the hydrogen bond. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Hydrogen bonding; Raman spectroscopy; Cooperative effects; Amides; Proteins
1. Introduction Time resolved spectroscopic techniques based on femtosecond laser pulses are modern methods to * Corresponding author. Tel.: 1 45-35-320321; fax: 1 45-35350609. E-mail address: [email protected] (O.F. Nielsen).
study fast molecular dynamics on a picosecond time scale and faster. A recent survey on ultra fast spectroscopy of protein dynamics is given by Hochstrasser [1]. In the present study, ordinary cw-lasers with excitation wavelengths in either the visible or the near infrared (NIR) region were used to obtain Raman spectra. Either Fourier transform (FT) or dispersive spectrometers were used. This is a rather conventional
0022-2860/00/$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0022-286 0(00)00477-4
72
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
instrumentation. However, it should be emphasized that the Raman spectrum re¯ects dynamics on the very fast time scale mentioned above. Thus, information on the fast dynamics is certainly present in the Raman spectrum. In this contribution examples are shown on Raman studies of both the region of fundamental vibrations above 200 cm 21 and of the lowfrequency part below 200 cm 21. The latter is the region for translations and librations. Examples from studies of hydrogen bonded liquid amides and proteins will be presented. The energy required to break a speci®c hydrogen bond can depend on the presence of other hydrogen bonds in the system. Compared to an isolated hydrogen bond this can make the hydrogen bond stronger. Recently, this cooperative effect has been investigated for peptides [2] and amides [3]. For peptides the cooperativity is described in terms of supramolecular properties [2]. The collective vibrational effects described in the present contribution for amides and proteins can be explained in analogy with a solid state phonon description of 12C/ 13C diamond mixtures. Accordingly, collective vibration is described in terms of a supramolecular hydrogen bonded lattice. Stronger hydrogen bonds give stronger vibrational couplings between the molecules. Thus the collective vibrational effects may be related to the cooperative effect in hydrogen bonded systems. Use of isotope substitution in studies of intermolecular interactions by Raman spectroscopy was presented at the ªSummer School on Isotope Effects as Tools in Basic and Environmental Researchº in Roskilde 1995 [4]. Further reference to this subject is found in two recent reviews [5,6]. The present contribution contains a survey of spectral phenomena in Raman caused by collective effects and new experimental results will be presented for hydrogen bonded amides and proteins.
2. Experimental 2.1. Chemicals N-methylformamide (purum, .99%) was obtained from FLUKA, Switzerland. H 13CONHCH3 and HC 18ONHCH3 were purchased from CAMPRO Scienti®c, The Netherlands. The speci®ed contents
of C-13 and O-18 were .99% and .95%, respectively. All N-methylforamide samples were distilled in vacuum prior to use. CH3C 18ONHCH3 was synthesized by a reaction between CH3C 18O 18OCH2CH3 and NH2CH3 with 95 at.% O-18. Tropomyosin was extracted from Rana esculenta leg muscles, as described elsewhere for another frog type [7]. The aa-tropomyosin form was separated from ab-tropomyosin by use of hydroxyapatite chromatography [8,9]. The puri®ed protein fractions were ®nally dialyzed against water, lyophilized, and stored frozen. The Human growth hormone (hGH) was supplied by Novo Nordisk A/S, Denmark. The hGH is produced by recombinant technology, and after several stages of column chromatographic puri®cation, the ®nal liquid can be lyophilized without addition of excipients, giving a pure, dry hGH product. This dry hGH powder was used without further puri®cation or other treatment. C-13 and N-15 labelled chymotrypsin inhibitor 2, CI 2, was prepared as described previously [10,11]. The molecular mass was measured by mass spectroscopy to be 7644:12 ^ 0:05 Da corresponding to an 81% isotope labelling. 2.2. Instrumental Raman spectra of the liquid amides were obtained at room temperature on a DILOR Z24 triple additive spectrometer using a Spectra Physics Millenia II laser operating at 532 nm with an output on 400 mW. Spectra were obtained by right angle scattering in a horizontal scattering plane. Spectra in Ivv- and Ivh-scattering con®gurations were recorded with 3 cm 21 spectral resolution. In order to avoid ¯uorescence NIR-FT-Raman spectra were obtained of the proteins in the solid state at room temperature. A BRUKER IFS66 interferometer equipped with a FRA106 Raman module was used. The exciting source was a Nd/YAG laser at 1064 nm with a maximum output on 300 mW. The detector was a Ge-diode cooled to liquid nitrogen temperature. All spectra were obtained at room temperature with a 6 cm 21 spectral slit width (after apodization) in a 1808 scattering con®guration.
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
Fig. 1. Simpli®ed description of the amide-I mode in amides and proteins.
3. Results and discussion 3.1. Liquid amides Band intensities in a Raman spectrum depend on the ®rst derivative of the polarizability tensor with regard to the normal coordinates. Usually the contributions for the isotropic and the anisotropic parts of the polarizability show up at the same frequency. Thus Raman spectra in Ivv- and Ivh-scattering con®gurations normally show different intensities, but the same band frequencies. Two anomalous observations in the Raman spectrum are caused by collectivity of vibrational modes:
73
(i) resonance energy transfer (RET); and (ii) coalescence of bands in mixtures of isotopomers (CBMI) [4±6]. The effect of RET is that the band frequency of a given normal mode depends on the scattering con®guration. CBMI is observed in binary mixtures of isotopomers, where the spectrum of a mixture is different from the weighted sum of the spectra of the two isotopomers. The amide-I band shows these peculiarities. The mode is mainly a carbonyl stretching mode as illustrated in Fig. 1, and the amide-I band is observed around 1650 cm 21 [12]. Fig. 2 shows Raman spectra in Ivv- and Ivh-scattering con®gurations of H 13CONHCH3. Evidently, the amide-I band has different intensities in the two scattering con®gurations, but the peak frequencies are also different. In the Ivv-scattering con®guration the maximum is found at 1617 cm 21, whereas the peak maximum in the Ivhcon®guration is around 1640 cm 21. This frequency shift for a given mode in the liquid state is typical for RET. The effect is caused by vibrational energy transfer in the vibrationally excited state from one molecule to another [5,6,13±15]. Fig. 3A illustrates a hydrogen bonded supramolecular chain of amide molecules. A short-term notation for this structure is
Fig. 2. Raman spectra of liquid H 13CONHCH3. The thicker curve is obtained in an Ivv-scattering con®guration and the thinner curve in an Ivhscattering con®guration.
