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Infrared and laser Raman spectroscopy in membrane analysis
Chem. Phys. Lipids $ (1972) 347-354 © North-Holland Publishing Company
INFRARED AND LASER RAMAN SPECTROSCOPY IN MEMBRANE ANALYSIS DONALD F. H. WALLACH
Division of Radiobiology, Dept. of Therapeutic Radiology, Tufts University School of Medicine, Boston, Mass. 02111, U.S.A.
I. Infrared spectroscopy
A. Introduction The stretching, twisting, etc., of chemical bonds at frequencies of 10 ' ° 1011 Hz may change their dipoles, allowing absorption of electromagnetic radiation of similar, i.e. infrared (IR) frequenciest-a). However, IR band frequencies reflect not only those of bond oscillations but also vibrational and environmental interactions. Thus peptide C----O, C-N, and N - H all absorb in the IR, but specific peptide IR bands, representing the combined contributions of these vibrations, may also indicate 2 ° structure. Because H20 layers >0.05 mm are IR-opaque, many IR studies require use of D20 (clear in most of the IR) or dried material on/in IR-transparent supports.
B. Soluble polypeptides and proteins Several IR bands reflect peptide conformation. E.g. poly-L-lysine can exist in one or a mixture of conformations, i.e. unordered at pH 7.0 and low A/2, or-helical at pH 11.3 at 20 ° and fl-structured (anti ][) at pH 11.3, 55°4). IR distinguishes the latter two because the Amide I band lies at 1652 cmin ~t-helix and near 1625 c m - ' in fl-structures. Antiparailel-fl also yields a shoulder at 1693 c m - l , 5, 6). The Amide I region cannot discriminate between ~t-helix and unordered conformations in globular proteins and in proteins of known 2 ° structure, containing both ~t-helical and unordered peptide, side chain absorption obscures the splitting expected since the Amide I bands of pure unordered peptides lie at 1656 cm- 1, and at 1652 cm- ' for pure or-helix. The conjoint presence of fl-structures plus or-helical and/or unordered conformations can, however, be recognized. Thus, lysozyme (10~ antiparallel-fl) 7) shows a 1650cm-' peak with 1632 c m - ' shoulder, while carboxypeptidase A (15~ fl-) has a 1650 peak and a 1637cm-' shoulder a). 347
348
DONALD F. H. WALLACH
The Amide V band also mirrors 2 ° structure; thus extended-unordered and ~-helical poly--~,-methyI-L-glutamate, absorb at 700, 650, and 620 erarespectively. Concordantly, iysozyme, a mixture of fl-structured, unordered and helical peptide has three bands in this region, at 690, 650 and 600 c m - 1,9).
C. Membrane proteins IR spectra of resting erythrocytel°-13), or other animal plasma membranes 14.15), dried from neutral aqueous solutions or suspended inneutral D20 show the Amide I bands at 1652 c m - l , due to or-helical and/or unordered conformations, but bacterial membranes seem to contain some fl-structure 15). In contrast, inner mitochondrial membranes contain significant antiparallel-fl peptide 17, 18). Their film or D20 spectra exhibit a peak around 1650 cm -~, a pronounced shoulder near 1635-1640 cm-~ and a smaller shoulder near 1690 cm -~. The presence in membranes of ordered conformations other than or-helix is not surprising, since most proteins are conformational mixtures, where even the ordered segments deviate from ideal synthetic models 6, 7, s, 19, z0).
D. IR detection of conformation change in membrane proteins For quick metabolic measurements, buffered membrane suspensions, on AgCI discs, are rapid frozen in liquid N2, lyophilized and their IR spectra measured. Several sensitive membrane functions can be recovered lz, is), provided the lyophilized membranes are first rehydrated at 100% Rel. Hum., prior to suspension in aqueous buffer. Erythrocyte ghosts, thus treated, reform biconcave discs, with full Na + - K + - A T P a s e activity and similar rehydration oflyophilized mitochondria indicates recovery of coupled oxidative phosphorylation zl). Addition of ATP and Mg 2+ to erythrocyte ghosts shifts the spectrum toward the fl-conformation according to the rate of ATP cleavage. This is ATP specific and is seen upon lyophilization and in D20. Inner mitochondrial membranes show large Amide I differences, depending upon their metabolic state at the instant of freezing 18). Electron transport increases antiparallel fl-structure, i.e. augmented absorption near 1635 cm - I and 1685 cm - t , which is blocked or reversed by inhibiting respiration, or inducing phosphorylation with ADP. An extreme shift towards the antiparallel-fl follows uncoupling with dinitropheno118). D20 spectra also demonstrate fl-structure in the peptides of mitochondrial membranes. Also, when erythrocyte ghosts are kept in D20 for 60 rain at 20°C, or more briefly at 35°C, the apparent proportion of parallel fl-structure increases, the absorbance in near 1640 cm- 1, gradually assuming prominence of the Amide I region. The ghosts simultaneously lose their
349
INFRARED AND LASER RAMAN SPECTROSCOPY
concave shape and turn into spheres. This is due in part to amide deuteration, but also because protein structure in general22) and /~-structures in particularZ3, 9.4) are stabilized by entropic effects, which change together with H-bonding, when D20 replaces H20. Large Amide I changes need not imply extensive peptide refolding. Thus, carboxypeptidase A s) and ~-chymotrypsin 2°) contain many antiparallel loops, but incomplete /i'-structured H-bonding. However, minor peptide rotation [such as occurs in these enzymes upon substrate addition s,24)], might increase H-bonding so as to augment the/~-signal markedly (fig. I).
