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Progress in Biophysics & Molecular Biology 74 (2000) 141–173

Review

The infrared absorption of amino acid side chains

Andreas Barth*

Institut fur Biophysik, Johann Wolfgang Goethe-Universitat, Theodor Stern-Kai 7, Haus 74,D-60590 Frankfurt am Main, Germany

Abstract

Amino acid side chains play fundamental roles in stabilising protein structures and in catalysingenzymatic reactions. These fields are increasingly investigated by infrared spectroscopy at the molecularlevel. To help the interpretation of the spectra, a review of the infrared absorption of amino acid side chainsin H2O and 2H2O is given. The spectral region of 2600–900 cm�1 is covered. # 2001 Elsevier Science Ltd.All rights reserved.

Keywords: Infrared; FTIR; Vibration; Amino acid; Protein

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

2. Infrared absorption of amino acid side chains . . . . . . . . . . . . . . . . . . . . . . . . . 144

2.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1442.2. Aliphatic side chain groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1532.3. Aromatic side chains: Tyr, Phe, Trp . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

2.4. Aliphatic side chains with hydroxyl or thiol groups: Ser, Thr, Cys . . . . . . . . . . . . 1552.5. Positively charged side chains: His, Arg, Lys . . . . . . . . . . . . . . . . . . . . . . . 1552.6. Side chains with amide groups: Asn, Gln . . . . . . . . . . . . . . . . . . . . . . . . . 160

2.7. Side chains with carboxyl groups: Asp, Glu . . . . . . . . . . . . . . . . . . . . . . . 161

3. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

*Corresponding author. Tel.: +49-69-6301-6087; fax: +49-69-6301-5838.

E-mail address: [email protected] (A. Barth).

0079-6107/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 7 9 - 6 1 0 7 ( 0 0 ) 0 0 0 2 1 - 3

1. Introduction

The infrared spectrum of a protein provides a wealth of information on structure andenvironment of the protein backbone and of the amino acid side chains. This makes infraredspectroscopy an extremely useful tool for the investigation of protein structure (Arrondo et al.,1993; Goormaghtigh et al., 1994a–c; Jackson and Mantsch, 1995; Siebert, 1995; Arrondo andGoni, 1999), of the molecular mechanism of protein reactions (Rothschild, 1992; Mantele, 1993,1995; Maeda, 1995; Siebert, 1995; Gerwert, 1999; Barth and Zscherp, 2000; Zscherp and Barth,2001) and of protein folding, unfolding and misfolding (Dyer et al., 1998; Arrondo and Goni,1999; Fabian et al., 1999; Reinstadler et al., 1999; Schultz, 2000; Troullier et al., 2000).

For the determination of secondary structure from infrared spectra, the absorption of aminoacid side chains presents a matter of concern. This is because the amide I vibration of thepolypeptide backbone used for this purpose absorbs in a spectral region (1610–1700 cm�1) whereside chains also absorb. It is estimated that 10–30% of the total absorption in that region derivesfrom side chains (Chirgadze et al., 1975; Venyaminov and Kalnin, 1990; Rahmelow et al., 1998)and attempts have been made to subtract the side chain contribution (Chirgadze et al., 1975;Venyaminov and Kalnin, 1990; Rahmelow et al., 1998) using spectra of model compounds inaqueous solution. However, this may be problematic for side chains not exposed to thesurrounding aqueous solvent.

In contrast to secondary structure determination, amino acid side chain absorption providesvery valuable information when the mechanism of protein reactions is investigated. This isbecause side chains are often at the heart of the molecular reaction mechanism. With infraredspectroscopy it is possible to follow in a single experiment the fate of the several individual groupsthat are involved in the reaction. The aim of this kind of research is to identify the catalyticallyimportant side chains and to deduce their environmental and structural changes from thespectrum in order to understand the molecular reaction mechanism. The problem here is theassignment of spectral features to specific amino acids.

Catalytically important residues can be identified by a combination of site-directed mutagenesisand infrared spectroscopy. The general strategy is to induce the protein reaction in the infraredcuvette, follow the infrared absorbance changes and identify crucial residues by repeating theexperiment with selected point mutants. An intensely studied example is bacteriorhodopsin. In

Nomenclature

d in plane bending vibrationFTIR Fourier transform infraredIR infraredgw wagging vibrationgt twisting vibrationgr rocking vibratione extinction coefficientm band of medium intensity

mw band of medium to weak intensityn stretching vibrationns symmetric stretching vibrationnas antisymmetric stretching vibrations band of strong intensitysh shouldervs very strong intensityw weak intensity

A. Barth / Progress in Biophysics & Molecular Biology 74 (2000) 141–173142

particular, those Asp and Glu residues that constitute the proton pumping pathway ofbacteriorhodopsin could be identified (Rothschild, 1992; Maeda, 1995; Gerwert, 1999; Heberle,1999).

Information on side chain properties stems from the physical principles of the absorptionprocess. Infrared light is absorbed by molecular vibrations that oscillate with the same frequency.The frequency of the vibration and the probability of absorption are influenced by intra- andintermolecular effects. Thus, information about structure and environment of amino acid sidechains can be deduced from the spectral parameters band position, band width and absorptioncoefficient.

In our particular case of amino acid side chains, protonation state, coordination of cations andhydrogen bonding are the dominating factors that determine the band position of a particularamino acid. Infrared spectroscopy is thus one of the few techniques that is able to define theprotonation state of side chains which has important consequences for the electrostaticinteractions in proteins. Protonation of Asp and Glu residues accompanies for example protonpumping by bacteriorhodopsin (Rothschild, 1992; Maeda, 1995; Gerwert, 1999; Heberle, 1999),electron transfer reactions (Mantele, 1995), Ca2+ release from the Ca2+-ATPase (Barth andZscherp, 2000) and seems to provide a mechanism of charge compensation when the negativelycharged ATP binds to the Ca2+-ATPase (Barth and Zscherp, 2000; von Germar et al., 2000).

From the environmental factors only hydrogen bonding will be mentioned here, whereas cationcoordination will be discussed in the section about Asp and Glu residues. As a general rule,hydrogen bonding lowers the frequency of stretching vibrations, since it lowers the restoring forcewhen the H-bonding partners are close, but increases the frequency of bending vibrations since itproduces an additional restoring force (Colthup et al., 1975). In the above-mentioned examples ofAsp and Glu protonation, the degree of hydrogen bonding to the protonated form could bedetermined. As for Asp and Glu residues, the protonation state and the environment of othercatalytically active side chains can be characterised, as done for example for His and Tyr residuesof photosystem II (Hienerwadel et al., 1997; Noguchi et al., 1999) and bacteriorhodopsin(Dollinger et al., 1986; Rothschild et al., 1986; Roepe et al., 1987; Rothschild, 1992).

The absorption coefficient increases with the change of dipole moment during the vibration.Often this is correlated with the polarity of the vibrating bonds (this is not the case when internalcoordinate contributions to the dipole moment of a normal mode cancel, as they do in the ns modeof CO2). Thus a change in environment that results in an altered bond polarity will lead to achange in band intensities.

The band width is a measure of conformational freedom with flexible structures giving broaderbands. This has been used for example to characterise the environment of the phosphorylated Aspresidue of the sarcoplasmic reticulum Ca2+-ATPase (Barth and Mantele, 1998). From the smallband width of the nðC¼OÞ band it was concluded that this group is not exposed to solvent waterbut exhibits defined interactions with the protein environment.

One advantage of infrared spectroscopy is that the protein backbone as well as the side chainscan be observed in the same experiment. Thus it is possible to compare the kinetics of backbonestructural changes with those of amino acid side chain signals. As an example, for the Ca2+-ATPase it was found the overall backbone conformational changes proceed at the same time asthe local perturbations of side chains (Barth et al., 1996). In contrast, in the complex refolding ofRibonuclease T1 the very late events due to the trans! cis isomerisation of a prolyl peptide bond

A. Barth / Progress in Biophysics & Molecular Biology 74 (2000) 141–173 143

lead to an increased compactness of the protein structure but not to an environmental change ofAsp, Glu and Tyr residues. They have therefore adopted their native environment already in thepreceding processes (Reinstadler et al., 1999).

The interpretation of the generally complicated spectral band profiles of protein reactions isoften guided by experience and is greatly helped by a condensation of present knowledge into alist of group frequencies. However, earlier collections of amino acid infrared spectra (Chirgadze etal., 1975; Venyaminov and Kalnin, 1990; Goormaghtigh et al., 1994a; Wright and Vanderkooi,1997; Rahmelow et al., 1998) were often restricted to the 1800–1500 cm�1 spectral region and donot make use of the whole mid infrared spectral region which is easily accessible to theexperimentalist. Thus it is attempted in this review to collect data from a variety of sources inorder to obtain a more complete picture. The review focuses on the 2600–900 cm�1 spectral regionsince it is the most useful for the structural interpretation and is easily accessible with commonexperimental set-ups. Compilations of amino acid side chain Raman frequencies can be found in(Lord and Yu, 1970a,b; Rava and Spiro, 1985; Asher et al., 1986; Lagant et al., 1998; Overmanand Thomas, 1999). I am aware that a collection of this amount of data cannot be complete andflawless. Thus, I am grateful if readers point me towards errors and studies I am unaware of.

2. Infrared absorption of amino acid side chains

2.1. Overview

Table 1 gives an overview of the infrared absorption of amino acid side chains in H2O and2H2O. Only the strongest bands are listed, or those in a spectral window free of overlap by bandsfrom other groups. Table 2 lists the infrared active vibrations of the aliphatic side chain groups,Tables 3–16 give more detailed information on the individual side chains. Here, observations ofside chain absorption in proteins are also listed. If available, parameters of infrared spectra ofamino acid side chains are given. If not, data are taken from infrared spectra of model compoundsor from Raman spectra. Band positions are given for H2O and 2H2O, the latter are indicated byitalic print. The shift upon H/2H exchange is given when a compound in both solvents iscompared in the original work. The listing of internal coordinate contributions to a normal modeis according to their contribution to the potential energy of the normal mode (if specified in theliterature): If the contribution of an internal coordinate to the potential energy of a normalvibration is 570% only that coordinate is listed. Two coordinates are listed if their contributiontogether is 570%. In all other cases those 3 coordinates that contribute strongest to the potentialenergy are listed. Vibrations dominated by amide group motions are not included.