74
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
Fig. 3. Schematical illustrations of hydrogen bonded chain structures in liquid amides. In (A) a idealized supramolecular entity is shown. A short notation for this structure is given in (B). (C) shows antiparallel structures of the chains. In real liquids these structures are much more disordered.
Fig. 4. Raman spectra obtained in the Ivv-scattering con®guration of carbon isotopomers of N-methylformamide: H 12CONHCH3 (broken curve), H 13CONHCH3 (dotted curve) and a 1:1 mixture of the two isotopomers (full curve).
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
75
Fig. 5. Raman spectra obtained in the Ivv-scattering con®gurations of oxygen isotopomers of N-methylformamide: HC 16ONHCH3 (broken curve), HC 18ONHCH3 (dotted curve) and a 1:1 mixture of the two isotopomers (full curve).
shown in Fig. 3B, and in Fig. 3C is shown two antiparallel amide chains. RET is a vibrationally excited state energy transfer between the adjacent carbonyl groups in the two chains in Fig. 3C, although RET also occurs between carbonyl groups in the hydrogen bonded chains [3,16]. The C-13 substitution in Nmethylformamide is not important for RET, but C13 and O-18 substituted amides are part of an ongoing study of binary mixtures of amide isotopomers. So far some binary mixtures of formamide isotopomers have been investigated including D, C-13, N-15 and O-18 substitution [4±6,17±21], and we are currently investigating binary mixtures with a similar isotope substitution of N-methylformamide and N-methylacetamide, because these amides are more relevant model compounds for hydrogen bonding in proteins. Some results for C-13 and O-18 substitution will be presented in this contribution. Fig. 4 shows Ivv-spectra of C-12 and C-13 Nmethylformamide isotopomers. In both cases, the isotope substitution is in the amide group. The amide-I band is as expected shifted down in frequency by the heavier carbon isotope. Two bands show up in the amide-I region of a 1:1 mixture of the two isotopes, but the spectrum is not just the sum of the individual isotopomers. The peak frequencies in the
mixture are shifted up in frequency as compared to the pure isotopomers. This is an effect often observed for RET by isotopic dilution [5,6]. In some cases binary mixtures of liquid amides show only one band with a frequency between the frequencies for the neat isotopomers. This is what we have called CBMI [4,6]. CBMI is illustrated for a mixture of HC 18ONHCH3 and HC 16ONHCH3 in Fig. 5. A peak maximum around 1641 cm 21 is found for O-18 substituted N-methylformamide and a maximum around 1657 cm 21 for N-methylformamide with O-16 in natural abundance. The 1:1 mixture shows only one band with a peak frequency between those for each of the two isotopomers. The exact position of the frequency of the band in the mixture depends on the mole fraction of the two isotopomers. Within the last 10 years several authors have observed a similar dependence of the Raman frequency on the 12 C/ 13C mole fraction in diamond [22±25]. The results were obtained for synthetic diamond with various ratios of the two carbon isotopes. Only one band is observed in the diamond containing a mixture of C-12 and C-13. The frequency for this band was found between the frequencies for the C-12 and C-13 isotopomers, and the exact frequency position were depending on the isotope composition. The diamond
76
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
Fig. 6. Raman spectra in the amide-I region of frog tropomyosin (upper curve, thin) and hGH (lower curve, thick).
results are explained in terms of a one phonon band in the crystal [22±24]. Transferred to the liquid amides a one-dimensional lattice can be obtained along the hydrogen bonded chains shown in Fig. 3. A phonon description in a crystal means that there are phase
relations between vibrational modes in different molecules. In the present case it means that there are phase relations between the carbonyl stretching modes along the hydrogen bonded amide chain, or one can say that there is a collectivity of the amide-I modes along this
Fig. 7. Raman spectra in the amide-I region of chymotrypsin inhibitor 2, CI 2. The thin curve is obtained from a sample with all isotopes in natural abundance, while the thick curve is recorded from a sample enriched 81% in C-13 and N-14.
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
77
Fig. 8. Low-frequency Raman spectrum of liquid CH3C 18ONHCH3 (thinner curve) and the Rn -representation calculated from the Raman data according to Eq. (1).