ANTIPARALLEL BETA-LOOP WITH INCOMPLETE H-BONDING
• =o I""-"
"
"
•
= O--H--N--O
•
= O--H--N--O
IMPROBABLE COMPLETION OF H-BONDING
POSSIBLE COMPLETION OF H-BONDING BY ROTATION ABOUT PEPTIDE AXIS
Fig. 1.
E. Lipids I. Table 1 lists some of the vibrations obtained from phospholipids. CH 2 rocking, (720 cm-l), indicates interaction between adjacent apolar chains, when at least 4 carbon atoms lie in an all-trans-planar configuration; in solids this band splits by as much as 6 cm- i, zs). P = O stretching ( 1250-1250 cm- a) can decrease in frequency and intensify upon H-bonding. The major lipid contribution near the Amide I region is due to sphingosine, whose amide absorbs at 1640-1540 c m - i , and phosphatidylserine - C O 0 - , absorbing near 1600 cm- 1 2. Aqueous dispersions of myelin 2s), as well as membrane lipid extracts 10), exhibit prominent CH2 rocking at 720 cm -a, but this is lacking in plasma membranes at room temperature x4,15), possibly by hydrophobic lipidprotein associations. In Micrococcus lysodekticus, CH2 rocking, as well as the ratios (asymmetric: symmetric methylene stretching), (at 2930 cm -l and 2855 cm-1 respectively), vary in whole membranes, their lipid extracts
350
DONALD F. H. WALLACH
and the extracted residues (0.3, 0.1 and 1.4, respectively at -180°C). Only extracted lipid shows significant CH2-rocking and the data all suggest apolar lipid-protein interactions 16). 3. Lipid extracted membranes, cast as a film, develop some E-structure 10), but this is lacking when the membranes are cast and dried before lipid extraction x0, 13, 14). However, exposure of intact or lipid-extracted membrane films to p H < 3 for 20 min, produces a/~-shoulder at 1630 cm -1 14). P = O stretching in dried red cell ghosts lies at 1225 cm -1, as does that of D20 dispersions of the membrane lipids 10). If these groups were ionically linked to membrane proteins, the P - O - phosphorus would be more electropositive, changing P = O stretching accordingly; however, this is not seen. The lower~ P = O stretching value then found in pure, isolated phospholipids 27), may reflect H-bonding. Pertinently, studies on the P : O stretching and CH 2 rocking amplitudes in cephalin and lecithin films 28) show intensification of these bands with applied electric fields, due to alignment of the phosphate-base dipoles and secondary rearrangement of the hydrocarbon chains; this shows that fatty acids may reorient with changing trans-membrane potentials. H. Laser-Raman spectroscopy
A. The Raman effect
,1%
Raman scattering also arises from molecular rotations and vibrations. When transparent substances are irradiated with monochromatic light, most of this is transmitted, some is scattered at the incident frequency, Rayleigh scattering, and traces are scattered at higher or lower frequencies, Raman scattering. The frequency shift between the incident light and a Raman line fits the vibrational frequency of the Raman-active bond. Raman scattering arises from changes in bond polarizability, i.e. induced dipole moments zg). Edsall z0) very early used Hg arcs for Raman analyses of amino acids and dipeptides, but polymer analysis by this method became feasible only when stable lasers emerged as intense, monochromatic sources.
