The Infrared Absorption of Amino Acid Side Chains

Progress in Biophysics & Molecular Biology 74 (2000) 141–173
Review
The infrared absorption of amino acid side chains

Andreas Barth*
Institut für Biophysik, Johann Wolfgang Goethe-Universität, 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 catalysing
enzymatic reactions. These fields are increasingly investigated by infrared spectroscopy at the molecular
level. To help the interpretation of the spectra, a review of the infrared absorption of amino acid side chains
in H2O and 2H2O is given. The spectral region of 2600–900 cm1 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
2.2. Aliphatic side chain groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
2.3. Aromatic side chains: Tyr, Phe, Trp . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
2.4. Aliphatic side chains with hydroxyl or thiol groups: Ser, Thr, Cys . . . . . . . . . . . . 155
2.5. Positively charged side chains: His, Arg, Lys . . . . . . . . . . . . . . . . . . . . . . . 155
2.6. 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: barth@biophysik.uni-frankfurt.de (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
142 A. Barth / Progress in Biophysics & Molecular Biology 74 (2000) 141–173
Nomenclature
d in plane bending vibration mw band of medium to weak intensity
FTIR Fourier transform infrared n stretching vibration
IR infrared ns symmetric stretching vibration
gw wagging vibration nas antisymmetric stretching vibration
gt twisting vibration s band of strong intensity
gr rocking vibration sh shoulder
e extinction coefficient vs very strong intensity
m band of medium intensity w weak intensity
1. Introduction
The infrared spectrum of a protein provides a wealth of information on structure and

environment of the protein backbone and of the amino acid side chains. This makes infrared
spectroscopy 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 and
Goñi, 1999), of the molecular mechanism of protein reactions (Rothschild, 1992; Mäntele, 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 Goñi,
1999; Fabian et al., 1999; Reinstädler et al., 1999; Schultz, 2000; Troullier et al., 2000).
For the determination of secondary structure from infrared spectra, the absorption of amino
acid side chains presents a matter of concern. This is because the amide I vibration of the
polypeptide backbone used for this purpose absorbs in a spectral region (1610–1700 cm1) where
side chains also absorb. It is estimated that 10–30% of the total absorption in that region derives
from 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 in
aqueous solution. However, this may be problematic for side chains not exposed to the
surrounding aqueous solvent.
In contrast to secondary structure determination, amino acid side chain absorption provides
very valuable information when the mechanism of protein reactions is investigated. This is
because side chains are often at the heart of the molecular reaction mechanism. With infrared
spectroscopy it is possible to follow in a single experiment the fate of the several individual groups
that are involved in the reaction. The aim of this kind of research is to identify the catalytically
important side chains and to deduce their environmental and structural changes from the
spectrum in order to understand the molecular reaction mechanism. The problem here is the
assignment of spectral features to specific amino acids.
Catalytically important residues can be identified by a combination of site-directed mutagenesis
and infrared spectroscopy. The general strategy is to induce the protein reaction in the infrared
cuvette, follow the infrared absorbance changes and identify crucial residues by repeating the
experiment with selected point mutants. An intensely studied example is bacteriorhodopsin. In
A. Barth / Progress in Biophysics & Molecular Biology 74 (2000) 141–173 143
particular, those Asp and Glu residues that constitute the proton pumping pathway of
bacteriorhodopsin could be identified (Rothschild, 1992; Maeda, 1995; Gerwert, 1999; Heberle,
1999).
Information on side chain properties stems from the physical principles of the absorption
process. 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- and
intermolecular effects. Thus, information about structure and environment of amino acid side
chains can be deduced from the spectral parameters band position, band width and absorption
coefficient.
In our particular case of amino acid side chains, protonation state, coordination of cations and
hydrogen bonding are the dominating factors that determine the band position of a particular
amino acid. Infrared spectroscopy is thus one of the few techniques that is able to define the
protonation state of side chains which has important consequences for the electrostatic
interactions in proteins. Protonation of Asp and Glu residues accompanies for example proton
pumping by bacteriorhodopsin (Rothschild, 1992; Maeda, 1995; Gerwert, 1999; Heberle, 1999),
electron transfer reactions (Mäntele, 1995), Ca2+ release from the Ca2+-ATPase (Barth and
Zscherp, 2000) and seems to provide a mechanism of charge compensation when the negatively
charged 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 cation
coordination 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 force
when the H-bonding partners are close, but increases the frequency of bending vibrations since it
produces an additional restoring force (Colthup et al., 1975). In the above-mentioned examples of
Asp and Glu protonation, the degree of hydrogen bonding to the protonated form could be
determined. As for Asp and Glu residues, the protonation state and the environment of other
catalytically active side chains can be characterised, as done for example for His and Tyr residues
of 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 internal
coordinate contributions to the dipole moment of a normal mode cancel, as they do in the ns mode
of CO2). Thus a change in environment that results in an altered bond polarity will lead to a
change in band intensities.
The band width is a measure of conformational freedom with flexible structures giving broader
bands. This has been used for example to characterise the environment of the phosphorylated Asp
residue of the sarcoplasmic reticulum Ca2+-ATPase (Barth and Mäntele, 1998). From the small
band width of the nðC¼OÞ band it was concluded that this group is not exposed to solvent water
but exhibits defined interactions with the protein environment.
One advantage of infrared spectroscopy is that the protein backbone as well as the side chains
can be observed in the same experiment. Thus it is possible to compare the kinetics of backbone
structural 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 as
the local perturbations of side chains (Barth et al., 1996). In contrast, in the complex refolding of
Ribonuclease T1 the very late events due to the trans ! cis isomerisation of a prolyl peptide bond
lead to an increased compactness of the protein structure but not to an environmental change of
Asp, Glu and Tyr residues. They have therefore adopted their native environment already in the
preceding processes (Reinstädler et al., 1999).
The interpretation of the generally complicated spectral band profiles of protein reactions is
often guided by experience and is greatly helped by a condensation of present knowledge into a
list of group frequencies. However, earlier collections of amino acid infrared spectra (Chirgadze et
al., 1975; Venyaminov and Kalnin, 1990; Goormaghtigh et al., 1994a; Wright and Vanderkooi,
1997; Rahmelow et al., 1998) were often restricted to the 1800–1500 cm1 spectral region and do
not make use of the whole mid infrared spectral region which is easily accessible to the
experimentalist. Thus it is attempted in this review to collect data from a variety of sources in
order to obtain a more complete picture. The review focuses on the 2600–900 cm1 spectral region
since it is the most useful for the structural interpretation and is easily accessible with common
experimental 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; Overman
and Thomas, 1999). I am aware that a collection of this amount of data cannot be complete and
flawless. 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 and
2
H2O. Only the strongest bands are listed, or those in a spectral window free of overlap by bands
from 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 of
side chain absorption in proteins are also listed. If available, parameters of infrared spectra of
amino acid side chains are given. If not, data are taken from infrared spectra of model compounds
or from Raman spectra. Band positions are given for H2O and 2H2O, the latter are indicated by
italic print. The shift upon H/2H exchange is given when a compound in both solvents is
compared in the original work. The listing of internal coordinate contributions to a normal mode
is according to their contribution to the potential energy of the normal mode (if specified in the
literature): If the contribution of an internal coordinate to the potential energy of a normal
vibration is 570% only that coordinate is listed. Two coordinates are listed if their contribution
together is 570%. In all other cases those 3 coordinates that contribute strongest to the potential
energy 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 from
their absorption in solution or in a crystal. The special environment provided by a protein is able
to modulate the electron density and the polarity of bonds, thus changing the vibrational
frequency and the absorption coefficient. Therefore, the band positions given in the tables should
be regarded only as guidelines for the interpretation of spectra. It may be mentioned here that also
the pKa of acidic residues in proteins may differ significantly from solution values. For D96 of
bacteriorhodopsin for example a pKa > 12 has been found (Zscherp et al., 1999).
Table 1
Overview of amino acid side chain infrared bandsa
Assignments Band position in cm1 Band position in cm1, References Remarks
(e in M1 cm1 ) in H2O (e in M1 cm1 ) in 2H2O
Cys, nðSHÞ 2551 1849 Susi et al. (1983)