hydrogen bonded chain. The hydrogen bonded structure in Fig. 3 can be considered as a supramolecular assembly, so our results seem to indicate that a solid state like description of the fast vibrational dynamics can be used in supramolecular hydrogen bonded structures. CBMI is observed for 16O/ 18O- but not for 12 C/ 13C-isotopomers of N-methylformamide. An explanation could be that the frequency shift is much larger for the 12C/ 13C-isotopomers than for the pair of 16O/ 18O-isotopomers (Figs. 4 and 5). 3.2. Proteins The secondary structures of proteins are characterized by narrow Raman frequency regions for the amide-I band [12]. Even proteins with different primary structures show similar amide-I band frequencies for a given secondary structure. An example is given in Fig. 6 where NIR-FT-Raman spectra are shown for hGH (191 amino acid residues) and frog tropomyosin (284 amino acid residues). Both molecules contain mostly a-helical secondary structures. The individual amide-I site frequencies for different amino acid residues might differ, depending on the amino acid residue. However, Fig. 6 shows only one sharp band with nearly coinciding frequencies for the two proteins, because the site frequencies
merge into one band by a CBMI effect along the hydrogen bonded spines in the a-helix in analogy with the results for mixtures of liquid amide isotopomers mentioned above. Chymotrypsin inhibitor 2 contains 83 amino acid residues with a main chain of 65 residues. Both helices, strands and turns are present. In this way, it is not the best model system for a study of collective vibrational effects by isotope substitution. However, it is very dif®cult to get real proteins with a mixture of C-12 and C-13. That is the reason for studying the chymotrypsin inhibitor 2 sample containing a total of 81% C-13 and N-15. Fig. 7 shows a comparison between NIR-FT-Raman spectra of this sample and a sample with carbon and nitrogen isotopes in natural abundance (C-12 and N-14). The sample with isotopes in natural abundance shows a strong band with a maximum around 1670 cm 21. Although the isotopically enriched sample still contains C-12, no signi®cant band appears at 1670 cm 21, but a strong band is observed around 1625 cm 21. This is very similar to the CBMI effect in liquid mixtures of 16O/ 18O-isotopically substituted N-methylforamides. The merging into one band was not observed for mixtures of 12 C/ 13C-isotopomers for N-methylformamides. This might indicate an even stronger coupling
78
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
3.3. Low-frequency modes
Fig. 9. An illustration of the mode giving rise to the band around 100 cm 21 in the low-frequency Raman spectrum. The upper part of the ®gure shows an in-plane libration. However, our results cannot distinguish between in-plane and out-of-plane librations. Thus, the illustration in the lower part covers both cases. Recent ab initio calculations seem to con®rm an out-of-plane-motion [29].
between the amide-I modes in the proteins as compared to the liquid amides. IR-spectroscopy is also very often used to characterize secondary structures of polypeptides and proteins, and also in this case transition dipole coupling is important in order to explain the main spectral features [26,27].
Low-frequency modes involve large entities of molecules and intermolecular forces, so due to the heavy masses and the weak forces these modes are expected at low frequencies (below 200 cm 21) [4± 6,28]. This region is referred to as the region for translations and librations. By isotopic substitution, it is very easy to distinguish between translations and librations because the ratio between the isotope frequencies is given as the square root of the ratio between the molecular masses for a translational motion. For a libration the ratio between the frequencies is given by the square root of the ratio between the molecular moments of inertia [4±6,28]. A serious problem in low-frequency Raman spectra is the enormous intensity of the Rayleigh line. This problem can be overcome by use of the R n -representation given in the following equation: R n / n 1 2 exp 2hn c=kTI n
1
where I n is the intensity in the ordinary Raman spectrum at a Raman shift on n cm21 ; h is Planck's constant and k is Boltzmann's constant and T is the absolute temperature [5,6]. A fourth power frequency correction was performed in calculating R n [5,6].
Fig. 10. R n -representations calculated according to Eq. (1) for frog tropomyosin (thinner curve) and hGH (thicker curve). The sharp band at 85 cm 21 is a spurious band from the laser. The spectrum is reliable at frequencies higher than 85 cm 21.
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80 18