As seen in the tables, the absorption of a side chain in a protein may deviate significantly fromtheir absorption in solution or in a crystal. The special environment provided by a protein is ableto modulate the electron density and the polarity of bonds, thus changing the vibrationalfrequency and the absorption coefficient. Therefore, the band positions given in the tables shouldbe regarded only as guidelines for the interpretation of spectra. It may be mentioned here that alsothe pKa of acidic residues in proteins may differ significantly from solution values. For D96 ofbacteriorhodopsin for example a pKa > 12 has been found (Zscherp et al., 1999).


Table 1Overview of amino acid side chain infrared bandsa

Assignments Band position in cm�1

(e in M�1cm�1) in H2OBand position in cm�1,(e in M�1cm�1) in 2H2O

References Remarks

Cys, nðSHÞ 2551 1849 Susi et al. (1983)

Asp, nðC¼OÞ 1716–1788 (280) 1713–1775 (290) Pinchas and Laulicht(1971), Chirgadze et al.(1975), Venyaminov andKalnin (1990)

Sensitive to H-bonding. Without H-bond up to 1762 cm�1 observed inproteins (Fahmy et al., 1993). SingleH-bond shifts 25 cm�1 down. Above�1740 cm�1 inverse correlation ofn(C=O) with the dielectric constant(Dioumaev and Braiman, 1995).Values for the unbonded C=Ogroup (1788 cm�1) are from theacetic acid spectrum (Pinchas andLaulicht, 1971)

Glu, nðC¼OÞ 1712–1788 (220) 1706–1775 (280) Pinchas and Laulicht(1971), Chirgadze et al.(1975), Venyaminov andKalnin (1990)

See Asp n(C=O)

Asn, nðC¼OÞ 1677–1678 (310–330) 1648 (570) Chirgadze et al. (1975),Venyaminov and Kalnin(1990), Rahmelow et al.(1998)

Up to 1704 cm�1 observedin proteins (Cao et al., 1993)

Arg, nasðCN3Hþ5 Þ 1672–1673 (420–490) 1608 (460) Chirgadze et al. (1975),

Venyaminov and Kalnin(1990), Rahmelow et al.(1998)

In proteins observed up to1695 cm�1 (H2O) and down to1595 cm�1 (2H2O) (Chirgadze et al.,1975; Berendzen and Braunstein,1990; Rudiger et al., 1995)

Gln, nðC¼OÞ 1668–1687 (360–380) 1635–1654 (550) Chirgadze et al. (1975),Venyaminov and Kalnin(1990), Dhamelincourtand Ramirez (1993),Rahmelow et al. (1998)

Arg, nsðCN3Hþ5 Þ 1633–1636 (300–340) 1586 (500) Chirgadze et al. (1975),

Venyaminov and Kalnin(1990), Rahmelow et al.(1998)

In deuterated proteins observedup to 10 cm�1 lower (Chirgadzeet al., 1975)

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HisH2+, nðC¼CÞ 1631 (250) 1600 (35), 1623 (16) Chirgadze et al. (1975),

Hienerwadel et al. (1997)Only one strong band observed for4-Methylimidazole at 1633 (H2O)and 1605 cm�1 (2H2O) (Hasegawaet al., 2000)

Lys, dasðNHþ3 Þ 1626–1629 (60–130) 1201 Pinchas and Laulicht

(1971), Venyaminov andKalnin (1990), Rahmelowet al. (1998)

2H2O band position based on theshift observed for CH3NH3Cl andCH3N

2H3Cl

Tyr–OH, n(CC)d(CH)

1614–1621 (85–150) 1612–1618 (�160) Chirgadze et al. (1975),Dollinger et al. (1986),Takeuchi et al. (1988),Venyaminov and Kalnin(1990), Hienerwadel et al.(1997), Rahmelow et al.(1998)

Tyr or p-cresol, e estimated relativeto 1517 cm�1 band

Asn, dðNH2Þ 1612–1622 (140–160) Venyaminov and Kalnin(1990), Rahmelow et al.(1998)

Trp, nðCCÞ,nðC¼CÞ

1622 1618 Takeuchi and Harada(1986), Lagant et al.(1998)

Tyr–O�, nðCCÞ 1599–1602 (160) 1603 (350) Chirgadze et al. (1975),Dollinger et al. (1986),Venyaminov and Kalnin(1990), Hienerwadelet al. (1997)

Tyr or p-cresol

Tyr–OH, nðCCÞ 1594–1602 (70–100) 1590–1591 (550) Chirgadze et al. (1975),Dollinger et al. (1986),Takeuchi et al. (1988),Venyaminov and Kalnin(1990), Hienerwadel et al.(1997), Rahmelow et al.(1998)

Tyr or p-cresol, e estimated relativeto 1517 cm�1 band

Gln, dðNH2Þ 1586–1610 (220–240) 1163 Venyaminov and Kalnin(1990), Dhamelincourtand Ramirez (1993),Rahmelow et al. (1998)

Table 1 (continued)


(e in M�1cm�1Þ in H2OBand position in cm�1,(e in M�1cm�1) in 2H2O

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HisH, nðC¼CÞ 1575,1594 (70) 1569, 1575 Venyaminov and Kalnin(1990), Hasegawa et al.(2000)

Doublet due to the two protonatedtautomers of His

Asp, nasðCOO�Þ 1574–1579 (290–380) 1584 (820) Chirgadze et al. (1975),Venyaminov and Kalnin(1990), Rahmelow et al.(1998)

May shift +60/�40 cm�1

(Tackett, 1989; Nara et al., 1994)upon cation chelation, in extremecases band position as forn(C=O) (Deacon and Phillips,1980)

Glu, nasðCOO�Þ 1556–1560 (450–470) 1567 (830) Chirgadze et al. (1975),Venyaminov and Kalnin(1990)

See Asp nas(COO�)

Lys, dsðNHþ3 Þ 1526–1527 (70–100) 1170 Pinchas and Laulicht

(1971), Venyaminov andKalnin (1990), Rahmelowet al. (1998)

2H2O band position based on theshift observed for CH3NH3Cl andCH3N

2H3Cl

Tyr–OH, nðCCÞ,dðCHÞ

1516–1518 (340–430) 1513–1517 (500) Chirgadze et al. (1975),Dollinger et al. (1986),Rothschild et al. (1986),Takeuchi et al. (1988),Venyaminov and Kalnin(1990), Hienerwadelet al. (1997), Rahmelowet al. (1998)

Tyr or p-cresol

Trp, nðCNÞ, dðCHÞ,dðNHÞ

1509 Lautie et al. (1980),Takeuchi and Harada(1986)

Indole IR spectrum

Tyr–O�, nðCCÞ,dðCHÞ

1498–1500 (700) 1498–1500 (650) Chirgadze et al. (1975),Rothschild et al. (1986),Venyaminov and Kalnin(1990), Hienerwadelet al. (1997)

Tyr or p-cresol

Trp, nðCCÞ,dðCHÞ

1496 Takeuchi and Harada(1986), Lagant et al.(1998)

Trp Raman spectrum, observed inthe indole infrared spectrum at1487 cm�1

Phe, nðCCringÞ 1494 (80) Venyaminov and Kalnin(1990)

dasðCH3Þ 1445–1480 Colthup et al. (1975)

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Trp, dðCHÞ, nðCCÞ,nðCNÞ

1462 1455 (200) Lautie et al. (1980),Takeuchi and Harada(1986), Fabian et al.(1994)

e estimated from comparison withthe 1517 cm�1 Tyr band (Fabianet al. (1994)

His�, dðCH3Þ,

nðCNÞ1439 1439 Hasegawa et al. (2000) Observed for 4-Methylimidazole

with a strong contribution ofd(CH3). Thus, position for Hismay differ

Pro, nðCNÞ 1400–1465 Caswell and Spiro (1987),Rothschild et al. (1989),Gerwert et al. (1990)

Sensitive to backbone confor-mation (Johnston and Krimm, 1971;Caswell and Spiro, 1987)

dðCH2Þ 1425–1475 Colthup et al. (1975) Good group frequency, normallyat 1463 cm�1. Near 1425 cm�1

and more intense when nextto a C=O group (Colthup et al.,1975)

Trp, dðNHÞ, nðCCÞ,dðCHÞ

1412–1435 1382 Lautie et al. (1980),Takeuchi and Harada(1986)

H2O: higher number forRaman spectrum of Trp, lowernumber for IR imidazolespectrum. 2H2O: Raman spectrumof Trp

Gln, nðCNÞ 1410 1409 Dhamelincourt andRamirez (1993)

Glu, nsðCOO�Þ 1404 (316) 1407 Venyaminov and Kalnin(1990)

See Asp ns(COO�)

Asp, nsðCOO�Þ 1402 (256) 1404 Venyaminov and Kalnin(1990)

May shift +60/�90 cm�1

upon cation chelation (Tackett,1989), in extreme cases bandposition as for n(C–O) of COOHgroup (Deacon and Phillips, 1980).Band position in 2H2O estimatedfrom the shift observed forCH3COO�

Table 1 (continued)



References Remarks

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dsðCH3Þ 1375 or 1368, 1385 Colthup et al. (1975) 1 band near 1375 cm�1 for 1 CH3

group, 2 bands at 1368 and1385 cm�1 for 2 adjacent groups(Val, Leu). The band is narrowerthan the das(CH3) band but has thesame intensity (Colthup et al., 1975).Insensitive to hydrocarbon chain con-formation (Lewis and McElhaney,1996) but to branching of the hydro-carbon chain (Colthup et al. (1975)

Trp 1352–1361 Takeuchi and Harada(1986), Lagant et al.(1998)

Higher number for Ramanspectrum of Trp, lower number forIR imidazole spectrum.