Asp, nðC¼OÞ 1716–1788 (280) 1713–1775 (290) Pinchas and Laulicht Sensitive to H-bonding. Without H-
A. Barth / Progress in Biophysics & Molecular Biology 74 (2000) 141–173

(1971), Chirgadze et al. bond up to 1762 cm1 observed in
(1975), Venyaminov and proteins (Fahmy et al., 1993). Single
Kalnin (1990) H-bond shifts 25 cm1 down. Above
1740 cm1 inverse correlation of
n(C=O) with the dielectric constant
(Dioumaev and Braiman, 1995).
Values for the unbonded C=O
group (1788 cm1) are from the
acetic acid spectrum (Pinchas and
Laulicht, 1971)
Glu, nðC¼OÞ 1712–1788 (220) 1706–1775 (280) Pinchas and Laulicht See Asp n(C=O)
(1971), Chirgadze et al.
(1975), Venyaminov and
Kalnin (1990)
Asn, nðC¼OÞ 1677–1678 (310–330) 1648 (570) Chirgadze et al. (1975), Up to 1704 cm1 observed
Venyaminov and Kalnin in proteins (Cao et al., 1993)
(1990), Rahmelow et al.
(1998)
Arg, nas ðCN3 Hþ
5Þ 1672–1673 (420–490) 1608 (460) Chirgadze et al. (1975), In proteins observed up to
Venyaminov and Kalnin 1695 cm1 (H2O) and down to
(1990), Rahmelow et al. 1595 cm1 (2H2O) (Chirgadze et al.,
(1998) 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), Dhamelincourt
and Ramirez (1993),
Rahmelow et al. (1998)
Arg, ns ðCN3 Hþ
5Þ 1633–1636 (300–340) 1586 (500) Chirgadze et al. (1975), In deuterated proteins observed
Venyaminov and Kalnin up to 10 cm1 lower (Chirgadze
(1990), Rahmelow et al. et al., 1975)
(1998)
145
continued overleaf
146
Table 1 (continued)
(e in M1 cm1 Þ in H2O (e in M1 cm1 ) in 2H2O
HisH+
2 , nðC¼CÞ 1631 (250) 1600 (35), 1623 (16) Chirgadze et al. (1975), Only one strong band observed for
Hienerwadel et al. (1997) 4-Methylimidazole at 1633 (H2O)
and 1605 cm1 (2H2O) (Hasegawa
et al., 2000)

Lys, das ðNHþ
3Þ 1626–1629 (60–130) 1201 Pinchas and Laulicht 2
H2O band position based on the
(1971), Venyaminov and shift observed for CH3NH3Cl and
Kalnin (1990), Rahmelow CH3N2H3Cl
et al. (1998)
Tyr–OH, n(CC) 1614–1621 (85–150) 1612–1618 (160) Chirgadze et al. (1975), Tyr or p-cresol, e estimated relative
d(CH) Dollinger et al. (1986), to 1517 cm1 band
Takeuchi et al. (1988),
(1990), Hienerwadel et al.
(1998)
Asn, dðNH2 Þ 1612–1622 (140–160) Venyaminov and Kalnin
(1998)
Trp, nðCCÞ, 1622 1618 Takeuchi and Harada
nðC¼CÞ (1986), Lagant et al.
(1998)
Tyr–O, nðCCÞ 1599–1602 (160) 1603 (350) Chirgadze et al. (1975), Tyr or p-cresol
Dollinger et al. (1986),
(1990), Hienerwadel
et al. (1997)
Tyr–OH, nðCCÞ 1594–1602 (70–100) 1590–1591 (550) Chirgadze et al. (1975), Tyr or p-cresol, e estimated relative
Dollinger et al. (1986), to 1517 cm1 band
(1990), Hienerwadel et al.
(1998)
Gln, dðNH2 Þ 1586–1610 (220–240) 1163 Venyaminov and Kalnin
(1990), Dhamelincourt
and Ramirez (1993),
Rahmelow et al. (1998)
HisH, nðC¼CÞ 1575,1594 (70) 1569, 1575 Venyaminov and Kalnin Doublet due to the two protonated
(1990), Hasegawa et al. tautomers of His
(2000)
Asp, nas ðCOO Þ 1574–1579 (290–380) 1584 (820) Chirgadze et al. (1975), May shift +60/40 cm1
Venyaminov and Kalnin (Tackett, 1989; Nara et al., 1994)
(1990), Rahmelow et al. upon cation chelation, in extreme
(1998) cases band position as for
n(C=O) (Deacon and Phillips,