B. Polypeptides and proteins The potency of laser Raman spectroscopy in the analysis of biopolymers is its versatility and manageable susceptibility to solvent interferences. H20 scatters neglibibly throughout most of the Raman spectrum and its OH band in the Amide I region is weak compared to OH absorption in the IR. Also H20 scattering can be overcome by high sample concentrations 31,32) and/or computer techniquesZZ), the latter allowing the use of low sample concentrations in H20. Table I lists important Raman bands of polypeptides.
351
INFRARED AND LASER RAMAN SPECTROSCOPY
TABLE l Some IR and Raman frequencies o f polypeptides and phospholipids 8 Assignment b
Frequency (cm- ~) Infrared Polypeptide b
Skeletal deformation Skeletal deformation N H and C = O out o f plane bending, (Amide IV) CHa rocking NH bending -~ skeletal modes; (Amide V) C-C stretch and CH rocking CHa stretch -t skeletal modes CH = CH bending P-O-C stretching C -. C stretching Methyl modes All-trans hydrocarbon ; extended Hydrocarbon chain; random Hydrocarbon ; all-trans Methyl-carbon stretching Hydrocarbon chain; all trans Methyl rocking CH2 twisting CH2 wagging P ~ O stretching ° CN stretching + N H in-plane bending; (Amide IIl) CH bending Methyl ; symmetric deformation Methyl; asymmetric deformation CH2 stretching and bending N H bending in plane + CN stretching; (Amide 1l) C ~ O stretching; amide; (Amide I). e C =- O stretching; ester CH2 asymmetrical stretching, (in plane) CH2 asymmetrical stretching, (out of plane) C = C - - H stretching
Phospholipid b
368s 528s
Raman PolYpeptide ~
Phospholipid b
375s d 527vs
625m
662w 720m
725w
756m 870-1150
907m
907vs 970m 980-1050s 1000-1620
1049vs
1048vw 1064m 1089m 1100m
1105s 1130s 1167s
1168m 1170-13OO 1180-1380 1250-1350
1300s 1328m
1310m 1331 s
1376vs
1371m
1453vs
1453vs 1460
1537-1560s
1549w
1635-1655vs
1654s 1720-1740s
2853s
2853s
2874m
2926s
2926s 3000
2927vs
a Selected data from Chapman27), Koenig and Suttona0, Wallach et al. 1:) and Lippert and Peticolasa4). a Assignments are not all absolute and some frequencies determined for polypeptides also apply to phospholipids, e The P - O stretching frequency and Amide I frequencies are very dependent on hydrogen bonding. a vs = very strong; s = strong; m ~ medium; w - weak; vw = very weak.
352
D O N A L D F. H . W A L L A C H
I m p o r t a n t l y , the technique seems to discriminate between s-helical,/3- and unordered-polypeptide conformations.
C. Lipids and membranes Th e R a m a n spectra o f fatty acids, fatty acid esters and p h o s p h o l i p i d micelles have also been studied 34, zs), Table 1; also, early data show this m e t h o d , particularly if j o i n e d to difference techniques, to be a unique tool for the analysis o f m e m b r a n e proteins and lipids in situ.
References 1) D. N. Kendall, in: Applied infrared spectroscopy, ed. by D. N. Kendall. Reinhold, New York (1966) 1 2) L. W. Herscher, in: Applied infrared spectroscopy, ed. by D. N. Kendall. Reinhold, New York (1966) 88 3) H. A. Szymanski, Theory and practice of infrared spectroscopy. Plenum Press, New York (1964) 4) R. Townend, T. F. Kumosinski, S. N. Timasheff, G.D. Fasman and B. Davidson, Biochem. Biophys. Res. Commun. 23 (1966) 163 5) H. Susi, S. N. Timasheff and L. Stevens, J. Biol. Chem. 242 (1967) 5460 6) S. N. Timasheff, H. Susi and L. Stevens, J. Biol. Chem. 242 (1967) 5407 7) D. C. Phillips, Sci. American 215 (5) (1966) 78 8) W. N. Lipscomb, Ace. Chem. Res. 3 (1970) 81 9) F. Fukushima and T. Miyazawa, Ann. Meeting of the Chem. So<:. of Japan, Tokyo (1964) 10) D. Chapman, V. B. Kamat and R. J. Levene, Science 160 (1968) 314 11) A. H. Maddy and B. R. Malcolm, Science 150 (1965) 1616 12) A. H. Maddy and B. R. Malcolm, Science 153 (1966) 213 13) J. M. Graham and D. F. H. Wallach, Biochem. Biophys. Acta 241 (1971) 180 14) D. F. H. Wallach and P. H. Zahler, Proc. Natl. Acad. Sci. U.S. 56 (1966) 1552 15) D. F. H. Wallach and P. H. Zahler, Biochem. Biophys. Acta 150 (1968) 186 16) D. H. Green and M. R. J. Salton, Biochem. Biophys. Acta 211 (1970) 139 17) D. F. H. Wallach, J. M. Graham and B. R. Fernbach, Arch. Biochem. Biophys. 