Trp 1334–1342 1334 (100) Takeuchi and Harada(1986), Fabian et al.(1994), Lagant et al.(1998)

H2O: higher number for Ramanspectrum of Trp, lower numberfor IR imidazole spectrum. 2H2O:IR spectrum of Trp in protein

dðCHÞ 1315–1350

Trp, dðNHÞ,nðCNÞ, dðCHÞ

1276 Lautie et al. (1980) Indole IR spectrum

Tyr–O�, nðC2OÞ,

nðCCÞ1269–1273 (580) Dollinger et al. (1986),

Venyaminov and Kalnin(1990), Hienerwadelet al. (1997)

Tyr or p-cresol

Asp, Glu, dðCOHÞ 1264–1450 955–1058 Pinchas and Laulicht(1971)

Hydrogen bonded (1058 and1450 cm�1) and free (955and 1264 cm�1) CH3COOH

Trp, dðCHÞ,nðCCÞ


Indole IR spectrum

Tyr–OH nðC2OÞ,nðCCÞ

1235–1270 (200) 1248–1265 (150) Dollinger et al. (1986),Rothschild et al. (1986),Takeuchi et al. (1988),Venyaminov and Kalnin(1990), Gerothanassiset al. (1992), Hienerwadelet al. (1997)

Tyr or p-cresol, band sensitive to H-bonding, 3–11 cm�1 lower in 2H2O,e in 2H2O estimated fromcomparison with the 1517 cm�1

band

His, dðCHÞ, nðCNÞ,dðNHÞ

1217, 1229, 1199 1217, 1223, 1239 Hasegawa et al. (2000) Values are for His�, HisH andHisH2

+, respectively

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Trp, nðCCÞ 1203 Lautie et al. (1980) Indole IR spectrum

Ser, dðCOHÞ ordðCO2 HÞ, nðCOÞ

1181–1420 875–985 Pinchas and Laulicht(1971), Colthup et al.(1975), Madec et al.(1978), Susi et al.(1983)

Band position sensitive to hydrogenbonding

gwðCH2Þ 1170–1382 Colthup et al. (1975) Couples with adjacent CH2

groups (Colthup et al., 1975).Sensitive to hydrocarbon chainconformation (Lewis andMcElhaney, 1996)

Tyr–OH, dðCOHÞ 1169–1260 (200) 913 Dollinger et al. (1986),Rothschild et al. (1986),Takeuchi et al. (1988),Venyaminov andKalnin (1990),Gerothanassis et al.(1992), Hienerwadelet al. (1997)

Tyr or p-cresol, band sensitive to H-bonding for OH group, 256 cm�1

lower for O2H group

Asp, Glu, nðC2OÞ 1120–1253 1270–1322 Pinchas and Laulicht(1971), Sengupta andKrimm (1985),Venyaminov and Kalnin(1990)

Range in H2O from band position inaqueous solution near 1250 cm�1

(Sengupta and Krimm, 1985;Venyaminov and Kalnin, 1990) andshift observed between hydrogenbonded and free CH3COOH spectra(Pinchas and Laulicht, 1971). Bandposition in 2H2O for hydrogenbonded and free CH3COOH. Aspand Glu absorption may be 25 cm�1

lower, since this is observed inH2O

His, nðCNÞ,dðCHÞ

1104,1090,1106,1094 1104, 1096, 1107, 1110 Noguchi et al. (1999) Values are for His�, N1-, N3-protonated HisH and HisH2

+,respectively

Trp, dðCHÞ,nðNCÞ


Indole IR spectrum

Table 1 (continued)



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Trp, nðNCÞ, dðCHÞ,nðCCÞ


Indole IR spectrum

gtðCH2Þ 1063–1295 Colthup et al. (1975) Weak, couples with adjacent CH2

groups, but the in phase mode at1300 cm�1 is a good groupfrequency (Colthup et al., 1975)

Thr, nðC2OÞ 1075–1150 Colthup et al. (1975) 2 bands expected

Ser, nðC2OÞ 1030 1023 Madec et al. (1978),Susi et al. (1983)

Trp, nðCCÞ,dðCHÞ

1012–1016 1012 Lautie et al. (1980),Takeuchi and Harada(1986)

Ser, nðCOÞ ornðCCÞ

983 Madec et al. (1978),Susi et al. (1983)

Ser, nðCOÞ,dðCO2HÞ

940 Susi et al. (1983)

Thr, dðCO2HÞ 865–942 Pinchas and Laulicht(1971)

grðCH2Þ 724–1174 Colthup et al. (1975) Couples with adjacent CH2 groups,the in phase mode at 724 cm�1 is themost intense (Colthup et al., 1975)

aListed are those side chain internal coordinates with the strongest contributions to the potential energy of the normal mode. Amide modes thatcontribute are omitted here but are listed in the tables for the individual amino acid side chains. If no assignment is listed, then multiple assignments

are given in the original publications and the reader is referred to the tables of the individual side chains for further information. Numbers in italicprint are for spectra in 2H2O or for SH-, OH- and NH-deuterated compounds. n: stretching vibration, ns: symmetric stretching vibration,nas: asymmetric stretching vibration, d: in plane bending vibration, gw: wagging vibration, gt: twisting vibration, gr: rocking vibration.

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Table 2CH3, CH2 and CH groupsa

Assignment

{1}

Band position in cm�1, (e in M�1cm�1) {1}

In

general

{2}

Ala

{3,4}

Cys

{4}

Gln Glu Gly

{5}

His Ile

{3}

Leu

{3}

Lys

{3}

Pro Ser

{4}

Thr Trp Tyr Val

{3}

dasðCH3Þ 1465 1470 1445 1445 1480 1460

1465 1448

(�100)1455

dðCH2Þ{6}

1425–

1475

1424

1432

(�120)

1451 1440 1441–1446

(120)

1465 1470 1445 1447–1472 1450

1467

1447–1454

(�80)

dsðCH3Þ{7}

1375 1378

(�100)1391

d(CH), 51382 1340 1341 1333 1323 1333–1337 1343–1345 1320 1345 1375 1375 1455 1376–1387 1355

1340 (�90) 1315 1130 (260) 1325 1317 (�100) 1352 (?) 1335 1320

g(CH2) {8} (�100) 1303 1310–1315 1268–1270 1290 1364 1334 (?) 1326

1290 1297 (40) (?) 1253 1352 1245 1179

(�50) (�70) 912–918 1110 1168 1340 1147 1100–1111

1269 (40) 1012 1083 (�100) 1119

1033 1312 1092

1248 (?)

aValues are for solid samples or samples in H2O. Numbers in italics are for 2H2O. In general upon H2O/2H2O exchange, band shifts of � 10 cm�1

are observed. Weak bands are not listed. Disputed assignments are marked with a question mark. See also legend of Table 1. {1} Colthup et al.(1975) or see Tables 3–16. {2} Colthup et al. (1975). {3} Raman spectra of the coat protein of phage fd. Bands were assigned to side chains by

selective deuteration (Overman and Thomas, 1999). {4} Numbers in italics are for amino acid spectra in 2H2O (Wright and Vanderkooi, 1997).e estimated from the spectra by comparison with the nas(COO�) band which has an e of 830M�1 cm�1 (Chirgadze et al., 1975). {5} Band positionsaccording to spectra of solid glycine and glycylglycine (Laulicht et al., 1966; Lagant et al., 1983; Kakihana et al., 1988). The extinction coefficient in

brackets was estimated from band intensities relative to the ns(COO�) band which has an extinction coefficient of e ¼ 200 M�1 cm�1 (Venyaminovand Kalnin, 1990). Assignment is according to normal mode calculations (Lagant et al., 1983; Kakihana et al., 1988). The mode near 1335 cm�1 maycontain a significant contribution of amide modes (Kakihana et al., 1988). Band positions in 2H2O are 1440–1446 cm�1, 1322–1324 cm�1 and1020 cm�1 (Lagant et al., 1983; Kakihana et al. (1988). {6} Good group frequency, normally at 1463; near 1425 cm�1 and more intense when next to

a C=O group (Colthup et al., 1975). {7} One band for one CH3 group, two bands for two CH3 groups in Val and Leu (Colthup et al., 1975),narrower than the dasðCH3Þ band but the same intensity (Colthup et al., 1975). Insensitive to hydrocarbon chain conformation (Lewis andMcElhaney, 1996). {8} These vibrations are often coupled to other modes. gwðCH2Þ (1170–1382 cm�1) couples with adjacent CH2 groups (Colthup et

al., 1975). The band position is sensitive to hydrocarbon chain conformation (Lewis and McElhaney, 1996). gt(CH2) (1063–1295 cm�1) gives weakbands, couples with adjacent CH2 groups, but the in phase mode at 1300 cm�1 is a good group frequency (Colthup et al., 1975). gr(CH2)(724–1174 cm�1) couples with adjacent CH2 groups, the in phase mode at 724 cm�1 gives the most intense infrared bands (Colthup et al., 1975).

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Only two side chain moieties absorb in spectral regions that are free from overlappingabsorption by other groups and thus allow the spectroscopist an unambiguous assignmentwithout further experiments. These are the SH group of Cys (2550–2600 cm�1) and the carbonylgroup of protonated carboxyl groups (1710–1790 cm�1). The latter proved to be particularlyuseful when protonation and deprotonation of carboxyl groups is of interest, for example whenproton pathways in proteins are explored (Rothschild, 1992; Maeda, 1995; Gerwert, 1999;Heberle, 1999).

All other side chain absorptions overlap with the absorption of other side chains or of thepolypeptide backbone and further experiments are needed to assign an absorption band to aspecific side chain moiety. These include H/2H exchange of the NH, OH and SH groups whichshifts the absorption bands in a characteristic way, uniform isotopic labelling of one type ofamino acids, site-directed isotope labelling of one specific amino acid in a protein and the use ofsite-directed mutants. The most powerful of these methods } site-directed isotope labelling(Sonar et al., 1994; Spudich, 1994) } is the one hardest to apply while the experimentallystraightforward H/2H exchange does not always help because there may be too many changes tothe spectrum or because amino acids deeply buried in the protein core do not exchange.A mutation may lead to more severe conformational effects than just the replacement of oneamino acid side chain by another and therefore may result in complicated alterations to thespectrum.

2.2. Aliphatic side chain groups

Fig. 1 shows the structure of the aliphatic amino acids. The aliphatic moieties of amino acidside chains give rise to several absorbance bands of medium to weak intensity which are compiledin Table 2. While the dasðCH3Þ, the dðCH2Þ and the dsðCH3Þ vibrations near 1465, 1450 and1375 cm�1 are relatively good group frequencies, the d(CH) and g(CH2) vibrations are oftencoupled to other modes. The frequency of the d(CH2) vibration of Asp and Glu residues isexpected to be sensitive to the protonation state and the ds(CH3) vibration to the branching of thehydrocarbon chain (Colthup et al., 1975). Of the aliphatic amino acids only Pro bands are listedseparately in Table 3. Fig. 1 shows the structure of Pro. Pro is remarkable in that is does not formthe usual amide group with the amino acid that precedes in the sequence but rather a N,N-disubstituted amide group due to the additional linkage of the side chain to the amide N-atom.This leads to an unusual amide I frequency (see legend of Table 3). The n(CN) band near1430 cm�1 is sensitive to backbone conformation (Johnston and Krimm, 1971) and has beenidentified in difference spectra of the photoreaction of bacteriorhodopsin upon 15N labelling(Rothschild et al., 1989; Gerwert et al., 1990).