Fig. 8 shows I n and R n spectra of O-N-methylacetamide. The bands around 200 and 300 cm 21 are from internal skeleton deformation modes. It is much easier to recognize the band around 100 cm 21 in the R n -representation than it is in the real Raman spectrum. This band shows a frequency maximum depending on the isotope substitution in N-methylacetamide. These shifts tell us how the atoms are involved in the mode as illustrated in Fig. 9. This mode is of the librational type, and the mode might be of importance for breaking of hydrogen bonds in liquid amides. Our results does not allow to distinguish between an in-plane or an out-of-plane mode, but recent ab initio molecular orbital calculations by Torii and Tasumi seems to favour an intense out-ofplane mode for the Raman band around 100 cm 21 [29]. A similar low-frequency band is observed in the R n -representation of proteins as illustrated for frog tropomyosin and hGH in Fig.10. In analogy with the amides the mode corresponding to this band can be of importance for breaking (or making) hydrogen bonds in proteins. 4. Conclusion Raman spectra obtained by conventional cw-laser excitation contain information about the dynamics on a picosecond and faster time scale in hydrogen bonded liquids. The hydrogen bonded chains can be considered as supramolecular entities, and collective amideI band vibrational effects (RET and CBMI) can be explained in terms of intermolecular couplings along or between the hydrogen bonded chains. Binary mixtures of isotopomers are very useful in order to understand the observed spectral features. The collective vibrational effects may be related to cooperativity of the hydrogen bonds. Observed amide-I bands in Raman spectra of proteins can be explained by a vibrational coupling within the hydrogen bonded secondary structures. A low-frequency mode with a band maximum around 100 cm 21 can be of importance for the breaking of hydrogen bonds in liquid amides and proteins. Results for isotopically substituted molecules are important in order to get a description of the atomic displacements for this mode. Breaking of a hydrogen bond destroys the supramolecular entity and in¯uences the vibrational
79
collective couplings along the hydrogen bonded chain. Thus, there is an interplay between the high frequency amide-I collective dynamics and the lowfrequency intermolecular dynamics. Making and breaking of hydrogen bonds occur during conformational changes of many biomolecules. Hydrogen bonding is also very often important for intermolecular interactions of biomolecules. Thus studies of fast dynamics on a picosecond time scale or faster can be of importance in the context of a better understanding of biomolecular reactions on a molecular level. Finally, it should be mentioned that the fast dynamics also may be of importance for the interaction between polypeptides and surrounding water molecules [30,31]. Acknowledgements The NIR-FT-Raman instrument was funded by the Danish Natural Science Research Program within the Material Science Program and by Haldor Topsùe A/S, Lyngby. O.F.N., D.H.C. and S.H. are grateful to the Danish Natural Science Research Council for general funding during this project. E. Philip is thanked for synthesizing the O-18 sample of N-methylacetamide. I. Blangsted and L. Ryelund are thanked for recording the Raman spectra. References [1] R.M. Hochstrasser, J. Chem. Ed. 75 (1998) 559. [2] H. Guo, M. Karplus, J. Phys. Chem. 98 (1994) 7104. [3] H. Torii, T. Tatsumi, T. Kanazawa, M. Tasumi, J. Phys. Chem. B 102 (1998) 309. [4] O. Faurskov Nielsen, A. Mortensen, J. Yarwood, V. Shelley, J. Mol. Struct. 378 (1996) 1. [5] O. Faurskov Nielsen, Ann. Rep. Prog. Chem., Sect. C, Phys. Chem. 90 (1993) 3. [6] O. Faurskov Nielsen, Ann. Rep. Prog. Chem., Sect. C, Phys. Chem. 93 (1997) 57. [7] S. Hvidt, Biophys. Chem. 24 (1986) 211. [8] S.S. Lehrer, Y. Qian, S. Hvidt, Science 246 (1989) 926. [9] S. Hvidt, S.S. Lehrer, Biophys. Chem. 45 (1992) 51. [10] J.C. Madsen, O.W. Sùrensen, P. Sùrensen, F.M. Poulsen, J. Biomol. NMR 3 (239) 1993. [11] P. Sùrensen, F.M. Poulsen, J. Biomol. NMR 2 (1992) 99. [12] A.T. Tu, in: R.J.H. Clark, R.E. Hester (Eds.), Spectroscopy of Biological Systems, vol. 13, Wiley, New York, 1986, p. 47. [13] J.L. McHale, J. Chem. Phys. 75 (1981) 30.
80 [14] [15] [16] [17] [18] [19] [20] [21] [22]
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80 D.E. Logan, Chem. Phys. 103 (1986) 215. H. Torii, M. Tasumi, J. Chem. Phys. 99 (1993) 8459. H. Torii, M. Tasumi, J. Phys. Chem. B 102 (1998) 315. A. Mortensen, O. Faurskov Nielsen, J. Yarwood, V. Shelley, J. Phys. Chem. 98 (1994) 5221. A. Mortensen, O. Faurskov Nielsen, J. Yarwood, V. Shelley, Vib. Spectrosc. 8 (1994) 37. A. Mortensen, O. Faurskov Nielsen, Spectrochim. Acta 51A (1995) 1345. A. Mortensen, O. Faurskov Nielsen, J. Yarwood, V. Shelley, J.Phys. Chem. 99 (1995) 4435. A. Mortensen, O. Faurskov Nielsen, J. Yarwood, V. Shelley, J. Raman Spectrosc. 26 (1995) 669. K. Hass, M.A. Tamor, T.R. Anthony, W.F. Banholzer, Phys. Rev. B 44 (1991) 12 046.
[23] K.C. Hass, M.A. Tamor, T.R. Anthony, W.F. Banholzer, Phys. Rev. B 45 (1992) 7171. [24] J. Spitzer, P. Etchegoin, M. Cardona, T.R. Anthony, W.F. Banholzer, Solid State Commun. 88 (1993) 509. [25] M. Nissum, E. Shabanova, O. Faurskov Nielsen, J. Chem. Ed. (2000) (in press). [26] S. Krimm, Y. Abe, Proc. Natl Acad. Sci. USA 69 (1972) 2788. [27] H. Torii, M. Tasumi, J. Chem. Phys. 96 (1992) 3379. [28] S.E.M. Colaianni, O. Faurskov Nielsen, J. Mol. Struct. 347 (1995) 267. [29] H. Torii, M. Tasumi, Int. J. Quantum Chem. 70 (1998) 241. [30] X.G. Chen, R. Schweitzer-Stenner, S. Krimm, N.G. Mirkin, S.A. Asher, J. Am. Chem. Soc. 116 (1994) 11 141. [31] L.D. Baron, L. Hecht, G. Wilson, Biochemistry 36 (1997) 13 143.