1980)
Glu, nas ðCOO Þ 1556–1560 (450–470) 1567 (830) Chirgadze et al. (1975), See Asp nas (COO)
(1990)
Lys, ds ðNHþ
3Þ 1526–1527 (70–100) 1170 Pinchas and Laulicht 2
H2O band position based on the
(1971), Venyaminov and shift observed for CH3NH3Cl and
Kalnin (1990), Rahmelow CH3N2H3Cl
et al. (1998)
Tyr–OH, nðCCÞ, 1516–1518 (340–430) 1513–1517 (500) Chirgadze et al. (1975), Tyr or p-cresol
dðCHÞ Dollinger et al. (1986),
Rothschild et al. (1986),
(1990), Hienerwadel
et al. (1997), Rahmelow
et al. (1998)
Trp, nðCNÞ, dðCHÞ, 1509 Lautié et al. (1980), Indole IR spectrum
dðNHÞ Takeuchi and Harada
(1986)
Tyr–O, nðCCÞ, 1498–1500 (700) 1498–1500 (650) Chirgadze et al. (1975), Tyr or p-cresol
dðCHÞ Rothschild et al. (1986),
(1990), Hienerwadel
et al. (1997)
Trp, nðCCÞ, 1496 Takeuchi and Harada Trp Raman spectrum, observed in
dðCHÞ (1986), Lagant et al. the indole infrared spectrum at
(1998) 1487 cm1
Phe, nðCCringÞ 1494 (80) Venyaminov and Kalnin
(1990)
das ðCH3 Þ 1445–1480 Colthup et al. (1975)
continued overleaf
147
148
Table 1 (continued)

Trp, dðCHÞ, nðCCÞ, 1462 1455 (200) Lautié et al. (1980), e estimated from comparison with
nðCNÞ Takeuchi and Harada the 1517 cm1 Tyr band (Fabian
(1986), Fabian et al. et al. (1994)
(1994)
His, dðCH3 Þ, 1439 1439 Hasegawa et al. (2000) Observed for 4-Methylimidazole
nðCNÞ with a strong contribution of
d(CH3). Thus, position for His
may differ
Pro, nðCNÞ 1400–1465 Caswell and Spiro (1987), Sensitive to backbone confor-
Rothschild et al. (1989), mation (Johnston and Krimm, 1971;
Gerwert et al. (1990) Caswell and Spiro, 1987)
dðCH2 Þ 1425–1475 Colthup et al. (1975) Good group frequency, normally
at 1463 cm1. Near 1425 cm1
and more intense when next
to a C=O group (Colthup et al.,
1975)
Trp, dðNHÞ, nðCCÞ, 1412–1435 1382 Lautié et al. (1980), H2O: higher number for
dðCHÞ Takeuchi and Harada Raman spectrum of Trp, lower
(1986) number for IR imidazole
spectrum. 2H2O: Raman spectrum
of Trp
Gln, nðCNÞ 1410 1409 Dhamelincourt and
Ramirez (1993)
Glu, ns ðCOO Þ 1404 (316) 1407 Venyaminov and Kalnin See Asp ns(COO)
(1990)
Asp, ns ðCOO Þ 1402 (256) 1404 Venyaminov and Kalnin May shift +60/90 cm1
(1990) upon cation chelation (Tackett,
1989), in extreme cases band
position as for n(C–O) of COOH
group (Deacon and Phillips, 1980).
Band position in 2H2O estimated
from the shift observed for
CH3COO
ds ðCH3 Þ 1375 or 1368, 1385 Colthup et al. (1975) 1 band near 1375 cm1 for 1 CH3
group, 2 bands at 1368 and
1385 cm1 for 2 adjacent groups
(Val, Leu). The band is narrower
than the das (CH3) band but has the
same 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 Higher number for Raman
(1986), Lagant et al. spectrum of Trp, lower number for
(1998) IR imidazole spectrum.
Trp 1334–1342 1334 (100) Takeuchi and Harada H2O: higher number for Raman
(1986), Fabian et al. spectrum of Trp, lower number
(1994), Lagant et al. for IR imidazole spectrum. 2H2O:
(1998) IR spectrum of Trp in protein
dðCHÞ 1315–1350
Trp, dðNHÞ, 1276 Lautié et al. (1980) Indole IR spectrum
nðCNÞ, dðCHÞ
Tyr–O, nðC2OÞ, 1269–1273 (580) Dollinger et al. (1986), Tyr or p-cresol
nðCCÞ Venyaminov and Kalnin
(1990), Hienerwadel
et al. (1997)
Asp, Glu, dðCOHÞ 1264–1450 955–1058 Pinchas and Laulicht Hydrogen bonded (1058 and
(1971) 1450 cm1) and free (955
and 1264 cm1) CH3COOH
Trp, dðCHÞ, 1245 Lautié et al. (1980), Indole IR spectrum
nðCCÞ Takeuchi and Harada
(1986)
Tyr–OH nðC2OÞ, 1235–1270 (200) 1248–1265 (150) Dollinger et al. (1986), Tyr or p-cresol, band sensitive to H-
nðCCÞ Rothschild et al. (1986), bonding, 3–11 cm1 lower in 2H2O,
Takeuchi et al. (1988), e in 2H2O estimated from
Venyaminov and Kalnin comparison with the 1517 cm1
(1990), Gerothanassis band
et al. (1992), Hienerwadel
et al. (1997)
His, dðCHÞ, nðCNÞ, 1217, 1229, 1199 1217, 1223, 1239 Hasegawa et al. (2000) Values are for His, HisH and
dðNHÞ HisH+2 , respectively
149
continued overleaf
150
Table 1 (continued)
Trp, nðCCÞ 1203 Lautié et al. (1980) Indole IR spectrum

Ser, dðCOHÞ or 1181–1420 875–985 Pinchas and Laulicht Band position sensitive to hydrogen
dðCO2 HÞ, nðCOÞ (1971), Colthup et al. bonding