Fig. 1. Structure of aliphatic amino acids.


2.3. Aromatic side chains: Tyr, Phe, Trp

Of the aromatic side chains shown in Fig. 2, Tyr has been most intensely studied due to itsinteresting properties: Tyr may take part in proton and electron transfer reactions (Dollingeret al., 1986; Rothschild et al., 1986; Roepe et al., 1987; Hienerwadel et al., 1997). The pKa valuein solution is 10.1 but may differ considerably from this value in proteins. Tyr is a relatively stronginfrared absorber due to its polar character and its bands are listed in Table 4. The most intensebands originate from a n(CC), the nðC2OÞ and the d(COH) mode near 1517, at 1235–1270 and at1169–1260 cm�1. The latter two are sensitive to H-bonding and merge to one broad band near1250 cm�1 in H2O (Hienerwadel et al., 1997). The ring mode near 1517 cm�1 is easily detected inprotein absorbance spectra, in particular when band narrowing procedures like Fourier self-

Table 3Proline side chaina

Assignment {1} Pro in H2O {2} Pro in 2H2O {3} Pro in proteins {4}Band position (cm�1) Band position, band shift (cm�1) Band position (cm�1)

dðCH2Þ 1472 s 1472, 0dðCH2Þ {5} 1447–1472 s 1456nðCNÞ {6} 1435–1465 1400–1454

dðCHÞ 1375 s, broadgtðCH2Þ 1340 sh 1328, �12dðCHÞ 1317 s 1306, �11gtðCH2Þ 1292 s 1296, +4gwðCH2Þ 1253m 1253, 0 and 1262, +9gtðCH2Þ 1168 s 1168, 0grðCH2Þ 1083m 1090, +7

gwðCH2Þ 1051w 1050, �1gwðCH2Þ 1033mnðCNÞ, nðCCÞ 979m 981, +2

945m 930, �15911m 902, �9

aA remark to the nðC¼OÞ backbone absorption: Proline absorption is unusual compared to the amide absorption ofother amino acids. It absorbs at 1623 cm�1 in an extended left-handed poly-Pro helix when the carbonyl groups areH-bonded, if not at 1640 cm�1 (Lazarev et al., 1985). In 2H2O and in unordered peptide chains the backboneabsorbance is at 1620–1623 cm�1 (Doyle et al., 1971). {1} Assignments are according to Herlinger and Long (1970),

except for the nðCNÞ vibration which was assigned according to Rothschild et al. (1989), Gerwert et al. (1990). See alsolegend of Table 1. {2} Solid l-proline (Herlinger and Long, 1970) or l-proline film (Rothschild et al., 1989), poly-Pro inH2O (Raman) (Caswell and Spiro (1987), band positions in aqueous solution are expected to be within 10 cm�1 of the

band position of solid l-proline (from Raman spectra of solid and dissolved proline (Herlinger and Long (1970)). {3}Band shifts upon H2O/2H2O exchange according to spectra of dl-proline (Herlinger and Long, 1970). Band positionsfor l-Pro are estimated from these shifts. {4} Bacteriorhodopsin: absorbance spectrum and difference spectrum of the

photoreaction (Rothschild et al., 1989; Gerwert et al., 1990). {5} Band position 26 cm�1 lower in [2H7]Pro (Rothschildet al., 1989) no (Rothschild et al., 1989) or –15 to –20 cm�1 (Gerwert et al., 1990) downshift for [15N]Pro. {6}Assignment according to Rothschild et al. (1989), Gerwert et al., (1990). The mode is sensitive to backboneconformation: Raman bands are observed at 1435 cm�1 for cis poly-Pro and at 1465 cm�1 for trans poly-Pro (Caswell

and Spiro, 1987, and the frequency depends upon the Ca–C0(=O) angle (Johnston and Krimm, 1971). �15 cm�1

downshift for [15N]Pro (Rothschild et al., 1989; Gerwert et al., 1990).


deconvolution (Kauppinen et al., 1981; Mantsch et al., 1988), 2nd derivative or fine-structureenhancement (Barth, 2000) are used which intensify the very narrow Tyr band. Besides its smallband width, the small downshift of 2 cm�1 in 2H2O is characteristic. The band can be used asmarker band for the protonation state of Tyr, since it is downshifted by 15 cm�1 in thedeprotonated state. This state is more polar than the neutral state and thus increases the intensityof several bands, in particular of the nðC2OÞ vibration.

Phe due to its apolar character is only weakly absorbing infrared radiation. However, a weakband at 1498 cm�1 can often be detected already in the absorbance spectrum of proteins(Berendzen and Braunstein, 1990; Fabian et al., 1996). Phe bands are listed in Table 5.

The only Trp bands (Table 6) with considerable infrared intensity seem to be those at 1334 and1455 cm�1. They have been detected in an elegant study where a difference spectrum could begenerated that showed the effect on the Ribonuclease T1 spectrum replacing two Tyr residues bytwo Trp residues. To generate the difference spectrum, the absorbance spectrum of point mutantW59Y was carefully subtracted from that of mutant Y45W (Fabian et al., 1994). The spectralrange covered here was 1300–1700 cm�1.

2.4. Aliphatic side chains with hydroxyl or thiol groups: Ser, Thr, Cys

Fig. 3 shows the structures of the aliphatic side chains containing an OH or an SH group.Tables 7 and 8 list the infrared bands of Ser and Thr, respectively. The nðC2OÞ and d(COH)vibrations are often coupled and contribute to several normal modes in the 1000–1420 cm�1

range. As observed for Tyr, they are sensitive to H-bonding. Those vibrations with largecontributions of the stretching vibration of the polar C–O bond have relatively strong infraredintensities.

For Cys (see Table 9), the large mass of the sulphur atom shifts the n(SH) vibration intoa spectral range that is free from overlap by other side chain modes. This has enabled its detectionfor several proteins (Alben and Bare, 1980; Moh et al., 1987; Baburina et al., 1996; Noguchi et al.,1997).

2.5. Positively charged side chains: His, Arg, Lys

His, Arg, Lys are positively charged near neutral pH in aqueous solution. Their structures areshown in Fig. 4. The imidazole group of His has two nitrogen atoms, which can be protonated or

Fig. 3. Structure of Ser, Thr and Cys.Fig. 2. Structure of the aromatic amino acids.


Table 4Tyrosine side chaina

Assignment {1} Tyr–OH {2} Tyr–O2 H {3} Tyr–O� {4} Tyr–O� in 2H2O {5} Tyr in proteins {6}Band position in

cm�1

(e in M�1cm�1)

Band position,

band shift in cm�1

(e in M�1cm�1)

Band position

in cm�1

(e in M�1cm�1)

Band position,


(e in M�1cm�1)

Band position in cm�1

nðCCÞ ring, dðCHÞ 1614–1621 (85–150) 1612–1618, 0 to �3 (160) 1615, 1612

n(CC) ring 1594–1602 (70–100) 1590–1591, �6 (550) 1599–1602 (160) 1603, (350) 1590–1597nðCCÞ ring, dðCHÞ 1516–1518 (340–430) 1513–1517, �1 to �2 (500) 1498–1500 (700) 1498–1500, �2(?) (650) 1510–1516, 1516–1518dðCH2Þ 1447–1454 (�80) 1442, �5 (�50) 1443 (�70) 1456

nðCCÞ ring, dðCHÞ 1415–1416 (�10) �1425, �+10 (�20)gwðCH2Þ 1376–1387 (�50) 1389, +2 (�30) 1355 (�70)

�1350 (�30) 1349, �1 (�15) 1330 (�70)

dðCHÞ, nðCCÞ 1326,1335 (530) 1307 (�15)nðCCÞ, dðCHÞ 1290–1295 (�50) 1281, �10 (�15)nðCOÞ, nðCCÞ 1235–1270 (200) {7} 1248–1265, �3 to �11 (�150) 1269–1273 (580) 1267–1277dðCHÞ 1170–1179 (�50) 1169, �1 (�30) 1174 (�150)

dðCOHÞ 1169–1260 (200) {7} 913, �256 1228–1250dðCHÞ 1100–1111 (�40) 1105–1106, +1 to +2 (�40) 1110 (�70)

a{1} Band assignment according to Takeuchi et al. (1988), Hienerwadel et al. (1997) and Lagant et al. (1998), assignment of CH2 vibrationsaccording to Colthup et al. (1975). See also legend of Table 1. {2} Band position and range according to Tyr spectra (Dollinger et al., 1986;Venyaminov and Kalnin (1990); Hienerwadel et al., 1997; Rahmelow et al., 1998) poly-l-Tyr spectra (Rothschild et al. (1986) and p-cresol spectra(Takeuchi et al., 1988; Gerothanassis et al., 1992; Hienerwadel et al., 1997), including studies of p-cresol in different solvents (Gerothanassis et al.,

1992; Hienerwadel et al., 1997). Extinction coefficient (in brackets) according to Venyaminov and Kalnin (1990); Rahmelow et al. (1998) or estimatedfrom absorbance spectra (Rothschild et al., 1986; Hienerwadel et al., 1997) by comparing band intensities to the intensity of the 1517 cm�1 band(indicated by ‘‘�’’). {3} Band positions in 2H2O and band shifts due to H2O/2H2O exchange according to spectra of Tyr (Chirgadze et al., 1975), poly-

l-Tyr (Rothschild et al., 1986) and p-cresol (Takeuchi et al., 1988; Hienerwadel et al., 1997). Shifts are given when the same compound has beeninvestigated in H2O and 2H2O. Extinction coefficient (in brackets) according to Chirgadze et al. (1975) or estimated from absorbance spectra(Rothschild et al., 1986) by comparing band intensities to the intensity of the 1515 cm�1 band (indicated by ‘‘�’’). {4} Band position and range

according to Tyr spectra (Dollinger et al., 1986; Rothschild et al., 1986; Venyaminov and Kalnin, 1990; Hienerwadel et al., 1997). Extinctioncoefficient (in brackets) according to Venyaminov and Kalnin (1990) or estimated from absorbance spectra (Hienerwadel et al., 1997) by comparingband intensities to the intensity of the 1499 cm�1 band (indicated by ‘‘�’’). {5} Band position and extinction coefficient (in brackets) according to Tyr

(Chirgadze et al., 1975) and poly-l-Tyr (Rothschild et al., 1986) spectra. The band shift of the �1500 cm�1 band is indicated with a question marksince it compares a Tyr� spectrum in H2O with a poly-l-Tyr� spectrum in 2H2O (Rothschild et al., 1986). {6} Ribonuclease T1 (1516, 1612 cm�1)(Fabian et al., 1994) and calmodulin in 2H2O (1516–1518 cm�1) (Berendzen and Braunstein, 1990; Fabian et al., 1996), bacteriorhodopsin (Dollingeret al., 1986; Rothschild et al., 1986; Liu et al., 1995) and photosystem II in H2O (Hienerwadel et al., 1997). {7} Both bands are sensitive to H-bonding.