www.elsevier.nl/locate/molstruc
Collective vibrational effects in hydrogen bonded liquid amides and proteins studied by isotopic substitution O.F. Nielsen a,*, C. Johansson a, D.H. Christensen a, S. Hvidt b, J. Flink c, S. Hùime Hansen c, F. Poulsen d a
Chemical Institute, University of Copenhagen, 5-Universitetsparken, DK-2100 Copenhagen, Denmark b Department of Chemistry, Roskilde University, DK-4000 Roskilde, Denmark c Novo Nordisk A/S, Novo AlleÂ, DK-2880 Bagsvñrd, Denmark d Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-1353 Copenhagen, Denmark Received 6 September 1999; received in revised form 1 November 1999; accepted 1 November 1999
Abstract Raman spectroscopy is used to study the fast dynamics of simple liquid amides and proteins. Raman spectra in the visible region of liquid amides are obtained with a triple additive scanning monochromator, whereas FT-Raman technique is used in the near-IR region in order to avoid ¯uorescence from impurities in the proteins. Raman spectra are shown in the amide-I region of HCONHCH3 (N-methylformamide with all isotopes in their natural abundance), H 13CONHCH3, HC 18ONHCH3, human growth hormone, frog tropomyosin and chymotrypsin inhibitor 2 including C-13 and N-15 enriched samples of the latter. Resonance energy transfer (RET) between amide molecules gives rise to a non-coincidence effect of the anisotropic and the isotropic components of the amide-I band. This effect in¯uences the band position in mixtures of liquid amide isotopomers. A further spectral feature caused by collective vibrational modes in the hydrogen bonded liquid amides is named coalescence of bands in mixtures of isotopomers (CBMI). The result of this effect is that only one band is found in mixtures of isotopomers where bands at different frequencies are observed for each of the isotopomers. A similar effect may account for the observation of protein amide-I bands with frequencies dependent only on the secondary structure of the protein and not on the amino acid residues. RET and CBMI are due to a collectivity of vibrational modes in different amide molecules. This collectivity may be related to a cooperativity of hydrogen bonds. A low-frequency band around 100 cm 21 is observed in hydrogen bonded liquid amides and proteins. Isotopic substitution shows that the mode corresponding to this band involves displacements of atoms in hydrogen bonds. This mode may drive a breaking of the hydrogen bond. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Hydrogen bonding; Raman spectroscopy; Cooperative effects; Amides; Proteins
1. Introduction Time resolved spectroscopic techniques based on femtosecond laser pulses are modern methods to * Corresponding author. Tel.: 1 45-35-320321; fax: 1 45-35350609. E-mail address: [email protected] (O.F. Nielsen).
study fast molecular dynamics on a picosecond time scale and faster. A recent survey on ultra fast spectroscopy of protein dynamics is given by Hochstrasser [1]. In the present study, ordinary cw-lasers with excitation wavelengths in either the visible or the near infrared (NIR) region were used to obtain Raman spectra. Either Fourier transform (FT) or dispersive spectrometers were used. This is a rather conventional
0022-2860/00/$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0022-286 0(00)00477-4
72
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
instrumentation. However, it should be emphasized that the Raman spectrum re¯ects dynamics on the very fast time scale mentioned above. Thus, information on the fast dynamics is certainly present in the Raman spectrum. In this contribution examples are shown on Raman studies of both the region of fundamental vibrations above 200 cm 21 and of the lowfrequency part below 200 cm 21. The latter is the region for translations and librations. Examples from studies of hydrogen bonded liquid amides and proteins will be presented. The energy required to break a speci®c hydrogen bond can depend on the presence of other hydrogen bonds in the system. Compared to an isolated hydrogen bond this can make the hydrogen bond stronger. Recently, this cooperative effect has been investigated for peptides [2] and amides [3]. For peptides the cooperativity is described in terms of supramolecular properties [2]. The collective vibrational effects described in the present contribution for amides and proteins can be explained in analogy with a solid state phonon description of 12C/ 13C diamond mixtures. Accordingly, collective vibration is described in terms of a supramolecular hydrogen bonded lattice. Stronger hydrogen bonds give stronger vibrational couplings between the molecules. Thus the collective vibrational effects may be related to the cooperative effect in hydrogen bonded systems. Use of isotope substitution in studies of intermolecular interactions by Raman spectroscopy was presented at the ªSummer School on Isotope Effects as Tools in Basic and Environmental Researchº in Roskilde 1995 [4]. Further reference to this subject is found in two recent reviews [5,6]. The present contribution contains a survey of spectral phenomena in Raman caused by collective effects and new experimental results will be presented for hydrogen bonded amides and proteins.
2. Experimental 2.1. Chemicals N-methylformamide (purum, .99%) was obtained from FLUKA, Switzerland. H 13CONHCH3 and HC 18ONHCH3 were purchased from CAMPRO Scienti®c, The Netherlands. The speci®ed contents
of C-13 and O-18 were .99% and .95%, respectively. All N-methylforamide samples were distilled in vacuum prior to use. CH3C 18ONHCH3 was synthesized by a reaction between CH3C 18O 18OCH2CH3 and NH2CH3 with 95 at.% O-18. Tropomyosin was extracted from Rana esculenta leg muscles, as described elsewhere for another frog type [7]. The aa-tropomyosin form was separated from ab-tropomyosin by use of hydroxyapatite chromatography [8,9]. The puri®ed protein fractions were ®nally dialyzed against water, lyophilized, and stored frozen. The Human growth hormone (hGH) was supplied by Novo Nordisk A/S, Denmark. The hGH is produced by recombinant technology, and after several stages of column chromatographic puri®cation, the ®nal liquid can be lyophilized without addition of excipients, giving a pure, dry hGH product. This dry hGH powder was used without further puri®cation or other treatment. C-13 and N-15 labelled chymotrypsin inhibitor 2, CI 2, was prepared as described previously [10,11]. The molecular mass was measured by mass spectroscopy to be 7644:12 ^ 0:05 Da corresponding to an 81% isotope labelling. 2.2. Instrumental Raman spectra of the liquid amides were obtained at room temperature on a DILOR Z24 triple additive spectrometer using a Spectra Physics Millenia II laser operating at 532 nm with an output on 400 mW. Spectra were obtained by right angle scattering in a horizontal scattering plane. Spectra in Ivv- and Ivh-scattering con®gurations were recorded with 3 cm 21 spectral resolution. In order to avoid ¯uorescence NIR-FT-Raman spectra were obtained of the proteins in the solid state at room temperature. A BRUKER IFS66 interferometer equipped with a FRA106 Raman module was used. The exciting source was a Nd/YAG laser at 1064 nm with a maximum output on 300 mW. The detector was a Ge-diode cooled to liquid nitrogen temperature. All spectra were obtained at room temperature with a 6 cm 21 spectral slit width (after apodization) in a 1808 scattering con®guration.