131 (1969) 322 18) J. M. Graham and D. F. H. Wallach, Biochem. Biophys. Acta 193 (1969) 225 19) J. C. Kendrew, R. E. Dickerson, B. E. Strandberg, R. G. Hart, D. R. Davies, D. C. Phillips and V. C. Shore, Nature 105 (1966) 422 20) D. M. Blow, Biochem. J. 112 (1969) 261 21) D. F. H. Wallach and J. M. Graham, in: Biochemistry and biophysics ofmitochondrial membranes, ed. by N. Siliprandi and G. F. Azzone. Academic Press, New York, in press 22) W. Kauzmann, Advan. Protein Chem. 14 (1959) 1 23) J. Lynn and G. Fasman, Biochem. Biophys. Res. Commun. 33 (1968) 327 24) B. Davidson and G. Fasman, Biochem. 6 (1967) 1616 25) L. J. Bellamy, Advances in infrared group frequencies. Methuen, London (1968) 26) T. J. Jenkinson, V. B. Kamat and D. Chapman, Biochem. Biophys. Acta 183 (1969) 427 27) D. Chapman, The structure of lipids. Methuen, London (1965) Chap. 4 28) L. May, A. B. Kamble and I. P. Acosta, J. Membrane Biol. 2 (1970) 192 29) L. A. Woodward, in: Raman spectroscopy - theory and practise, ed. by H.A. Szymanski. Plenum Press, New York (1967) 1
INFRARED A N D LASER RAMAN SPECTROSCOPY
353
30) J. Edsall, in: Proteins, amino acids and peptides, ed. by E. J. Cohn and J. Edsall. Reinhold, New York (1943) 9 31) J. L. Koenig and P. L. Sutton, Biopolymers 8 (1969) 167 32) R. C. Lord and N.-T. Yu, J. Mol. Biol. 50 (1970) 509 33) D. F. H. Wallach, J. M. Graham and A. R. Oseroff, FEBS Letters 7 (1970) 330 34) J. L. Lipport and W. L. Peticolas, Proc. Natl. Acad. Sci. U.S. 68 (1971) 1572
Discussion
WALLACH also showed how optical activity measurements were at present inadequate for the quantitative analysis of secondary peptide structure in globular proteins, whether soluble or membrane located, and refuted contentions that the optical activities of membranes were necessarily distorted by light scattering, citing the work of Ottaway and Wettlaufer, Arch. Biochem. Biophys. 139 (1970) 257; Straus et al., Europ. J. Biochem. l l (1969) 201; and Wallach et al., J. Cell. Biol. 30 (1966) 601. He anticipates resolution of many of the problems involved through technical improvements and neu mathematical approaches, e.g. Saxena and Wettlaufer, Proc. Natl. Acad. Sci. U.S. 68 (1971) 969. In reply to BANGHAM'Squestion, he pointed out that mitochondrial membranes are particularly difficult to analyse by optical activity because of their high content of fl-structured polypeptide and variations in the proportion of fl-structure with metabolic state. Particularly for these reasons, they are unsuitable for the computation of light-scattering effects. In reply to CHERRY'S question, WALLACHcontended that most membranes must be treated optically as shells with an overall refractive index a little higher than water but with a wall refractive index of about i.55. ALLISON queried whether recent spectroscopic evidence from Wallach's and other laboratories was inconsistent with membrane penetration of proteins. WALLACH replied that the data still indicated the average membrane protein to be rather highly helical with a substantial proportion of peptide in an apolar environment such as envisaged in the membrane core. METCALFE enquired whether there was any technique by which one could determine the orientation of fl-structured segments with respect to the membrane surface, and WALLACH referred to infrared dichroism as such an approach, but that it would be useful only if the membranes could be oriented very precisely with regard to the incident infrared beam. In reply to CHAPMAN'S question whether the infrared assignments of various amide bands have been adequately tested, Wallach pointed to the consistency in the location of various conformationally-sensitive Amide bands in numerous synthetic polypeptides and various proteins where second-
354
D O N A L D F. H. W A L L A C H
ary structure had been established by X-ray crystallography. However, he stressed the persistent but resolvable problem of quantifying fl-structure by infrared techniques. WALLACH also alluded to the consistency between the location of the pertinent Raman scattering lines and the infrared bands as support for the correctness of the assignments and finally cited the theoretical studies of Miyazawa and others in support for the fundamental validity of the infraredapproach.