(1975), Madec et al.
(1978), Susi et al.
(1983)
gw ðCH2 Þ 1170–1382 Colthup et al. (1975) Couples with adjacent CH2
groups (Colthup et al., 1975).
Sensitive to hydrocarbon chain
conformation (Lewis and
McElhaney, 1996)
Tyr–OH, dðCOHÞ 1169–1260 (200) 913 Dollinger et al. (1986), Tyr or p-cresol, band sensitive to H-
Rothschild et al. (1986), bonding for OH group, 256 cm1
Takeuchi et al. (1988), lower for O2H group
Venyaminov and
Kalnin (1990),
Gerothanassis et al.
(1992), Hienerwadel
et al. (1997)
Asp, Glu, nðC2OÞ 1120–1253 1270–1322 Pinchas and Laulicht Range in H2O from band position in
(1971), Sengupta and aqueous solution near 1250 cm1
Krimm (1985), (Sengupta and Krimm, 1985;
Venyaminov and Kalnin Venyaminov and Kalnin, 1990) and
(1990) shift observed between hydrogen
bonded and free CH3COOH spectra
(Pinchas and Laulicht, 1971). Band
position in 2H2O for hydrogen
bonded and free CH3COOH. Asp
and Glu absorption may be 25 cm1
lower, since this is observed in
H2O
His, nðCNÞ, 1104,1090,1106,1094 1104, 1096, 1107, 1110 Noguchi et al. (1999) Values are for His, N1-, N3-
dðCHÞ protonated HisH and HisH+ 2 ,
respectively
Trp, dðCHÞ, 1092 Lautié et al. (1980), Indole IR spectrum
nðNCÞ Takeuchi and Harada
(1986)
Trp, nðNCÞ, dðCHÞ, 1064 Lautié et al. (1980), Indole IR spectrum
nðCCÞ Takeuchi and Harada
(1986)
gt ðCH2 Þ 1063–1295 Colthup et al. (1975) Weak, couples with adjacent CH2
groups, but the in phase mode at
1300 cm1 is a good group
frequency (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, 1012–1016 1012 Lautié et al. (1980),
dðCHÞ Takeuchi and Harada
(1986)
Ser, nðCOÞ or 983 Madec et al. (1978),
nðCCÞ Susi et al. (1983)
Ser, nðCOÞ, 940 Susi et al. (1983)
dðCO2 HÞ
Thr, dðCO2 HÞ 865–942 Pinchas and Laulicht
(1971)
gr ðCH2 Þ 724–1174 Colthup et al. (1975) Couples with adjacent CH2 groups,
the in phase mode at 724 cm1 is the
most intense (Colthup et al., 1975)
a
Listed are those side chain internal coordinates with the strongest contributions to the potential energy of the normal mode. Amide modes that
contribute 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 italic
print 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.
151
152
Table 2
CH3, CH2 and CH groupsa
Assignment Band position in cm1, (e in M1cm1) {1}
{1}
In Ala Cys Gln Glu Gly His Ile Leu Lys Pro Ser Thr Trp Tyr Val
general {3,4} {4} {5} {3} {3} {3} {4} {3}
{2}

das ðCH3 Þ 1465 1470 1445 1445 1480 1460
1465 1448
(100)
1455
dðCH2 Þ 1425– 1424 1451 1440 1441–1446 1465 1470 1445 1447–1472 1450 1447–1454
{6} 1475 1432 (120) 1467 (80)
(120)
ds ðCH3 Þ 1375 1378 1391

{7} (100)
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 (?)
a
Values are for solid samples or samples in H2O. Numbers in italics are for 2H2O. In general upon H2O/2H2O exchange, band shifts of 10 cm1
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 830 M1 cm1 (Chirgadze et al., 1975). {5} Band positions
according 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 M1 cm1 (Venyaminov
and Kalnin, 1990). Assignment is according to normal mode calculations (Lagant et al., 1983; Kakihana et al., 1988). The mode near 1335 cm1 may
contain a significant contribution of amide modes (Kakihana et al., 1988). Band positions in 2H2O are 1440–1446 cm1, 1322–1324 cm1 and
1020 cm1 (Lagant et al., 1983; Kakihana et al. (1988). {6} Good group frequency, normally at 1463; near 1425 cm1 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 das ðCH3 Þ band but the same intensity (Colthup et al., 1975). Insensitive to hydrocarbon chain conformation (Lewis and
McElhaney, 1996). {8} These vibrations are often coupled to other modes. gw ðCH2 Þ (1170–1382 cm1) 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 cm1) gives weak
bands, couples with adjacent CH2 groups, but the in phase mode at 1300 cm1 is a good group frequency (Colthup et al., 1975). gr (CH2)
(724–1174 cm1) couples with adjacent CH2 groups, the in phase mode at 724 cm1 gives the most intense infrared bands (Colthup et al., 1975).
Only two side chain moieties absorb in spectral regions that are free from overlapping
absorption by other groups and thus allow the spectroscopist an unambiguous assignment
without further experiments. These are the SH group of Cys (2550–2600 cm1) and the carbonyl
group of protonated carboxyl groups (1710–1790 cm1). The latter proved to be particularly
useful when protonation and deprotonation of carboxyl groups is of interest, for example when
proton 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 the
polypeptide backbone and further experiments are needed to assign an absorption band to a
specific side chain moiety. These include H/2H exchange of the NH, OH and SH groups which
shifts the absorption bands in a characteristic way, uniform isotopic labelling of one type of
amino acids, site-directed isotope labelling of one specific amino acid in a protein and the use of
site-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 experimentally
straightforward H/2H exchange does not always help because there may be too many changes to
the 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 one
amino acid side chain by another and therefore may result in complicated alterations to the
spectrum.
2.2. Aliphatic side chain groups
Fig. 1 shows the structure of the aliphatic amino acids. The aliphatic moieties of amino acid
side chains give rise to several absorbance bands of medium to weak intensity which are compiled
in Table 2. While the das ðCH3 Þ, the dðCH2 Þ and the ds ðCH3 Þ vibrations near 1465, 1450 and
1375 cm1 are relatively good group frequencies, the d(CH) and g(CH2) vibrations are often
coupled to other modes. The frequency of the d(CH2) vibration of Asp and Glu residues is
expected to be sensitive to the protonation state and the ds(CH3) vibration to the branching of the
hydrocarbon chain (Colthup et al., 1975). Of the aliphatic amino acids only Pro bands are listed
separately in Table 3. Fig. 1 shows the structure of Pro. Pro is remarkable in that is does not form
the 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 near
1430 cm1 is sensitive to backbone conformation (Johnston and Krimm, 1971) and has been
identified 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.