For p-cresol in an aprotic and apolar solvent two bands are observed at 1175 and 1255 cm�1. Both bands merge to one broad band near 1250 cm�1 inH2O. nðCOÞ shifts to higher frequencies when the COH group is a H-bond donor (Hienerwadel et al., 1997), dðCOHÞ absorbs at 1210 to 1225 cm�1

when the group is an H-bond donor and at 1235 to 1245 cm�1 when it is both an H-bond donor and an acceptor (Gerothanassis et al., 1992).

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deprotonated with pKa values of 6 and 14 or coordinated to metal ions. In the fully deprotonatedimidazolate anion, the infrared spectrum is not sensitive to the solvent being H2O or 2H2O(Hasegawa et al., 2000). Singly protonated, the neutral imidazole group adopts two tautomericforms in which either N1 or N3 is protonated. In aqueous solution the ratio betweenthe N1- and the N3-protonated tautomer is 8:2 (l-His at 258C pH 11) (Ashikawa and Itoh, 1979)and thus the N1-protonated tautomer is shown in Fig. 4. At pH values below 6, thefully protonated imidazolium cation is formed. There are several useful marker bands inthe infrared spectrum for the protonation state of the imidazole group (see Table 10). Inparticular, the highest frequency band of the nðC¼CÞ vibration produces a shift of more than30 cm�1 upon formation of the fully protonated imidazolium cation (Hasegawa et al., 2000). Itssensitivity on H2O/2H2O exchange facilitates the identification of this band in the spectrum.Unfortunately, the nðC¼CÞ stretching vibration has a very low intensity for the imidazolate anion.The anion can probably be best detected by the strong band that is observed at 1439 cm�1 bandfor 4-Methylimidazole (Hasegawa et al., 2000). However, the CH3 group of 4 Methylimidazole,not present in His, contributes strongly to the respective normal mode and therefore questions itsuse as marker band for His deprotonation. Another useful band is the n(CN) band near 1100 cm�1

(Noguchi et al., 1999; Hasegawa et al., 2000), in particular when its sensitivity towards H2O/2H2Oexchange is taken into account. In the fully protonated form HisH2

+ it is most sensitive, whereasin the deprotonated form His� it is unaffected. Both marker bands can also serve to distinguishbetween the N1- and the N3-protonated tautomer (see Table 10). They have been identified inprotein infrared difference spectra of photosystem II at 1617 cm�1 on the basis of 13C labelling(Hienerwadel et al., 1997) and at 1094–1113 cm�1 as compiled in Noguchi et al. (1999).

In Arg and Lys the functional group is linked to the backbone by a relatively long aliphaticspacer of 3 or 4 methylene groups, respectively (see Fig. 4). The guanidyl group of Arg leads totwo relatively strong bands near 1633 and 1672 cm�1 which exhibit strong shifts of 50–70 cm�1

upon deuteration (see Table 11). These shifts distinguish Arg bands on the one hand from otherside chains absorbing in that region and on the other hand from amide I absorption of thepolypeptide backbone.

Table 5Phenylalanine side chaina

Assignment {1} Phe in H2O {2} Phe in 2H2O {3} Phe in proteins {4}Band position in cm�1

(e in M�1cm�1)


(e in M�1cm�1)


nðCCringÞ �1605 (30) 1607 (10)nðCCringÞ 1585 1596 (10)

nðCCringÞ 1494 (80) 1498

a {1} Assignments are according to Colthup et al. (1975). See also legend of Table 1. {2} Toluene in H2O (spectralrange covered: 1800–1450 cm�1) (Venyaminov and Kalnin, 1990) or monosubstituted benzenes (Colthup et al., 1975).

{3} Phe in 2H2O (spectral range covered: 1800–1500 cm�1) (Chirgadze et al., 1975). {4} Calmodulin in 2H2O (Berendzenand Braunstein, 1990; Fabian et al., 1996).


The Lys side chain amino group gives rise to only weak infrared bands, in particular inthe neutral state (Table 12). Two vibrations near 1526 and 1626 cm�1 can be assigned to theasymmetric and symmetric deformation vibration of the NH3

+ group.

Table 6Tryptophan side chaina

Assignment {1} Indole–NH {2} Trp–NH {3} Trp–N2 in H {4} Trp in proteins {5}Band position

in cm�1

(e in M�1cm�1)

Band position

in cm�1Band position,


(e in M�1cm�1)


nðCCbÞ, nðC¼CpÞ 1612 m 1622 1618, �4nðCCbÞ, nðC¼CpÞ 1576 w 1578–1579 1574, �5nðCCbÞ, dðCHbÞ 1552–1555 1545–1550, �2 (10)nðCNÞ, dðCHpÞ, dðNHÞ 1509 s

nðCCbÞ, dðCHbÞ 1487 s 1496dðCHbÞ, nðCCbÞ, nðCNÞ 1455 vs 1462 1455 (�200) 1455dðNHÞ, nðCCpÞ, dðCHbÞ 1412 s 1435 1383, –52n(CCp), n(CN), d(CHb) {6}

or d(CHb), gw(CH2),d(CHp) {1b}

1352 vs 1360–1361 1352–1353, �8

nðCCpÞ, nðCNÞ,dðCHbÞ {1a,6}or gwðCH2Þ, nðCCH2Þ {1b}

1334 vs 1342 1334, �8 (�100) 1334

nðCCbÞ 1305

dðNHÞ, nðCNÞ, dðCHbÞ 1276 sdðCHbÞ, nðCCpÞ 1245 sgtðCH2Þ, dðCHbÞ, dðCHpÞ 1238

nðCCbÞ, nðCCpÞ 1203 mnðCCbÞ, nðCCpÞ, dðCHbÞ 1191 wdðCHbÞ 1147 wdðCHbÞ, nðCCbÞ 1119 m 1127

dðCHpÞ 1092 vsnðNCÞ, dðCHpÞ 1064 snðCCbÞ, dðCHbÞ 1010 s 1012–1016 1012, � 0

970 sh930 w

a{1} Assignments are for indole (Takeuchi and Harada, 1986) {1a} or 3-Ethylindole (Lagant et al., 1998) {1b}. ‘‘b’’

and ‘‘p’’ indicate vibrations of the benzene or pyrole moieties, respectively. See also legend of Table 1. {2} Bandpositions and relative intensities according to infrared spectra of indole (Lautie et al., 1980). {3} According to Ramanspectra of Trp in aqueous solutions cited in Takeuchi and Harada (1986) and Lagant et al. (1998). {4} Raman spectra

cited in Takeuchi and Harada (1986) and infrared spectra (Chirgadze et al., 1975) (band at 1545 cm�1) of Trp inaqueous solutions, Trp in Ribonuclease T1 (1455, 1334 cm�1) (Fabian et al., 1994). Extinction coefficient (in brackets)from the infrared spectrum of a Trp solution in 2H2O (1550 cm�1) (Chirgadze et al., 1975) or estimated from the band

intensities relative to the Tyr 1515 cm�1 band in the difference spectrum that shows the effect of replacing two Tyr bytwo Trp in Ribonuclease T1 mutants (1334 and 1455 cm�1) (Fabian et al., 1994). {5} Ribonuclease T1 in 2H2O (Fabianet al., 1994). {6} The 1334 and 1352 cm�1 bands form a Fermi resonance doublet according to Takeuchi and Harada

(1986).


Table 7Serine side chaina

Assignment {1} Ser–OH {1} Ser–O2 H {2}Band position in cm�1 Band position,


dðCH2Þ 1450 1467, +2dðCHÞ, nðCCÞ {1a} or gwðCH2Þ {1b} 1364 1375, +6 (�100)gt(CH2), ns(COO�), d(CH) {1a} or d(CH) {1b} 1352 1340, �12 (�100)gwðCH2Þ, dðCHÞ {1a} or dðCHÞ {1b} 1312gtðCH2Þ {1b} 1248 {1b}

In H2O: dðCOHÞ, in 2H2O: dðCO2HÞ, nðCOÞ 1181 {1b}or 1248 {1a} – 1420 {3}

875–985, �263{4}

nðCOÞ, grðNHþ3 Þ 1030 s {5} 1023, �7

nðCOÞ, grðNHþ3 Þ, nðCCamideÞ {1a}

or nðCCÞ {1b}

983 {1a}

nðCOÞ, dðCO2HÞ 940

a {1} According to Raman spectra of l-serine crystals and normal mode analysis {1a} (Susi et al., 1983) and IRspectra of dl-serine crystals {1b} (Madec et al., 1978). See also legend of Table 1. {2} Infrared spectra of serine in 2H2O(Wright and Vanderkooi, 1997). Band shifts as observed upon deuteration from Raman spectra of l-serine crystals

(Susi et al., 1983). e estimated from infrared spectra of serine in 2H2O (Wright and Vanderkooi, 1997) by comparisonwith the nasðCOO�Þ band which has an e of 830 M�1 cm�1 (Chirgadze et al., 1975). {3} Band is expected at 1420 cm�1

(broad, weak) if the –CH2–OH group is hydrogen bonded (Pinchas and Laulicht, 1971; Colthup et al., 1975). {4} Bandis expected at 875 cm�1 if the –CH2–OH group is not hydrogen bonded (Pinchas and Laulicht, 1971), it is observed at

985 cm�1 in crystals (Susi et al., 1983). {5} Intensity according to Colthup et al. (1975), band slightly sensitive tohydrogen bonding (Colthup et al., 1975).