INFRARED AND LASER RAMAN SPECTROSCOPY IN MEMBRANE ANALYSIS DONALD F. H. WALLACH
Division of Radiobiology, Dept. of Therapeutic Radiology, Tufts University School of Medicine, Boston, Mass. 02111, U.S.A.
I. Infrared spectroscopy
A. Introduction The stretching, twisting, etc., of chemical bonds at frequencies of 10 ' ° 1011 Hz may change their dipoles, allowing absorption of electromagnetic radiation of similar, i.e. infrared (IR) frequenciest-a). However, IR band frequencies reflect not only those of bond oscillations but also vibrational and environmental interactions. Thus peptide C----O, C-N, and N - H all absorb in the IR, but specific peptide IR bands, representing the combined contributions of these vibrations, may also indicate 2 ° structure. Because H20 layers >0.05 mm are IR-opaque, many IR studies require use of D20 (clear in most of the IR) or dried material on/in IR-transparent supports.
B. Soluble polypeptides and proteins Several IR bands reflect peptide conformation. E.g. poly-L-lysine can exist in one or a mixture of conformations, i.e. unordered at pH 7.0 and low A/2, or-helical at pH 11.3 at 20 ° and fl-structured (anti ][) at pH 11.3, 55°4). IR distinguishes the latter two because the Amide I band lies at 1652 cmin ~t-helix and near 1625 c m - ' in fl-structures. Antiparailel-fl also yields a shoulder at 1693 c m - l , 5, 6). The Amide I region cannot discriminate between ~t-helix and unordered conformations in globular proteins and in proteins of known 2 ° structure, containing both ~t-helical and unordered peptide, side chain absorption obscures the splitting expected since the Amide I bands of pure unordered peptides lie at 1656 cm- 1, and at 1652 cm- ' for pure or-helix. The conjoint presence of fl-structures plus or-helical and/or unordered conformations can, however, be recognized. Thus, lysozyme (10~ antiparallel-fl) 7) shows a 1650cm-' peak with 1632 c m - ' shoulder, while carboxypeptidase A (15~ fl-) has a 1650 peak and a 1637cm-' shoulder a). 347
348
DONALD F. H. WALLACH
The Amide V band also mirrors 2 ° structure; thus extended-unordered and ~-helical poly--~,-methyI-L-glutamate, absorb at 700, 650, and 620 erarespectively. Concordantly, iysozyme, a mixture of fl-structured, unordered and helical peptide has three bands in this region, at 690, 650 and 600 c m - 1,9).
C. Membrane proteins IR spectra of resting erythrocytel°-13), or other animal plasma membranes 14.15), dried from neutral aqueous solutions or suspended inneutral D20 show the Amide I bands at 1652 c m - l , due to or-helical and/or unordered conformations, but bacterial membranes seem to contain some fl-structure 15). In contrast, inner mitochondrial membranes contain significant antiparallel-fl peptide 17, 18). Their film or D20 spectra exhibit a peak around 1650 cm -~, a pronounced shoulder near 1635-1640 cm-~ and a smaller shoulder near 1690 cm -~. The presence in membranes of ordered conformations other than or-helix is not surprising, since most proteins are conformational mixtures, where even the ordered segments deviate from ideal synthetic models 6, 7, s, 19, z0).
D. IR detection of conformation change in membrane proteins For quick metabolic measurements, buffered membrane suspensions, on AgCI discs, are rapid frozen in liquid N2, lyophilized and their IR spectra measured. Several sensitive membrane functions can be recovered lz, is), provided the lyophilized membranes are first rehydrated at 100% Rel. Hum., prior to suspension in aqueous buffer. Erythrocyte ghosts, thus treated, reform biconcave discs, with full Na + - K + - A T P a s e activity and similar rehydration oflyophilized mitochondria indicates recovery of coupled oxidative phosphorylation zl). Addition of ATP and Mg 2+ to erythrocyte ghosts shifts the spectrum toward the fl-conformation according to the rate of ATP cleavage. This is ATP specific and is seen upon lyophilization and in D20. Inner mitochondrial membranes show large Amide I differences, depending upon their metabolic state at the instant of freezing 18). Electron transport increases antiparallel fl-structure, i.e. augmented absorption near 1635 cm - I and 1685 cm - t , which is blocked or reversed by inhibiting respiration, or inducing phosphorylation with ADP. An extreme shift towards the antiparallel-fl follows uncoupling with dinitropheno118). D20 spectra also demonstrate fl-structure in the peptides of mitochondrial membranes. Also, when erythrocyte ghosts are kept in D20 for 60 rain at 20°C, or more briefly at 35°C, the apparent proportion of parallel fl-structure increases, the absorbance in near 1640 cm- 1, gradually assuming prominence of the Amide I region. The ghosts simultaneously lose their
349
INFRARED AND LASER RAMAN SPECTROSCOPY
concave shape and turn into spheres. This is due in part to amide deuteration, but also because protein structure in general22) and /~-structures in particularZ3, 9.4) are stabilized by entropic effects, which change together with H-bonding, when D20 replaces H20. Large Amide I changes need not imply extensive peptide refolding. Thus, carboxypeptidase A s) and ~-chymotrypsin 2°) contain many antiparallel loops, but incomplete /i'-structured H-bonding. However, minor peptide rotation [such as occurs in these enzymes upon substrate addition s,24)], might increase H-bonding so as to augment the/~-signal markedly (fig. I).