Table 3
Proline side chaina
Assignment {1} Pro in H2O {2} Pro in 2H2O {3} Pro in proteins {4}
Band position (cm1) Band position, band shift (cm1) Band position (cm1)
dðCH2 Þ 1472 s 1472, 0
dðCH2 Þ {5} 1447–1472 s 1456
nðCNÞ {6} 1435–1465 1400–1454
dðCHÞ 1375 s, broad
gt ðCH2 Þ 1340 sh 1328, 12
dðCHÞ 1317 s 1306, 11
gt ðCH2 Þ 1292 s 1296, +4
gw ðCH2 Þ 1253 m 1253, 0 and 1262, +9
gt ðCH2 Þ 1168 s 1168, 0
gr ðCH2 Þ 1083 m 1090, +7
gw ðCH2 Þ 1051 w 1050, 1
gw ðCH2 Þ 1033 m
nðCNÞ, nðCCÞ 979 m 981, +2
945 m 930, 15
911 m 902, 9
a
A remark to the nðC¼OÞ backbone absorption: Proline absorption is unusual compared to the amide absorption of
other amino acids. It absorbs at 1623 cm1 in an extended left-handed poly-Pro helix when the carbonyl groups are
H-bonded, if not at 1640 cm1 (Lazarev et al., 1985). In 2H2O and in unordered peptide chains the backbone
absorbance is at 1620–1623 cm1 (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 also
legend of Table 1. {2} Solid l-proline (Herlinger and Long, 1970) or l-proline film (Rothschild et al., 1989), poly-Pro in
H2O (Raman) (Caswell and Spiro (1987), band positions in aqueous solution are expected to be within 10 cm1 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 positions
for 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 cm1 lower in [2H7]Pro (Rothschild
et al., 1989) no (Rothschild et al., 1989) or –15 to –20 cm1 (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 backbone
conformation: Raman bands are observed at 1435 cm1 for cis poly-Pro and at 1465 cm1 for trans poly-Pro (Caswell
and Spiro, 1987, and the frequency depends upon the Ca–C0 (=O) angle (Johnston and Krimm, 1971). 15 cm1
downshift for [15N]Pro (Rothschild et al., 1989; Gerwert et al., 1990).
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 its
interesting properties: Tyr may take part in proton and electron transfer reactions (Dollinger
et al., 1986; Rothschild et al., 1986; Roepe et al., 1987; Hienerwadel et al., 1997). The pKa value
in solution is 10.1 but may differ considerably from this value in proteins. Tyr is a relatively strong
infrared absorber due to its polar character and its bands are listed in Table 4. The most intense
bands originate from a n(CC), the nðC2OÞ and the d(COH) mode near 1517, at 1235–1270 and at
1169–1260 cm1. The latter two are sensitive to H-bonding and merge to one broad band near
1250 cm1 in H2O (Hienerwadel et al., 1997). The ring mode near 1517 cm1 is easily detected in
protein absorbance spectra, in particular when band narrowing procedures like Fourier self-
Fig. 2. Structure of the aromatic amino acids. Fig. 3. Structure of Ser, Thr and Cys.
deconvolution (Kauppinen et al., 1981; Mantsch et al., 1988), 2nd derivative or fine-structure
enhancement (Barth, 2000) are used which intensify the very narrow Tyr band. Besides its small
band width, the small downshift of 2 cm1 in 2H2O is characteristic. The band can be used as
marker band for the protonation state of Tyr, since it is downshifted by 15 cm1 in the
deprotonated state. This state is more polar than the neutral state and thus increases the intensity
of several bands, in particular of the nðC2OÞ vibration.
Phe due to its apolar character is only weakly absorbing infrared radiation. However, a weak
band at 1498 cm1 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 and
1455 cm1. They have been detected in an elegant study where a difference spectrum could be
generated that showed the effect on the Ribonuclease T1 spectrum replacing two Tyr residues by
two Trp residues. To generate the difference spectrum, the absorbance spectrum of point mutant
W59Y was carefully subtracted from that of mutant Y45W (Fabian et al., 1994). The spectral
range covered here was 1300–1700 cm1.
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 cm1
range. As observed for Tyr, they are sensitive to H-bonding. Those vibrations with large
contributions of the stretching vibration of the polar C–O bond have relatively strong infrared
intensities.
For Cys (see Table 9), the large mass of the sulphur atom shifts the n(SH) vibration into
a spectral range that is free from overlap by other side chain modes. This has enabled its detection
for 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 are
shown in Fig. 4. The imidazole group of His has two nitrogen atoms, which can be protonated or
156
Table 4
Tyrosine 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 Band position, Band position Band position, Band position in cm1
cm1 band shift in cm1 in cm1 band shift in cm1
(e in M1 cm1 ) (e in M1 cm1 ) (e in M1 cm1 ) (e in M1 cm1 )
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) 1590–1597
nðCCÞ ring, dðCHÞ 1516–1518 (340–430) 1513–1517, 1 to 2 (500) 1498–1500 (700) 1498–1500, 2(?) (650) 1510–1516, 1516–1518
dðCH2 Þ 1447–1454 (80) 1442, 5 (50) 1443 (70) 1456
nðCCÞ ring, dðCHÞ 1415–1416 (10) 1425, +10 (20)
gw ðCH2 Þ 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) 1267–1277
dðCHÞ 1170–1179 (50) 1169, 1 (30) 1174 (150)
dðCOHÞ 1169–1260 (200) {7} 913, 256 1228–1250
dðCHÞ 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 vibrations
according 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 estimated
from absorbance spectra (Rothschild et al., 1986; Hienerwadel et al., 1997) by comparing band intensities to the intensity of the 1517 cm1 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 been
investigated 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 cm1 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). Extinction
coefficient (in brackets) according to Venyaminov and Kalnin (1990) or estimated from absorbance spectra (Hienerwadel et al., 1997) by comparing
band intensities to the intensity of the 1499 cm1 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 cm1 band is indicated with a question mark
since it compares a Tyr spectrum in H2O with a poly-l-Tyr spectrum in 2H2O (Rothschild et al., 1986). {6} Ribonuclease T1 (1516, 1612 cm1)
(Fabian et al., 1994) and calmodulin in 2H2O (1516–1518 cm1) (Berendzen and Braunstein, 1990; Fabian et al., 1996), bacteriorhodopsin (Dollinger
et 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 cm1. Both bands merge to one broad band near 1250 cm1 in
H2O. nðCOÞ shifts to higher frequencies when the COH group is a H-bond donor (Hienerwadel et al., 1997), dðCOHÞ absorbs at 1210 to 1225 cm1
when the group is an H-bond donor and at 1235 to 1245 cm1 when it is both an H-bond donor and an acceptor (Gerothanassis et al., 1992).
Table 5
Phenylalanine side chaina
Assignment {1} Phe in H2O {2} Phe in 2H2O {3} Phe in proteins {4}
Band position in cm1 Band position in cm1 Band position in cm1
(e in M1 cm1 ) (e in M1 cm1 )
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 (spectral
range covered: 1800–1450 cm1) (Venyaminov and Kalnin, 1990) or monosubstituted benzenes (Colthup et al., 1975).
{3} Phe in 2H2O (spectral range covered: 1800–1500 cm1) (Chirgadze et al., 1975). {4} Calmodulin in 2H2O (Berendzen
and Braunstein, 1990; Fabian et al., 1996).
deprotonated with pKa values of 6 and 14 or coordinated to metal ions. In the fully deprotonated
imidazolate 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 tautomeric
forms in which either N1 or N3 is protonated. In aqueous solution the ratio between
the 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, the
fully protonated imidazolium cation is formed. There are several useful marker bands in
the infrared spectrum for the protonation state of the imidazole group (see Table 10). In
particular, the highest frequency band of the nðC¼CÞ vibration produces a shift of more than
30 cm1 upon formation of the fully protonated imidazolium cation (Hasegawa et al., 2000). Its
sensitivity 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 cm1 band
for 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 its
use as marker band for His deprotonation. Another useful band is the n(CN) band near 1100 cm1
(Noguchi et al., 1999; Hasegawa et al., 2000), in particular when its sensitivity towards H2O/2H2O
exchange is taken into account. In the fully protonated form HisH+ 2 it is most sensitive, whereas
in the deprotonated form His it is unaffected. Both marker bands can also serve to distinguish
between the N1- and the N3-protonated tautomer (see Table 10). They have been identified in
protein infrared difference spectra of photosystem II at 1617 cm1 on the basis of 13C labelling
(Hienerwadel et al., 1997) and at 1094–1113 cm1 as compiled in Noguchi et al. (1999).
In Arg and Lys the functional group is linked to the backbone by a relatively long aliphatic
spacer of 3 or 4 methylene groups, respectively (see Fig. 4). The guanidyl group of Arg leads to
two relatively strong bands near 1633 and 1672 cm1 which exhibit strong shifts of 50–70 cm1
upon deuteration (see Table 11). These shifts distinguish Arg bands on the one hand from other
side chains absorbing in that region and on the other hand from amide I absorption of the
polypeptide backbone.
Table 6
Tryptophan side chaina
Assignment {1} Indole–NH {2} Trp–NH {3} Trp–N2 in H {4} Trp in proteins {5}
Band position Band position Band position, Band position in cm1
in cm1 in cm1 band shift in cm1
nðCCbÞ, nðC¼CpÞ 1612 m 1622 1618, 4
nðCCbÞ, nðC¼CpÞ 1576 w 1578–1579 1574, 5
nðCCbÞ, dðCHbÞ 1552–1555 1545–1550, 2 (10)
nðCNÞ, dðCHpÞ, dðNHÞ 1509 s
nðCCbÞ, dðCHbÞ 1487 s 1496
dðCHbÞ, nðCCbÞ, nðCNÞ 1455 vs 1462 1455 (200) 1455
dðNHÞ, nðCCpÞ, dðCHbÞ 1412 s 1435 1383, –52
n(CCp), n(CN), d(CHb) {6} 1352 vs 1360–1361 1352–1353, 8
or d(CHb), gw (CH2),
d(CHp) {1b}
nðCCpÞ, nðCNÞ, 1334 vs 1342 1334, 8 (100) 1334
dðCHbÞ {1a,6}
or gw ðCH2 Þ, nðCCH2 Þ {1b}
nðCCbÞ 1305
dðNHÞ, nðCNÞ, dðCHbÞ 1276 s
dðCHbÞ, nðCCpÞ 1245 s
gt ðCH2 Þ, dðCHbÞ, dðCHpÞ 1238
nðCCbÞ, nðCCpÞ 1203 m
nðCCbÞ, nðCCpÞ, dðCHbÞ 1191 w
dðCHbÞ 1147 w
dðCHbÞ, nðCCbÞ 1119 m 1127
dðCHpÞ 1092 vs
nðNCÞ, dðCHpÞ 1064 s
nðCCbÞ, dðCHbÞ 1010 s 1012–1016 1012, 0
970 sh
930 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} Band
positions and relative intensities according to infrared spectra of indole (Lautié et al., 1980). {3} According to Raman
spectra 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 cm1) of Trp in
aqueous solutions, Trp in Ribonuclease T1 (1455, 1334 cm1) (Fabian et al., 1994). Extinction coefficient (in brackets)
from the infrared spectrum of a Trp solution in 2H2O (1550 cm1) (Chirgadze et al., 1975) or estimated from the band
intensities relative to the Tyr 1515 cm1 band in the difference spectrum that shows the effect of replacing two Tyr by
two Trp in Ribonuclease T1 mutants (1334 and 1455 cm1) (Fabian et al., 1994). {5} Ribonuclease T1 in 2H2O (Fabian
et al., 1994). {6} The 1334 and 1352 cm1 bands form a Fermi resonance doublet according to Takeuchi and Harada
(1986).
The Lys side chain amino group gives rise to only weak infrared bands, in particular in
the neutral state (Table 12). Two vibrations near 1526 and 1626 cm1 can be assigned to the
asymmetric and symmetric deformation vibration of the NH+ 3 group.
Table 7
Serine side chaina
Assignment {1} Ser–OH {1} Ser–O2 H {2}
Band position in cm1 Band position,
band shift in cm1
dðCH2 Þ 1450 1467, +2
dðCHÞ, nðCCÞ {1a} or gw ðCH2 Þ {1b} 1364 1375, +6 (100)
gt (CH2), ns (COO), d(CH) {1a} or d(CH) {1b} 1352 1340, 12 (100)
gw ðCH2 Þ, dðCHÞ {1a} or dðCHÞ {1b} 1312
gt ðCH2 Þ {1b} 1248 {1b}
In H2 O: dðCOHÞ, in 2H2 O: dðCO2 HÞ, nðCOÞ 1181 {1b} 875–985, 263{4}
or 1248 {1a} – 1420 {3}
nðCOÞ, gr ðNHþ
3Þ 1030 s {5} 1023, 7
nðCOÞ, gr ðNHþ
3 Þ, nðCCamideÞ {1a} 983 {1a}
or nðCCÞ {1b}
nðCOÞ, dðCO2 HÞ 940
a
{1} According to Raman spectra of l-serine crystals and normal mode analysis {1a} (Susi et al., 1983) and IR
spectra 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 comparison
with the nas ðCOO Þ band which has an e of 830 M1 cm1 (Chirgadze et al., 1975). {3} Band is expected at 1420 cm1
(broad, weak) if the –CH2–OH group is hydrogen bonded (Pinchas and Laulicht, 1971; Colthup et al., 1975). {4} Band
is expected at 875 cm1 if the –CH2–OH group is not hydrogen bonded (Pinchas and Laulicht, 1971), it is observed at
985 cm1 in crystals (Susi et al., 1983). {5} Intensity according to Colthup et al. (1975), band slightly sensitive to
hydrogen bonding (Colthup et al., 1975).
Table 8
Threonine side chaina
Assignment {1} Thr in H2O {2} Thr in 2H2O {3}
Band position in cm1 Band position in cm1
das ðCH3 Þ 1448 m, 1458 sh, 1480 sh
ds ðCH3 Þ 1391 w
1360 w
dðCOHÞ, dðCHÞ 1385–1420 mw {4}
dðCOHÞ, dðCHÞ 1225–1330 mw {4}
nðCOÞ 1075–1150 s
910–950 {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 aliphatic
alcohols (Colthup et al., 1975), relative intensities are from ethanol spectra (not shown). {3} Poly-l-Thr in 2H2O
(spectral range covered: 1800–1300 cm1) (Kubota and Fasman, 1975). The position of the dðCOHÞ band was taken
from 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} Weaker
than the nðC2OÞ band.
Table 9
Cysteine side chaina
Assignment {1} Cys–SH {2} Cys–S2 H {2} Cys in proteins {3}
Band position in cm1 Band position, Band position in cm1
band shift in cm1
(e in M1 cm1 )
nðSHÞ or nðS2 HÞ 2551 1849, 702 2550–2592, 1854–1861
dðCH2 Þ 1424 1432, +2 (120)
d(CH), ns (COO), 1303 1341, +27 (90)
gt (CH2)
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 cm1) (Wright and Vanderkooi, 1997). Only those Raman bands were listed for H2O where
the corresponding bands upon deuteration matched closely the infrared bands in 2H2O. Band shifts upon deuteration
are from Raman spectra of l-cysteine crystals (Susi et al., 1983) using the bands of the deuterated crystals at 1285, 1330
and 1426 cm1 that matched the infrared bands within 12 cm1. e estimated from infrared spectra of cysteine in 2H2O
(Wright and Vanderkooi, 1997) by comparison with the nas ðCOO Þ band which has an e of 830 M1 cm1 (Chirgadze
et 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.
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 absorption
bands of Asn and Gln, respectively. The nðC¼OÞ vibration of Asn and Gln side chains near
1680 cm1 is a relatively strong infrared absorber. Due to coupling with the d(NH2) vibration
it shows a strong sensitivity towards deuteration. The downshift of 30 cm1 is considerably larger
than that of the amide I bands of the protein backbone and helps distinguish the amide side chain
absorption from the amide backbone absorption. If spectra are recorded under identical
conditions, Asn absorbs 10 cm1 lower than Gln due to intramolecular interactions (Venyaminov
and Kalnin, 1990). nðC¼OÞ bands are sensitive to H-bonding and the band position is the lower
the stronger the H-bond is. The observation of an Asn band at 1704 cm1 in a bacteriorhodopsin
mutant (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 cm1 gives rise to
weaker absorption bands. Upon deuteration they are shifted by several hundred cm1 to
1160 cm1.
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 vibrational
spectroscopist with a unique monitor to follow proton pathways in proteins. An intensely studied
example is the proton pump bacteriorhodopsin (Rothschild, 1992; Maeda, 1995; Gerwert, 1999;
Heberle, 1999). Of particular interest here is the nðC¼OÞ vibration of the protonated carboxyl
group that has the particular advantage of absorbing in a spectral region that is generally free
from overlap by other amino acid absorptions (1710–1760 cm1). Overlap may occur however
with the nðC¼OÞ vibration of lipids. The nðC¼OÞ vibration is sensitive to H-bonding with shifts of
approximately 50 cm1 observed upon formation of strong hydrogen bonds. The deprotonated
carboxylate group shows two strong bands near 1400 and 1570 cm1 for the symmetric and the
antisymmetric stretching vibration, respectively. The band position of these may shift
considerably 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 and
this 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 of
the carboxylate group coordinates the cation), the two CO bonds are no longer equivalent, with
the bond of the chelating oxygen having more single bond character and that of the non-chelating
oxygen more double bond character.
3. Outlook
As the discussion above has shown, the infrared spectrum encodes a lot of important
information on amino acid side chains, like protonation state, charge, accessibility to H-
bonding partners and conformational freedom. Often marker bands can be found that
monitor these properties in the course of a protein reaction. The information deduced
from the infrared spectra is thus part of the jigsaw puzzle that builds up the complete
molecular picture of the protein reaction mechanism. As there is an increasing number of
infrared spectroscopic techniques to monitor protein reactions (Mäntele, 1993; Backmann et al.,
1995; Siebert, 1995; White et al., 1995; Gerwert, 1999; Barth and Zscherp, 2000), it can be
expected that its application range will constantly widen. Thus infrared spectroscopy will
continue to provide important contributions to the understanding of side chain action in protein
function.
162
Table 10
Histidine side chaina