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
Fig. 1. Simpli®ed description of the amide-I mode in amides and proteins.
3. Results and discussion 3.1. Liquid amides Band intensities in a Raman spectrum depend on the ®rst derivative of the polarizability tensor with regard to the normal coordinates. Usually the contributions for the isotropic and the anisotropic parts of the polarizability show up at the same frequency. Thus Raman spectra in Ivv- and Ivh-scattering con®gurations normally show different intensities, but the same band frequencies. Two anomalous observations in the Raman spectrum are caused by collectivity of vibrational modes:
73
(i) resonance energy transfer (RET); and (ii) coalescence of bands in mixtures of isotopomers (CBMI) [4±6]. The effect of RET is that the band frequency of a given normal mode depends on the scattering con®guration. CBMI is observed in binary mixtures of isotopomers, where the spectrum of a mixture is different from the weighted sum of the spectra of the two isotopomers. The amide-I band shows these peculiarities. The mode is mainly a carbonyl stretching mode as illustrated in Fig. 1, and the amide-I band is observed around 1650 cm 21 [12]. Fig. 2 shows Raman spectra in Ivv- and Ivh-scattering con®gurations of H 13CONHCH3. Evidently, the amide-I band has different intensities in the two scattering con®gurations, but the peak frequencies are also different. In the Ivv-scattering con®guration the maximum is found at 1617 cm 21, whereas the peak maximum in the Ivhcon®guration is around 1640 cm 21. This frequency shift for a given mode in the liquid state is typical for RET. The effect is caused by vibrational energy transfer in the vibrationally excited state from one molecule to another [5,6,13±15]. Fig. 3A illustrates a hydrogen bonded supramolecular chain of amide molecules. A short-term notation for this structure is
Fig. 2. Raman spectra of liquid H 13CONHCH3. The thicker curve is obtained in an Ivv-scattering con®guration and the thinner curve in an Ivhscattering con®guration.
74
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
Fig. 3. Schematical illustrations of hydrogen bonded chain structures in liquid amides. In (A) a idealized supramolecular entity is shown. A short notation for this structure is given in (B). (C) shows antiparallel structures of the chains. In real liquids these structures are much more disordered.
Fig. 4. Raman spectra obtained in the Ivv-scattering con®guration of carbon isotopomers of N-methylformamide: H 12CONHCH3 (broken curve), H 13CONHCH3 (dotted curve) and a 1:1 mixture of the two isotopomers (full curve).
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
75
Fig. 5. Raman spectra obtained in the Ivv-scattering con®gurations of oxygen isotopomers of N-methylformamide: HC 16ONHCH3 (broken curve), HC 18ONHCH3 (dotted curve) and a 1:1 mixture of the two isotopomers (full curve).
shown in Fig. 3B, and in Fig. 3C is shown two antiparallel amide chains. RET is a vibrationally excited state energy transfer between the adjacent carbonyl groups in the two chains in Fig. 3C, although RET also occurs between carbonyl groups in the hydrogen bonded chains [3,16]. The C-13 substitution in Nmethylformamide is not important for RET, but C13 and O-18 substituted amides are part of an ongoing study of binary mixtures of amide isotopomers. So far some binary mixtures of formamide isotopomers have been investigated including D, C-13, N-15 and O-18 substitution [4±6,17±21], and we are currently investigating binary mixtures with a similar isotope substitution of N-methylformamide and N-methylacetamide, because these amides are more relevant model compounds for hydrogen bonding in proteins. Some results for C-13 and O-18 substitution will be presented in this contribution. Fig. 4 shows Ivv-spectra of C-12 and C-13 Nmethylformamide isotopomers. In both cases, the isotope substitution is in the amide group. The amide-I band is as expected shifted down in frequency by the heavier carbon isotope. Two bands show up in the amide-I region of a 1:1 mixture of the two isotopes, but the spectrum is not just the sum of the individual isotopomers. The peak frequencies in the
mixture are shifted up in frequency as compared to the pure isotopomers. This is an effect often observed for RET by isotopic dilution [5,6]. In some cases binary mixtures of liquid amides show only one band with a frequency between the frequencies for the neat isotopomers. This is what we have called CBMI [4,6]. CBMI is illustrated for a mixture of HC 18ONHCH3 and HC 16ONHCH3 in Fig. 5. A peak maximum around 1641 cm 21 is found for O-18 substituted N-methylformamide and a maximum around 1657 cm 21 for N-methylformamide with O-16 in natural abundance. The 1:1 mixture shows only one band with a peak frequency between those for each of the two isotopomers. The exact position of the frequency of the band in the mixture depends on the mole fraction of the two isotopomers. Within the last 10 years several authors have observed a similar dependence of the Raman frequency on the 12 C/ 13C mole fraction in diamond [22±25]. The results were obtained for synthetic diamond with various ratios of the two carbon isotopes. Only one band is observed in the diamond containing a mixture of C-12 and C-13. The frequency for this band was found between the frequencies for the C-12 and C-13 isotopomers, and the exact frequency position were depending on the isotope composition. The diamond
76
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
Fig. 6. Raman spectra in the amide-I region of frog tropomyosin (upper curve, thin) and hGH (lower curve, thick).
results are explained in terms of a one phonon band in the crystal [22±24]. Transferred to the liquid amides a one-dimensional lattice can be obtained along the hydrogen bonded chains shown in Fig. 3. A phonon description in a crystal means that there are phase
relations between vibrational modes in different molecules. In the present case it means that there are phase relations between the carbonyl stretching modes along the hydrogen bonded amide chain, or one can say that there is a collectivity of the amide-I modes along this
Fig. 7. Raman spectra in the amide-I region of chymotrypsin inhibitor 2, CI 2. The thin curve is obtained from a sample with all isotopes in natural abundance, while the thick curve is recorded from a sample enriched 81% in C-13 and N-14.