ANTIPARALLEL BETA-LOOP WITH INCOMPLETE H-BONDING
• =o I""-"
"
"
•
= O--H--N--O
•
= O--H--N--O
IMPROBABLE COMPLETION OF H-BONDING
POSSIBLE COMPLETION OF H-BONDING BY ROTATION ABOUT PEPTIDE AXIS
Fig. 1.
E. Lipids I. Table 1 lists some of the vibrations obtained from phospholipids. CH 2 rocking, (720 cm-l), indicates interaction between adjacent apolar chains, when at least 4 carbon atoms lie in an all-trans-planar configuration; in solids this band splits by as much as 6 cm- i, zs). P = O stretching ( 1250-1250 cm- a) can decrease in frequency and intensify upon H-bonding. The major lipid contribution near the Amide I region is due to sphingosine, whose amide absorbs at 1640-1540 c m - i , and phosphatidylserine - C O 0 - , absorbing near 1600 cm- 1 2. Aqueous dispersions of myelin 2s), as well as membrane lipid extracts 10), exhibit prominent CH2 rocking at 720 cm -a, but this is lacking in plasma membranes at room temperature x4,15), possibly by hydrophobic lipidprotein associations. In Micrococcus lysodekticus, CH2 rocking, as well as the ratios (asymmetric: symmetric methylene stretching), (at 2930 cm -l and 2855 cm-1 respectively), vary in whole membranes, their lipid extracts
350
DONALD F. H. WALLACH
and the extracted residues (0.3, 0.1 and 1.4, respectively at -180°C). Only extracted lipid shows significant CH2-rocking and the data all suggest apolar lipid-protein interactions 16). 3. Lipid extracted membranes, cast as a film, develop some E-structure 10), but this is lacking when the membranes are cast and dried before lipid extraction x0, 13, 14). However, exposure of intact or lipid-extracted membrane films to p H < 3 for 20 min, produces a/~-shoulder at 1630 cm -1 14). P = O stretching in dried red cell ghosts lies at 1225 cm -1, as does that of D20 dispersions of the membrane lipids 10). If these groups were ionically linked to membrane proteins, the P - O - phosphorus would be more electropositive, changing P = O stretching accordingly; however, this is not seen. The lower~ P = O stretching value then found in pure, isolated phospholipids 27), may reflect H-bonding. Pertinently, studies on the P : O stretching and CH 2 rocking amplitudes in cephalin and lecithin films 28) show intensification of these bands with applied electric fields, due to alignment of the phosphate-base dipoles and secondary rearrangement of the hydrocarbon chains; this shows that fatty acids may reorient with changing trans-membrane potentials. H. Laser-Raman spectroscopy
A. The Raman effect
,1%
Raman scattering also arises from molecular rotations and vibrations. When transparent substances are irradiated with monochromatic light, most of this is transmitted, some is scattered at the incident frequency, Rayleigh scattering, and traces are scattered at higher or lower frequencies, Raman scattering. The frequency shift between the incident light and a Raman line fits the vibrational frequency of the Raman-active bond. Raman scattering arises from changes in bond polarizability, i.e. induced dipole moments zg). Edsall z0) very early used Hg arcs for Raman analyses of amino acids and dipeptides, but polymer analysis by this method became feasible only when stable lasers emerged as intense, monochromatic sources.