Table 8Threonine side chaina

Assignment {1} Thr in H2O {2} Thr in 2H2O {3}

Band position in cm�1 Band position in cm�1

dasðCH3Þ �1448m, �1458 sh, �1480 shdsðCH3Þ �1391w

�1360wdðCOHÞ, dðCHÞ 1385–1420mw {4}dðCOHÞ, dðCHÞ 1225–1330mw {4}

nðC�OÞ 1075–1150 s910–950 {5}

dðCO2 HÞ 865–942 s

a {1} Assignments are according to Colthup et al. (1975). See also legend of Table 1. {2} Absorption of aliphaticalcohols (Colthup et al., 1975), relative intensities are from ethanol spectra (not shown). {3} Poly-l-Thr in 2H2O

(spectral range covered: 1800–1300 cm�1) (Kubota and Fasman, 1975). The position of the dðCOHÞ band was takenfrom the spectrum of deuterated methanol. It is in H2O close to that of secondary alcohols (Pinchas and Laulicht,1971). {4} Higher value for hydrogen bonded OH group, lower value for free group (Colthup et al., 1975). {5} Weakerthan the nðC2OÞ band.


2.6. Side chains with amide groups: Asn, Gln

The structures of Asn and Gln are shown in Fig. 5, Tables 13 and 14 list the absorptionbands of Asn and Gln, respectively. The nðC¼OÞ vibration of Asn and Gln side chains near1680 cm�1 is a relatively strong infrared absorber. Due to coupling with the d(NH2) vibrationit shows a strong sensitivity towards deuteration. The downshift of 30 cm�1 is considerably largerthan that of the amide I bands of the protein backbone and helps distinguish the amide side chainabsorption from the amide backbone absorption. If spectra are recorded under identical

Table 9Cysteine side chaina

Assignment {1} Cys–SH {2} Cys–S2 H {2} Cys in proteins {3}Band position in cm�1 Band position,


(e in M�1cm�1)


nðSHÞ or nðS2 HÞ 2551 1849, �702 2550–2592, 1854–1861

dðCH2Þ 1424 1432, +2 (�120)d(CH), ns(COO�),gt(CH2)

1303 1341, +27 (�90)

gw(CH2), ns(COO�), 1269 1297, +16 (�70)a {1} According to Susi et al. (1983). See also legend of Table 1. {2} Band positions according to Raman spectra of

crystals grown from a H2O or 2H2O solution of l-cysteine (Susi et al., 1983), infrared spectra of cysteine in 2H2O

(spectral range 1800–1200 cm�1) (Wright and Vanderkooi, 1997). Only those Raman bands were listed for H2O wherethe corresponding bands upon deuteration matched closely the infrared bands in 2H2O. Band shifts upon deuterationare from Raman spectra of l-cysteine crystals (Susi et al., 1983) using the bands of the deuterated crystals at 1285, 1330

and 1426 cm�1 that matched the infrared bands within 12 cm�1. e estimated from infrared spectra of cysteine in 2H2O(Wright and Vanderkooi, 1997) by comparison with the nasðCOO�Þ band which has an e of 830M�1 cm�1 (Chirgadzeet al., 1975). {3} Hemoglobin (Alben and Bare, 1980; Moh et al., 1987), pyruvat decarboxylase (Baburina et al., 1996),

bacterial reaction centre (Noguchi et al., 1997).

Fig. 4. Structure of His, Arg and Lys. For His, protonation at the N1-atom is indicated.


conditions, Asn absorbs 10 cm�1 lower than Gln due to intramolecular interactions (Venyaminovand Kalnin, 1990). nðC¼OÞ bands are sensitive to H-bonding and the band position is the lowerthe stronger the H-bond is. The observation of an Asn band at 1704 cm�1 in a bacteriorhodopsinmutant (Cao et al., 1993) shows that this C¼O group is only weakly H-bonded.

The deformation vibration of the side chain amino group near 1610 cm�1 gives rise toweaker absorption bands. Upon deuteration they are shifted by several hundred cm�1 to1160 cm�1.

2.7. Side chains with carboxyl groups: Asp, Glu

The structure of the carboxyl group containing amino acids Asp and Glu is shown in Fig. 6.Tables 15 and 16 list their infrared bands. The carboxyl group provides the vibrationalspectroscopist with a unique monitor to follow proton pathways in proteins. An intensely studiedexample is the proton pump bacteriorhodopsin (Rothschild, 1992; Maeda, 1995; Gerwert, 1999;Heberle, 1999). Of particular interest here is the nðC¼OÞ vibration of the protonated carboxylgroup that has the particular advantage of absorbing in a spectral region that is generally freefrom overlap by other amino acid absorptions (1710–1760 cm�1). Overlap may occur howeverwith the nðC¼OÞ vibration of lipids. The nðC¼OÞ vibration is sensitive to H-bonding with shifts ofapproximately 50 cm�1 observed upon formation of strong hydrogen bonds. The deprotonatedcarboxylate group shows two strong bands near 1400 and 1570 cm�1 for the symmetric and theantisymmetric stretching vibration, respectively. The band position of these may shiftconsiderably upon cation chelation (Deacon and Phillips, 1980; Tackett, 1989; Nara et al.,1994) which changes bond lengths and angles. The effects depend upon the mode of chelation andthis has been used to study several Ca2+ binding proteins (Nara et al., 1994; Fabian et al., 1996;Mizuguchi et al., 1997a). For example upon unidentate chelation (where only one oxygen atom ofthe carboxylate group coordinates the cation), the two CO bonds are no longer equivalent, withthe bond of the chelating oxygen having more single bond character and that of the non-chelatingoxygen more double bond character.

3. Outlook

As the discussion above has shown, the infrared spectrum encodes a lot of importantinformation on amino acid side chains, like protonation state, charge, accessibility to H-bonding partners and conformational freedom. Often marker bands can be found thatmonitor these properties in the course of a protein reaction. The information deducedfrom the infrared spectra is thus part of the jigsaw puzzle that builds up the completemolecular picture of the protein reaction mechanism. As there is an increasing number ofinfrared spectroscopic techniques to monitor protein reactions (Mantele, 1993; Backmann et al.,1995; Siebert, 1995; White et al., 1995; Gerwert, 1999; Barth and Zscherp, 2000), it can beexpected that its application range will constantly widen. Thus infrared spectroscopy willcontinue to provide important contributions to the understanding of side chain action in proteinfunction.


Table 10

Histidine side chaina

Assignment {1} His� in H2Oand 2H2O {2}

HisH {3} His2 H {3} HisH2+ {4} His2H2

+ {4} His in proteins {5}

Band positionin cm�1

(e in M�1cm�1)


(e in M�1cm�1)

Band position,band shiftin cm�1

(e in M�1cm�1)


(e in M�1cm�1)

Band position,band shiftin cm�1

(e in M�1cm�1)


nðC¼CÞ, nðCCÞ 1575 s, 1594 s (70){6}

1569, �6 sand 1575,

�19m {6}

1631 (250) vs {13} 1600 (35),1623 (16) {11}

1617 (?)

nðC¼NÞ, dðCHÞ 1490 s 1485, �5 sdðCHxÞ, nðCNÞ 1439 vs 1408m

dðNHÞ, nðCNÞ, dsðCHxÞ 1423mnðCNÞ 1363, 1369m {6} 1366mnðC¼NÞ, nðCNÞ 1304m {7}{9} 1305, +1m {7}

dðCHÞ, nðCNÞ 1255mnðCNÞ, nðCCÞ {1a,c}or gtðCH2Þ,dðCHÞ, n ring {1b}

1300m 1265 s {8} 1259, �6 s {7} 1267m

nðCNÞ 1232mdðCHÞ, nðCNÞ, dNH,nðCCÞ {12}

1217 s 1229 s 1223, �6 s {7} 1199m 1239m

nðCNÞ, dðNHÞ 1153m, 1161 m {10} 1182mnðCNÞ, dðCHÞ 1104 s {14} 1090 s, 1106 s {7}

{14}1096,+6mand 1107,

+1 s {7}{14}

1094 s {14} 1110, +16 s {14} 1094–1114,1093–1114

d ring, nðC¼CÞ 1010mdðCHÞ {1a} or nðCCÞ,nðNCÞ, dðCHÞ {1b}or grðCHxÞ, nðC¼CÞ {1c}

995m {7} 1017, +22 s {7} 1022m

nðCNÞ, dðCHxÞ, d ring 975m 978, +3m 973md ring {1b,c}, nðCCÞ {1b} 950m 941 s 943, +2 s 925m

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a {1} Assignments are for 4-Ethylimidazole according to Ashikawa and Itoh (1979) {1a} and Lagant et al. (1998) {1b}, for 4-Methylimidazole

according to Hasegawa et al. (2000) {1c}. CHx denotes the CH2 group in 4-Ethylimidazole and the CH3 group in 4-Methylimidazole. See also legendof Table 1. {2} 4-Methylimidazole in NaOH and NaO2H (Hasegawa et al., 2000) for all bands except for the 1104 cm�1 band which was observed fordl-His (Noguchi et al., 1999). Absorption bands with a strong contribution of the CH3 group in 4-Methylimidazole are not listed since the

corresponding group in His is CH2. {3} Band positions are for 4-Methylimidazole in H2O and 2H2O (Hasegawa et al., 2000) with the exception of thebands at �1100 cm�1 which are for dl-His (Noguchi et al., 1999). Relative intensities are from Hasegawa et al., (2000), e from the respective band ofimidazole in H2O at 1596 cm�1 (Venyaminov and Kalnin, 1990). {4} Band positions and relative intensities are for 4-Methylimidazole in HCl or2HCl Hasegawa et al., 2000), except for the highest frequency band which is taken from His spectra in HCl (Hienerwadel et al., 1997) and 2HCl(Chirgadze et al., 1975) and the band near 1100 cm�1 taken from dl-His spectra in HCl and 2HCl (Noguchi et al., 1999). {5} Photosystem II in H2O(Hienerwadel et al., 1997) and H2O and 2H2O as compiled in Noguchi et al. (1999). {6} Doublet due to the two protonated tautomers (Ashikawa and