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
77
Fig. 8. Low-frequency Raman spectrum of liquid CH3C 18ONHCH3 (thinner curve) and the Rn -representation calculated from the Raman data according to Eq. (1).
hydrogen bonded chain. The hydrogen bonded structure in Fig. 3 can be considered as a supramolecular assembly, so our results seem to indicate that a solid state like description of the fast vibrational dynamics can be used in supramolecular hydrogen bonded structures. CBMI is observed for 16O/ 18O- but not for 12 C/ 13C-isotopomers of N-methylformamide. An explanation could be that the frequency shift is much larger for the 12C/ 13C-isotopomers than for the pair of 16O/ 18O-isotopomers (Figs. 4 and 5). 3.2. Proteins The secondary structures of proteins are characterized by narrow Raman frequency regions for the amide-I band [12]. Even proteins with different primary structures show similar amide-I band frequencies for a given secondary structure. An example is given in Fig. 6 where NIR-FT-Raman spectra are shown for hGH (191 amino acid residues) and frog tropomyosin (284 amino acid residues). Both molecules contain mostly a-helical secondary structures. The individual amide-I site frequencies for different amino acid residues might differ, depending on the amino acid residue. However, Fig. 6 shows only one sharp band with nearly coinciding frequencies for the two proteins, because the site frequencies
merge into one band by a CBMI effect along the hydrogen bonded spines in the a-helix in analogy with the results for mixtures of liquid amide isotopomers mentioned above. Chymotrypsin inhibitor 2 contains 83 amino acid residues with a main chain of 65 residues. Both helices, strands and turns are present. In this way, it is not the best model system for a study of collective vibrational effects by isotope substitution. However, it is very dif®cult to get real proteins with a mixture of C-12 and C-13. That is the reason for studying the chymotrypsin inhibitor 2 sample containing a total of 81% C-13 and N-15. Fig. 7 shows a comparison between NIR-FT-Raman spectra of this sample and a sample with carbon and nitrogen isotopes in natural abundance (C-12 and N-14). The sample with isotopes in natural abundance shows a strong band with a maximum around 1670 cm 21. Although the isotopically enriched sample still contains C-12, no signi®cant band appears at 1670 cm 21, but a strong band is observed around 1625 cm 21. This is very similar to the CBMI effect in liquid mixtures of 16O/ 18O-isotopically substituted N-methylforamides. The merging into one band was not observed for mixtures of 12 C/ 13C-isotopomers for N-methylformamides. This might indicate an even stronger coupling
78
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80
3.3. Low-frequency modes
Fig. 9. An illustration of the mode giving rise to the band around 100 cm 21 in the low-frequency Raman spectrum. The upper part of the ®gure shows an in-plane libration. However, our results cannot distinguish between in-plane and out-of-plane librations. Thus, the illustration in the lower part covers both cases. Recent ab initio calculations seem to con®rm an out-of-plane-motion [29].
between the amide-I modes in the proteins as compared to the liquid amides. IR-spectroscopy is also very often used to characterize secondary structures of polypeptides and proteins, and also in this case transition dipole coupling is important in order to explain the main spectral features [26,27].
Low-frequency modes involve large entities of molecules and intermolecular forces, so due to the heavy masses and the weak forces these modes are expected at low frequencies (below 200 cm 21) [4± 6,28]. This region is referred to as the region for translations and librations. By isotopic substitution, it is very easy to distinguish between translations and librations because the ratio between the isotope frequencies is given as the square root of the ratio between the molecular masses for a translational motion. For a libration the ratio between the frequencies is given by the square root of the ratio between the molecular moments of inertia [4±6,28]. A serious problem in low-frequency Raman spectra is the enormous intensity of the Rayleigh line. This problem can be overcome by use of the R n -representation given in the following equation: R n / n 1 2 exp 2hn c=kTI n
1
where I n is the intensity in the ordinary Raman spectrum at a Raman shift on n cm21 ; h is Planck's constant and k is Boltzmann's constant and T is the absolute temperature [5,6]. A fourth power frequency correction was performed in calculating R n [5,6].
Fig. 10. R n -representations calculated according to Eq. (1) for frog tropomyosin (thinner curve) and hGH (thicker curve). The sharp band at 85 cm 21 is a spurious band from the laser. The spectrum is reliable at frequencies higher than 85 cm 21.