B. Polypeptides and proteins The potency of laser Raman spectroscopy in the analysis of biopolymers is its versatility and manageable susceptibility to solvent interferences. H20 scatters neglibibly throughout most of the Raman spectrum and its OH band in the Amide I region is weak compared to OH absorption in the IR. Also H20 scattering can be overcome by high sample concentrations 31,32) and/or computer techniquesZZ), the latter allowing the use of low sample concentrations in H20. Table I lists important Raman bands of polypeptides.
351
INFRARED AND LASER RAMAN SPECTROSCOPY
TABLE l Some IR and Raman frequencies o f polypeptides and phospholipids 8 Assignment b
Frequency (cm- ~) Infrared Polypeptide b
Skeletal deformation Skeletal deformation N H and C = O out o f plane bending, (Amide IV) CHa rocking NH bending -~ skeletal modes; (Amide V) C-C stretch and CH rocking CHa stretch -t skeletal modes CH = CH bending P-O-C stretching C -. C stretching Methyl modes All-trans hydrocarbon ; extended Hydrocarbon chain; random Hydrocarbon ; all-trans Methyl-carbon stretching Hydrocarbon chain; all trans Methyl rocking CH2 twisting CH2 wagging P ~ O stretching ° CN stretching + N H in-plane bending; (Amide IIl) CH bending Methyl ; symmetric deformation Methyl; asymmetric deformation CH2 stretching and bending N H bending in plane + CN stretching; (Amide 1l) C ~ O stretching; amide; (Amide I). e C =- O stretching; ester CH2 asymmetrical stretching, (in plane) CH2 asymmetrical stretching, (out of plane) C = C - - H stretching
Phospholipid b
368s 528s
Raman PolYpeptide ~
Phospholipid b
375s d 527vs
625m
662w 720m
725w
756m 870-1150
907m
907vs 970m 980-1050s 1000-1620
1049vs
1048vw 1064m 1089m 1100m
1105s 1130s 1167s
1168m 1170-13OO 1180-1380 1250-1350
1300s 1328m
1310m 1331 s
1376vs
1371m
1453vs
1453vs 1460
1537-1560s
1549w
1635-1655vs
1654s 1720-1740s
2853s
2853s
2874m
2926s
2926s 3000
2927vs
a Selected data from Chapman27), Koenig and Suttona0, Wallach et al. 1:) and Lippert and Peticolasa4). a Assignments are not all absolute and some frequencies determined for polypeptides also apply to phospholipids, e The P - O stretching frequency and Amide I frequencies are very dependent on hydrogen bonding. a vs = very strong; s = strong; m ~ medium; w - weak; vw = very weak.
352
D O N A L D F. H . W A L L A C H
I m p o r t a n t l y , the technique seems to discriminate between s-helical,/3- and unordered-polypeptide conformations.
C. Lipids and membranes Th e R a m a n spectra o f fatty acids, fatty acid esters and p h o s p h o l i p i d micelles have also been studied 34, zs), Table 1; also, early data show this m e t h o d , particularly if j o i n e d to difference techniques, to be a unique tool for the analysis o f m e m b r a n e proteins and lipids in situ.
References 1) D. N. Kendall, in: Applied infrared spectroscopy, ed. by D. N. Kendall. Reinhold, New York (1966) 1 2) L. W. Herscher, in: Applied infrared spectroscopy, ed. by D. N. Kendall. Reinhold, New York (1966) 88 3) H. A. Szymanski, Theory and practice of infrared spectroscopy. Plenum Press, New York (1964) 4) R. Townend, T. F. Kumosinski, S. N. Timasheff, G.D. Fasman and B. Davidson, Biochem. Biophys. Res. Commun. 23 (1966) 163 5) H. Susi, S. N. Timasheff and L. Stevens, J. Biol. Chem. 242 (1967) 5460 6) S. N. Timasheff, H. Susi and L. Stevens, J. Biol. Chem. 242 (1967) 5407 7) D. C. Phillips, Sci. American 215 (5) (1966) 78 8) W. N. Lipscomb, Ace. Chem. Res. 3 (1970) 81 9) F. Fukushima and T. Miyazawa, Ann. Meeting of the Chem. So<:. of Japan, Tokyo (1964) 10) D. Chapman, V. B. Kamat and R. J. Levene, Science 160 (1968) 314 11) A. H. Maddy and B. R. Malcolm, Science 150 (1965) 1616 12) A. H. Maddy and B. R. Malcolm, Science 153 (1966) 213 13) J. M. Graham and D. F. H. Wallach, Biochem. Biophys. Acta 241 (1971) 180 14) D. F. H. Wallach and P. H. Zahler, Proc. Natl. Acad. Sci. U.S. 56 (1966) 1552 15) D. F. H. Wallach and P. H. Zahler, Biochem. Biophys. Acta 150 (1968) 186 16) D. H. Green and M. R. J. Salton, Biochem. Biophys. Acta 211 (1970) 139 17) D. F. H. Wallach, J. M. Graham and B. R. Fernbach, Arch. Biochem. Biophys. 