Assignment {1} His in H2O HisH {3} His2 H {3} HisH+
2 {4} His2H+
2 {4} His in proteins {5}
and 2H2O {2}
Band position Band position Band position, Band position Band position, Band position in cm1
in cm1 in cm1 band shift in cm1 band shift
(e in M1 cm1 ) (e in M1 cm1 ) in cm1 (e in M1 cm1 ) in cm1
nðC¼CÞ, nðCCÞ 1575 s, 1594 s (70) 1569, 6 s 1631 (250) vs {13} 1600 (35), 1617 (?)
{6} and 1575, 1623 (16) {11}
19 m {6}
nðC¼NÞ, dðCHÞ 1490 s 1485, 5 s
dðCHx Þ, nðCNÞ 1439 vs 1408 m
dðNHÞ, nðCNÞ, ds ðCHx Þ 1423 m
nðCNÞ 1363, 1369 m {6} 1366 m
nðC¼NÞ, nðCNÞ 1304 m {7}{9} 1305, +1 m {7}
dðCHÞ, nðCNÞ 1255 m
nðCNÞ, nðCCÞ {1a,c} 1300 m 1265 s {8} 1259, 6 s {7} 1267 m
or gt ðCH2 Þ,
dðCHÞ, n ring {1b}
nðCNÞ 1232 m
dðCHÞ, nðCNÞ, dNH, 1217 s 1229 s 1223, 6 s {7} 1199 m 1239 m
nðCCÞ {12}
nðCNÞ, dðNHÞ 1153 m, 1161 m {10} 1182 m
nðCNÞ, dðCHÞ 1104 s {14} 1090 s, 1106 s {7} 1096,+6 m 1094 s {14} 1110, +16 s {14} 1094–1114,
{14} and 1107, 1093–1114
+1 s {7}{14}
d ring, nðC¼CÞ 1010 m
dðCHÞ {1a} or nðCCÞ, 995 m {7} 1017, +22 s {7} 1022 m
nðNCÞ, dðCHÞ {1b}
or gr ðCHx Þ, nðC¼CÞ {1c}
nðCNÞ, dðCHx Þ, d ring 975 m 978, +3 m 973 m
d ring {1b,c}, nðCCÞ {1b} 950 m 941 s 943, +2 s 925 m
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 legend
of Table 1. {2} 4-Methylimidazole in NaOH and NaO2H (Hasegawa et al., 2000) for all bands except for the 1104 cm1 band which was observed for
dl-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 the
bands at 1100 cm1 which are for dl-His (Noguchi et al., 1999). Relative intensities are from Hasegawa et al., (2000), e from the respective band of
imidazole in H2O at 1596 cm1 (Venyaminov and Kalnin, 1990). {4} Band positions and relative intensities are for 4-Methylimidazole in HCl or