Itoh, 1979; Hasegawa et al., 2000), lower frequency band is due to the N1 protonated tautomer. {7} Only for the N1 protonated tautomer (Ashikawaand Itoh, 1979; Hasegawa et al. 2000). {8} 7 cm�1 variation depending on which nitrogen atom is protonated (Ashikawa and Itoh, 1979; Hasegawaet al., 2000). The higher frequency band at 1265 cm�1 for the N1 protonated tautomer has a higher intensity in the infrared spectrum (Hasegawaet al., 2000). {9} 20�35 cm�1 lower in Raman spectra of l-His or Poly-l-His (Ashikawa and Itoh, 1979) as compared to Raman spectra (Ashikawa

and Itoh, 1979) and the IR data listed here (Hasegawa et al., 2000) of 4-Methylimidazole. {10} Doublet due to the two protonated tautomers, lowerfrequency band for N3 protonated tautomer (Hasegawa et al., 2000). {11} Two bands with very weak intensity observed for His at 1600 and1623 cm�1 (Chirgadze et al., 1975), only one very strong band for 4-Methylimidazole at 1605 cm�1 (Hasegawa et al., 2000). {12} dðNHÞ contribution

calculated only for HisH2+ in Hasegawa et al. (2000) or for HisH and HisH2

+ in Ashikawa and Itoh (1979), nðCCÞ only for N3-protonated4-Methylimidazole (Hasegawa et al., 2000). {13} Shifts �38 cm�1 upon 13C labeling (Hienerwadel et al., 1997). The respective band of4-Methylimidazole is at 1633 cm�1 (Hasegawa et al., 2000). {14} Band positions and H2O/2H2O exchange induced band shifts are for dl-His

(Noguchi et al., 1999), intensities from the respective 4-Methylimidazole bands (Hasegawa et al., 2000) at 1101 (MeIm�), 1087, 1104 (MeImH), 1097,1104 (MeIm2H), 1088 (MeImH2

+) and 1106 (MeIm2H2+).

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Table 11Arginine side chaina

Assignment {1} Arg {2} Arg in 2H2O {3} Arg in proteins {4}


(e in M�1cm�1)


(e in M�1cm�1)


nasðCN3Hþ5 Þ 1672–1673 (420–490) 1608 (460) 1688–1695, 1595–1605

nsðCN3Hþ5 Þ 1633–1636 (300–340) 1586 (500) 1576–1577

a {1} Assignments are according to Chirgadze et al. (1975) and Venyaminov and Kalnin (1990). See also legend ofTable 1. {2} Arg in H2O (1800–1500 cm�1) (Venyaminov and Kalnin, 1990; Rahmelow et al., 1998). {3} Arg in2H2O (1800–1500 cm�1) (Chirgadze et al., 1975). {4} Observed for deuterated lysozyme and a-Keratin (two bands at1576–1577 and 1595–1600 cm�1) (Chirgadze et al., 1975), deuterated calmodulin (1605 cm�1) (Berendzen and

Braunstein, 1990), halorhodopsin (1688–1695 cm�1) in H2O (Rudiger et al., 1995).

Table 12Lysine side chaina

Assignment {1} Lys–NH3+ {2} Lys–N2H3

+ {3} Lys in proteins {4}


(e in M�1cm�1)

Band position,

band shift in cm�1Band position in cm�1

dasðNHþ3 Þ 1626–1629 (60–130) �1201, �428

dsðNHþ3 Þ 1526–1527 (70–100) �1170, �356

dðCH2Þ 1445

gwðCH2Þ; grðCH2Þ 1325,1345

a{1} Assignments are according to Pinchas and Laulicht (1971) and Venyaminov and Kalnin (1990). See also legendof Table 1. In the neutral form the absorption of the e-NH2 group is weak with extinction coefficients less than40M�1 cm�1 between 1800 and 1500 cm�1 (Venyaminov and Kalnin, 1990). {2} Lys in H2O (1800–1500 cm�1)(Venyaminov and Kalnin, 1990; Rahmelow et al., 1998) {3} Band shifts according to those observed for crystals of

CH3NH3Cl and CH3N2H3Cl (Pinchas and Laulicht, 1971). Band positions are estimated from these shifts. {4} Raman

spectra in H2O of the coat protein of phage fd, identified by selective deuteration (Overman and Thomas, 1999).

Fig. 5. Structure of Asn and Gln.


Table 13Asparagine side chaina

Assignment {1} Asn–NH2 {2} Asn–N2H2 {3} Asn in proteins {4}Band position in cm�1

(e in M�1cm�1)


(e in M�1cm�1)


nðC¼OÞ 1677–1678 (310–330) {5} �1650 (570) 1704dðNH2Þ 1612–1622 (140–160) 1622 (?)

a {1} Assignments are according to Chirgadze et al. (1975) and Venyaminov and Kalnin (1990). See also legend ofTable 1. {2} Band positions and extinction coefficients (in brackets) of Asn in H2O according to Venyaminov andKalnin (1990) and Rahmelow et al. (1998). {3} Band positions and extinction coefficients (in brackets) of Asn in 2H2O

according to Chirgadze et al. (1975) and Wright and Vanderkooi (1997). {4} Photoreaction of a bacteriorhodopsinmutant in H2O (Cao et al., 1993). {5} 10 cm�1 lower than for Gln due to intramolecular interactions in Asn(Venyaminov and Kalnin, 1990).

Table 14Glutamine side chaina

Assignment {1} Gln–NH2 {2} Gln–N2H2 {2} Gln in proteins {3}Band position in cm�1

(e in M�1cm�1)Band position,band shift in cm�1

(e in M�1cm�1)


nðC¼OÞ 1668–1687 (360–380) s 1635–1654, �33 (550) 1659–1696dðNH2Þ 1586–1611 (220–240) m 1163, �410mdðCH2Þ 1451 m 1453, +2mdðCH2Þ 1442mnðCNÞ 1410 s 1409, �1msdðCHÞ 1359 w 1361, +2wmdðCHÞ 1333 ms 1333, � 0msgwðCH2Þ 1315 m 1317, +2m

gwðCH2Þ 1281 wm 1279, –2wmgtðCH2Þ 1256 wm 1257, +1wgtðCH2Þ 1202 w 1204, +2w

grðNH2Þ 1104 w 810, �296wnðCNÞ 1084 w 1071, �13 vwnðCCÞ 1052 m 1040, �12wnðCCÞ 999 wm

nðCCÞ 926 w

a{1} According to Dhamelincourt and Ramirez (1993). See also legend of Table 1. {2} Band positions are

from spectra of Gln crystals (Dhamelincourt and Ramirez, 1993) except for the 1668–1687, the 1635–1654 and the1586–1611 cm�1 bands which are from spectra of Gln in aqueous solution (Venyaminov and Kalnin, 1990; Rahmelowet al., 1998), extinction coefficient in brackets according to Venyaminov and Kalnin (1990); Rahmelow et al. (1998)or relative intensities and band shifts according to Dhamelincourt and Ramirez (1993). Band shifts are according

to Dhamelincourt and Ramirez (1993). {3} Photoreaction of a photosystem II mutant in H2O (Hienerwadel et al.,1997).


Table 15Aspartate side chaina

Assignment {1} Asp–COO�

in H2O {2}Asp–COO�

in 2H2O {3}Asp–COOH {4} Asp–COO2 H {5} Asp in proteins {6}

Band position

in cm�1

(e in M�1cm�1)

Band position,


(e in M�1cm�1)

Band position

in cm�1

(e in M�1cm�1)

Band position,


(e in M�1cm�1)

Band position

in cm�1

nðC ¼ OÞ {7} 1716–1788 (280) 1713–1775,�13 (290)

1732–1762

nasðCOO�Þ {8} 1574–1579(290–380)

1584–1586,+9 (820)

1578–1595

nsðCOO�Þ {9} 1402 (256) 1404, +2 (�450) 1392–1425dðCOHÞ 1264–1450 955–1058,

�309mwnðC2OÞ {10} 1120–1250

(100–200)1270–1322,+88m

a {1} According to Pinchas and Laulicht (1971), Chirgadze et al. (1975), Colthup et al. (1975) and Venyaminov andKalnin (1990). See also legend of Table 1. {2} Asp in H2O (Venyaminov and Kalnin, 1990; Rahmelow et al., 1998). {3}Shifts upon H2O/2H2O exchange are for sodium acetate (Tackett, 1989). Band position near 1585 cm�1 is for Asp in2H2O (Chirgadze et al., 1975; Nara et al., 1994; Wright and Vanderkooi, 1997) and at 1404 cm�1 estimated from the

shift observed for sodium acetate (Tackett, 1989). e of the 1404 cm�1 band estimated from the comparison with thenasðCOO�Þ band which has an e of 830 M�1 cm�1 (Chirgadze et al., 1975) using spectra of sodium acetate (Tackett,1989; Wright and Vanderkooi, 1997). {4} Band positions and extinction coefficient for Asp in H2O (Venyaminov and

Kalnin, 1990) (1716 and 1250 cm�1), hydrogen bonded (1450 cm�1, spectra not shown) and free CH3COOH (1264,1788 cm�1) (Pinchas and Laulicht, 1971). Range for the 1120–1250 cm�1 band estimated from the band observed forAsp in H2O at 1250 cm�1 and from the shift upon hydrogen bonding observed for hydrogen bonded and free

C2H3COOH (Pinchas and Laulicht, 1971). The corresponding band of aqueous CH3COOH is at 1274 cm�1 (data notshown). {5} Asp in 2H2O (Chirgadze et al., 1975) (1713 cm�1), hydrogen bonded (1058, 1322 cm�1) and freeCH3COO2H (955, 1270, 1775 cm�1) (Pinchas and Laulicht, 1971). The frequency of the nðC2OÞ band may be �25 cm�1

lower for Asp than given here for acetic acid, since this is observed in H2O. Shifts upon deuteration are for free

CH3COOH (Pinchas and Laulicht, 1971). Relative intensities are from spectra of acetic acid (data not shown). {6}Bacteriorhodopsin (1392 and 1732–1762 cm�1) (Fahmy et al., 1993; Sasaki et al., 1994), pike parvalbumin in 2H2O(1584 cm�1) (Nara et al., 1994), calmodulin in 2H2O (1584 cm�1) (Fabian et al., 1996), Ca2+ free form of a-lactalbumin

at 1585 cm�1 (2H2O) and 1393 cm�1 (H2O), Ca2+-loaded form of a-lactalbumin at 1578 and 1592 cm�1 (2H2O), and at1406 and 1425 cm�1 (H2O) (Mizuguchi et al., 1997a), Ca2+-loaded form of lysozyme at 1578 and 1595 cm�1 (2H2O),and at 1403 and 1423 cm�1 (H2O) (Mizuguchi et al., 1997b). {7} Sensitive to H-bonding: –25 cm�1 shift upon formation

of a single H-bond, above �1740 cm�1 inverse correlation of nðC¼OÞ and the dielectric constant e (Dioumaev andBraiman, 1995). For esters, hydrogen bonding not only to the carbonyl oxygen but also to the ether oxygen was found.