131 (1969) 322 18) J. M. Graham and D. F. H. Wallach, Biochem. Biophys. Acta 193 (1969) 225 19) J. C. Kendrew, R. E. Dickerson, B. E. Strandberg, R. G. Hart, D. R. Davies, D. C. Phillips and V. C. Shore, Nature 105 (1966) 422 20) D. M. Blow, Biochem. J. 112 (1969) 261 21) D. F. H. Wallach and J. M. Graham, in: Biochemistry and biophysics ofmitochondrial membranes, ed. by N. Siliprandi and G. F. Azzone. Academic Press, New York, in press 22) W. Kauzmann, Advan. Protein Chem. 14 (1959) 1 23) J. Lynn and G. Fasman, Biochem. Biophys. Res. Commun. 33 (1968) 327 24) B. Davidson and G. Fasman, Biochem. 6 (1967) 1616 25) L. J. Bellamy, Advances in infrared group frequencies. Methuen, London (1968) 26) T. J. Jenkinson, V. B. Kamat and D. Chapman, Biochem. Biophys. Acta 183 (1969) 427 27) D. Chapman, The structure of lipids. Methuen, London (1965) Chap. 4 28) L. May, A. B. Kamble and I. P. Acosta, J. Membrane Biol. 2 (1970) 192 29) L. A. Woodward, in: Raman spectroscopy - theory and practise, ed. by H.A. Szymanski. Plenum Press, New York (1967) 1
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30) J. Edsall, in: Proteins, amino acids and peptides, ed. by E. J. Cohn and J. Edsall. Reinhold, New York (1943) 9 31) J. L. Koenig and P. L. Sutton, Biopolymers 8 (1969) 167 32) R. C. Lord and N.-T. Yu, J. Mol. Biol. 50 (1970) 509 33) D. F. H. Wallach, J. M. Graham and A. R. Oseroff, FEBS Letters 7 (1970) 330 34) J. L. Lipport and W. L. Peticolas, Proc. Natl. Acad. Sci. U.S. 68 (1971) 1572
Discussion
WALLACH also showed how optical activity measurements were at present inadequate for the quantitative analysis of secondary peptide structure in globular proteins, whether soluble or membrane located, and refuted contentions that the optical activities of membranes were necessarily distorted by light scattering, citing the work of Ottaway and Wettlaufer, Arch. Biochem. Biophys. 139 (1970) 257; Straus et al., Europ. J. Biochem. l l (1969) 201; and Wallach et al., J. Cell. Biol. 30 (1966) 601. He anticipates resolution of many of the problems involved through technical improvements and neu mathematical approaches, e.g. Saxena and Wettlaufer, Proc. Natl. Acad. Sci. U.S. 68 (1971) 969. In reply to BANGHAM'Squestion, he pointed out that mitochondrial membranes are particularly difficult to analyse by optical activity because of their high content of fl-structured polypeptide and variations in the proportion of fl-structure with metabolic state. Particularly for these reasons, they are unsuitable for the computation of light-scattering effects. In reply to CHERRY'S question, WALLACHcontended that most membranes must be treated optically as shells with an overall refractive index a little higher than water but with a wall refractive index of about i.55. ALLISON queried whether recent spectroscopic evidence from Wallach's and other laboratories was inconsistent with membrane penetration of proteins. WALLACH replied that the data still indicated the average membrane protein to be rather highly helical with a substantial proportion of peptide in an apolar environment such as envisaged in the membrane core. METCALFE enquired whether there was any technique by which one could determine the orientation of fl-structured segments with respect to the membrane surface, and WALLACH referred to infrared dichroism as such an approach, but that it would be useful only if the membranes could be oriented very precisely with regard to the incident infrared beam. In reply to CHAPMAN'S question whether the infrared assignments of various amide bands have been adequately tested, Wallach pointed to the consistency in the location of various conformationally-sensitive Amide bands in numerous synthetic polypeptides and various proteins where second-
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ary structure had been established by X-ray crystallography. However, he stressed the persistent but resolvable problem of quantifying fl-structure by infrared techniques. WALLACH also alluded to the consistency between the location of the pertinent Raman scattering lines and the infrared bands as support for the correctness of the assignments and finally cited the theoretical studies of Miyazawa and others in support for the fundamental validity of the infraredapproach.