2
HCl 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 cm1 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 (Ashikawa
and Itoh, 1979; Hasegawa et al. 2000). {8} 7 cm1 variation depending on which nitrogen atom is protonated (Ashikawa and Itoh, 1979; Hasegawa
et al., 2000). The higher frequency band at 1265 cm1 for the N1 protonated tautomer has a higher intensity in the infrared spectrum (Hasegawa
et al., 2000). {9} 2035 cm1 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, lower
frequency band for N3 protonated tautomer (Hasegawa et al., 2000). {11} Two bands with very weak intensity observed for His at 1600 and
1623 cm1 (Chirgadze et al., 1975), only one very strong band for 4-Methylimidazole at 1605 cm1 (Hasegawa et al., 2000). {12} dðNHÞ contribution
calculated only for HisH+ +
2 in Hasegawa et al. (2000) or for HisH and HisH2 in Ashikawa and Itoh (1979), nðCCÞ only for N3-protonated
1 13
4-Methylimidazole (Hasegawa et al., 2000). {13} Shifts 38 cm upon C labeling (Hienerwadel et al., 1997). The respective band of
4-Methylimidazole is at 1633 cm1 (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 (MeImH+ 2 +
2 ) and 1106 (MeIm H2 ).
163
Table 11
Arginine side chaina
Assignment {1} Arg {2} Arg in 2H2O {3} Arg in proteins {4}
nas ðCN3 Hþ5Þ 1672–1673 (420–490) 1608 (460) 1688–1695, 1595–1605