Fig. 6. Structure of Asp and Glu.


Both exhibit opposite effects on the nðC¼OÞ band position, with the latter leading to an upshift of 10 cm�1 (Maes andZeegers-Huyskens, 1983; Maes et al., 1988). While an interaction like this is not observed in aqueous solution, it mayoccur in proteins. In aqueous solution, the nðC¼OÞ band position is an indicator of the intramolecular effects that

determine the pKa value, when different compounds are compared, with a higher band position indicating a lower pKa

(Wright and Vanderkooi, 1997). For proteins however, the environment plays probably the dominant role for a givenAsp or Glu residue in stabilising either the protonated, neutral form or the deprotonated, charged form. A hydrophobicenvironment leads to a higher band position due to the lack of hydrogen bonding and a higher pKa value due to

stabilisation of the neutral form. It seems that the band shift upon deuteration is larger for the monomer than inaqueous solution. For the monomer a shift of �13 cm�1 was found (Pinchas and Laulicht, 1971) but only a shift of�3 cm�1 is derived from the comparison of the data in aqueous solution of Venyaminov and Kalnin (1990) and

Chirgadze et al. (1975). 13C labeling shifts the band by �40 cm�1 (Fahmy et al., 1993). {8} May shift +60/�40 cm�1

upon cation chelation (Tackett, 1989; Nara et al., 1994). In extreme cases, a band position as for nðC¼OÞ of the COOHgroup is possible (Deacon and Phillips, 1980). In aqueous solution is the band position an indicator of the

intramolecular effects that determine the pKa value, when different compounds are compared, with a higher bandposition indicating a lower pKa (Wright and Vanderkooi, 1997). {9} Band position in 2H2O estimated from the shiftobserved for sodium acetate (Tackett, 1989). May shift +60/�90 cm�1 upon cation chelation (Tackett, 1989). In

extreme cases, a band position as for nðC2OÞ of the COOH group is possible (Deacon and Phillips, 1980). e for 2H2Oestimated from the comparison with the nasðCOO�Þ band which has an e of 830M�1 cm�1 (Chirgadze et al., 1975) usingspectra of sodium acetate (Tackett, 1989; Wright and Vanderkooi, 1997). �20 cm�1 shift upon 13C labelling (Fahmyet al., 1993). {10} For esters it was found that a hydrogen bond to the carbonyl oxygen leads to an upshift of 21 cm�1

and a hydrogen bond to the ether oxygen to a downshift of 17 cm�1 (Maes and Zeegers-Huyskens, 1983).

Table 16

Glutamate side chaina

Assignment {1} Glu–COO�

in H2O {2}

Glu–COO�

in 2H2O {3}

Glu–COOH {4} Glu–COO2H {5} Glu in proteins {6}


(e in M�1cm�1)

Band position,band shift in cm�1

(e in M�1cm�1)


(e in M�1cm�1)


(e in M�1cm�1)


nðC¼OÞ {7} 1712–1788 (220) 1706–1775,�13 (280)

nasðCOO�Þ {8} 1556–1560(450–470)

1567–1568,+9 (830)

1553–1575

dðCH2Þ 1452 sh 1451 wdðCH2Þ 1440 s 1417 m

nsðCOO�Þ {9} 1404 (316) 1406, +2 (�450) 1397–1424gwðCH2Þ,dðCHÞ, nðCCÞ

1388 w

gwðCH2Þ 1359 wgtðCH2Þ, gwðCH2Þ 1343 shgtðCH2Þ 1323 m

gwðCH2Þ, dðCHÞ 1313 wdðCHÞ, dðNHÞ 1283 wgwðCH2Þ 1292 w

dðCOHÞ 1264–1450 955–1058, �309 mwnðNCÞ, dðCHÞ 1260 mwnðC2OÞ {10} 1120–1253

(100–200)ms1270–1322, +88 m

Table 15 ( footnote continued )


Table 16 (continued)

Assignment {1} Glu–COO�

in H2O {2}

Glu–COO�

in 2H2O {3}

Glu–COOH {4} Glu–COO2H {5} Glu in proteins {6}


(e in M�1cm�1)


(e in M�1cm�1)


(e in M�1cm�1)


(e in M�1cm�1)


gtðCH2Þ, dðCHÞ 1225 shgwðCH3Þ 1212 w

gtðCH2Þ, dðCHÞ 1187 mw 1181 wgtðCH2Þ, dðCHÞ 1130 mnðNCÞ, nðCCÞ 1118 m

nðCCÞ 1074 vw 1083 wnðCCÞ 1040 vw 1060 wnðCCÞ 1018 vw

1028 wnðCCÞ 982 w

a{1} According to Sengupta and Krimm (1984, 1985), for COO� and COOH group according to Pinchas and

Laulicht (1971), Chirgadze et al. (1975), Colthup et al. (1975) and Venyaminov and Kalnin (1990). See also legend ofTable 1. {2} Glu in H2O (Venyaminov and Kalnin, 1990; Rahmelow et al., 1998) (1560 and 1404 cm�1) and poly-l-glutamate (Sengupta and Krimm, 1984). {3} Band position and extinction coefficient of the �1567 cm�1 band are for

Glu in 2H2O (Chirgadze et al., 1975; Nara et al., 1994; Wright and Vanderkooi, 1997). Band position at 1406 cm�1

estimated from the shift observed for CH3COO� in H2O and 2H2O (Tackett, 1989), e of this band estimated from thecomparison with the nasðCOO�Þ band which has an e of 830M�1 cm�1 (Chirgadze et al., 1975) using spectra of sodium

acetate (Tackett, 1989; Wright and Vanderkooi, 1997). Band shifts upon H2O/2H2O exchange as observed forCH3COO� (Tackett, 1989). {4} Band positions are for poly-l-glutamate (Sengupta and Krimm, 1984), Glu in H2O(Venyaminov and Kalnin, 1990) (1712 and 1250 cm�1), hydrogen bonded (1450 cm�1, spectra not shown) and freeCH3COOH (1264, 1788 cm�1) (Pinchas and Laulicht, 1971). Range for the 1120–1253 cm�1 band estimated from the

band observed for Glu in H2O at 1250 cm�1 or poly-l-Glu at 1253 cm�1 and from the shift upon hydrogen bondingobserved for hydrogen bonded and free CH3COOH (Pinchas and Laulicht, 1971). {5} Glu in 2H2O (Chirgadze et al.,1975) (1706 cm�1), hydrogen bonded (1058, 1322 cm�1) and free CH3COO2H (955, 1270, 1775 cm�1). The frequency of

the nðC2OÞ band may be �25 cm�1 lower for Glu than given here for acetic acid, since this is observed in H2O. Shiftsupon deuteration are for free CH3COOH (Pinchas and Laulicht, 1971). Relative intensities are from spectra of aceticacid (data not shown). {6} 1553–1575 cm�1 band: pike parvalbumin with bidentate Ca2+ coordination (1553 cm�1) or

unidentate Mn2+ coordination (�1575 cm�1) (Nara et al., 1994), Ca2+ loaded calmodulin (1555 cm�1) (Fabian et al.,1996) in 2H2O. 1397–1424 cm�1 band: Asp or Glu of pike parvalbumin in 2H2O with bound Ca2+, Mn2+ or Mg2+. {7}Sensitive to H-bonding: –25 cm�1 shift upon formation of a single H-bond, above �1740 cm�1 inverse correlation of

nðC¼OÞ and the dielectric constant e (Dioumaev and Braiman, 1995). For esters, hydrogen bonding not only to thecarbonyl oxygen but also to the ether oxygen was found. Both exhibit opposite effects on the nðC¼OÞ band position,with the latter leading to an upshift of 10 cm�1 (Maes and Zeegers–Huyskens, 1983; Maes et al., 1988). While aninteraction like this is not observed in aqueous solution, it may occur in proteins. In aqueous solution, the nðC¼OÞband position is an indicator of the intramolecular effects that determine the pKa value, when different compounds arecompared, with a higher band position indicating a lower pKa (Wright and Vanderkooi, 1997). For proteins however,the environment plays probably the dominant role for a given Asp or Glu residue in stabilising either the protonated,

neutral form or the deprotonated, charged form. A hydrophobic environment leads to a higher band position due to thelack of hydrogen bonding and a higher pKa value due to stabilisation of the neutral form. It seems that the band shiftupon deuteration is larger for the monomer than in aqueous solution. For the monomer a shift of �13 cm�1 was found

(Pinchas and Laulicht, 1971) but only a shift of �3 cm�1 is derived from the comparison of the data in aqueous solutionof Venyaminov and Kalnin (1990) and Chirgadze et al. (1975). 13C labeling shifts the band by �40 cm�1 (Fahmy et al.,1993). {8} May shift +60/�40 cm�1 upon cation chelation (Tackett, 1989; Nara et al., 1994). In extreme cases, a bandposition as for nðC¼OÞ of the COOH group is possible (Deacon and Phillips, 1980). In aqueous solution is the band


Acknowledgements

The author gratefully acknowledges continuous support by W. Mantele. Current work issupported by grants Ba 1887/1-1 and 2-1 of the Deutsche Forschungsgemeinschaft.

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position an indicator of the intramolecular effects that determine the pKa value, when different compounds arecompared, with a higher band position indicating a lower pKa (Wright and Vanderkooi, 1997). {9} Band positionestimated from the shift observed for CH3COO� in H2O and 2H2O (Tackett, 1989). May shift +60/�90 cm�1 upon

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carbonyl oxygen leads to an upshift of 21 cm�1 and a hydrogen bond to the ether oxygen to a downshift of 17 cm�1

(Maes and Zeegers-Huyskens, 1983).

Table 16 ( footnote continued)


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