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80 18
Fig. 8 shows I n and R n spectra of O-N-methylacetamide. The bands around 200 and 300 cm 21 are from internal skeleton deformation modes. It is much easier to recognize the band around 100 cm 21 in the R n -representation than it is in the real Raman spectrum. This band shows a frequency maximum depending on the isotope substitution in N-methylacetamide. These shifts tell us how the atoms are involved in the mode as illustrated in Fig. 9. This mode is of the librational type, and the mode might be of importance for breaking of hydrogen bonds in liquid amides. Our results does not allow to distinguish between an in-plane or an out-of-plane mode, but recent ab initio molecular orbital calculations by Torii and Tasumi seems to favour an intense out-ofplane mode for the Raman band around 100 cm 21 [29]. A similar low-frequency band is observed in the R n -representation of proteins as illustrated for frog tropomyosin and hGH in Fig.10. In analogy with the amides the mode corresponding to this band can be of importance for breaking (or making) hydrogen bonds in proteins. 4. Conclusion Raman spectra obtained by conventional cw-laser excitation contain information about the dynamics on a picosecond and faster time scale in hydrogen bonded liquids. The hydrogen bonded chains can be considered as supramolecular entities, and collective amideI band vibrational effects (RET and CBMI) can be explained in terms of intermolecular couplings along or between the hydrogen bonded chains. Binary mixtures of isotopomers are very useful in order to understand the observed spectral features. The collective vibrational effects may be related to cooperativity of the hydrogen bonds. Observed amide-I bands in Raman spectra of proteins can be explained by a vibrational coupling within the hydrogen bonded secondary structures. A low-frequency mode with a band maximum around 100 cm 21 can be of importance for the breaking of hydrogen bonds in liquid amides and proteins. Results for isotopically substituted molecules are important in order to get a description of the atomic displacements for this mode. Breaking of a hydrogen bond destroys the supramolecular entity and in¯uences the vibrational
79
collective couplings along the hydrogen bonded chain. Thus, there is an interplay between the high frequency amide-I collective dynamics and the lowfrequency intermolecular dynamics. Making and breaking of hydrogen bonds occur during conformational changes of many biomolecules. Hydrogen bonding is also very often important for intermolecular interactions of biomolecules. Thus studies of fast dynamics on a picosecond time scale or faster can be of importance in the context of a better understanding of biomolecular reactions on a molecular level. Finally, it should be mentioned that the fast dynamics also may be of importance for the interaction between polypeptides and surrounding water molecules [30,31]. Acknowledgements The NIR-FT-Raman instrument was funded by the Danish Natural Science Research Program within the Material Science Program and by Haldor Topsùe A/S, Lyngby. O.F.N., D.H.C. and S.H. are grateful to the Danish Natural Science Research Council for general funding during this project. E. Philip is thanked for synthesizing the O-18 sample of N-methylacetamide. I. Blangsted and L. Ryelund are thanked for recording the Raman spectra. References [1] R.M. Hochstrasser, J. Chem. Ed. 75 (1998) 559. [2] H. Guo, M. Karplus, J. Phys. Chem. 98 (1994) 7104. [3] H. Torii, T. Tatsumi, T. Kanazawa, M. Tasumi, J. Phys. Chem. B 102 (1998) 309. [4] O. Faurskov Nielsen, A. Mortensen, J. Yarwood, V. Shelley, J. Mol. Struct. 378 (1996) 1. [5] O. Faurskov Nielsen, Ann. Rep. Prog. Chem., Sect. C, Phys. Chem. 90 (1993) 3. [6] O. Faurskov Nielsen, Ann. Rep. Prog. Chem., Sect. C, Phys. Chem. 93 (1997) 57. [7] S. Hvidt, Biophys. Chem. 24 (1986) 211. [8] S.S. Lehrer, Y. Qian, S. Hvidt, Science 246 (1989) 926. [9] S. Hvidt, S.S. Lehrer, Biophys. Chem. 45 (1992) 51. [10] J.C. Madsen, O.W. Sùrensen, P. Sùrensen, F.M. Poulsen, J. Biomol. NMR 3 (239) 1993. [11] P. Sùrensen, F.M. Poulsen, J. Biomol. NMR 2 (1992) 99. [12] A.T. Tu, in: R.J.H. Clark, R.E. Hester (Eds.), Spectroscopy of Biological Systems, vol. 13, Wiley, New York, 1986, p. 47. [13] J.L. McHale, J. Chem. Phys. 75 (1981) 30.
80 [14] [15] [16] [17] [18] [19] [20] [21] [22]
O.F. Nielsen et al. / Journal of Molecular Structure 552 (2000) 71±80 D.E. Logan, Chem. Phys. 103 (1986) 215. H. Torii, M. Tasumi, J. Chem. Phys. 99 (1993) 8459. H. Torii, M. Tasumi, J. Phys. Chem. B 102 (1998) 315. A. Mortensen, O. Faurskov Nielsen, J. Yarwood, V. Shelley, J. Phys. Chem. 98 (1994) 5221. A. Mortensen, O. Faurskov Nielsen, J. Yarwood, V. Shelley, Vib. Spectrosc. 8 (1994) 37. A. Mortensen, O. Faurskov Nielsen, Spectrochim. Acta 51A (1995) 1345. A. Mortensen, O. Faurskov Nielsen, J. Yarwood, V. Shelley, J.Phys. Chem. 99 (1995) 4435. A. Mortensen, O. Faurskov Nielsen, J. Yarwood, V. Shelley, J. Raman Spectrosc. 26 (1995) 669. K. Hass, M.A. Tamor, T.R. Anthony, W.F. Banholzer, Phys. Rev. B 44 (1991) 12 046.
[23] K.C. Hass, M.A. Tamor, T.R. Anthony, W.F. Banholzer, Phys. Rev. B 45 (1992) 7171. [24] J. Spitzer, P. Etchegoin, M. Cardona, T.R. Anthony, W.F. Banholzer, Solid State Commun. 88 (1993) 509. [25] M. Nissum, E. Shabanova, O. Faurskov Nielsen, J. Chem. Ed. (2000) (in press). [26] S. Krimm, Y. Abe, Proc. Natl Acad. Sci. USA 69 (1972) 2788. [27] H. Torii, M. Tasumi, J. Chem. Phys. 96 (1992) 3379. [28] S.E.M. Colaianni, O. Faurskov Nielsen, J. Mol. Struct. 347 (1995) 267. [29] H. Torii, M. Tasumi, Int. J. Quantum Chem. 70 (1998) 241. [30] X.G. Chen, R. Schweitzer-Stenner, S. Krimm, N.G. Mirkin, S.A. Asher, J. Am. Chem. Soc. 116 (1994) 11 141. [31] L.D. Baron, L. Hecht, G. Wilson, Biochemistry 36 (1997) 13 143.