ns ðCN3 Hþ5Þ 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 of
Table 1. {2} Arg in H2O (1800–1500 cm1) (Venyaminov and Kalnin, 1990; Rahmelow et al., 1998). {3} Arg in
2
H2O (1800–1500 cm1) (Chirgadze et al., 1975). {4} Observed for deuterated lysozyme and a-Keratin (two bands at
1576–1577 and 1595–1600 cm1) (Chirgadze et al., 1975), deuterated calmodulin (1605 cm1) (Berendzen and
Braunstein, 1990), halorhodopsin (1688–1695 cm1) in H2O (Rudiger et al., 1995).
Table 12
Lysine side chaina
Assignment {1} Lys–NH+ 3 {2} Lys–N2H+ 3 {3} Lys in proteins {4}
(e in M1 cm1 ) band shift in cm1
das ðNHþ3Þ 1626–1629 (60–130) 1201, 428

ds ðNHþ3Þ 1526–1527 (70–100) 1170, 356
dðCH2 Þ 1445
gw ðCH2 Þ; gr ðCH2 Þ 1325,1345
a
{1} Assignments are according to Pinchas and Laulicht (1971) and Venyaminov and Kalnin (1990). See also legend
of Table 1. In the neutral form the absorption of the e-NH2 group is weak with extinction coefficients less than
40 M1 cm1 between 1800 and 1500 cm1 (Venyaminov and Kalnin, 1990). {2} Lys in H2O (1800–1500 cm1)
(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 13
Asparagine side chaina
Assignment {1} Asn–NH2 {2} Asn–N2H2 {3} Asn in proteins {4}
nðC¼OÞ 1677–1678 (310–330) {5} 1650 (570) 1704
dðNH2 Þ 1612–1622 (140–160) 1622 (?)
a
{1} Assignments are according to Chirgadze et al. (1975) and Venyaminov and Kalnin (1990). See also legend of
Table 1. {2} Band positions and extinction coefficients (in brackets) of Asn in H2O according to Venyaminov and
Kalnin (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 bacteriorhodopsin
mutant in H2O (Cao et al., 1993). {5} 10 cm1 lower than for Gln due to intramolecular interactions in Asn
(Venyaminov and Kalnin, 1990).
Table 14
Glutamine side chaina
Assignment {1} Gln–NH2 {2} Gln–N2H2 {2} Gln in proteins {3}
(e in M1 cm1 ) band shift in cm1
(e in M1 cm1 )
nðC¼OÞ 1668–1687 (360–380) s 1635–1654, 33 (550) 1659–1696
dðNH2 Þ 1586–1611 (220–240) m 1163, 410 m
dðCH2 Þ 1451 m 1453, +2 m
dðCH2 Þ 1442 m
nðCNÞ 1410 s 1409, 1 ms
dðCHÞ 1359 w 1361, +2 wm
dðCHÞ 1333 ms 1333, 0 ms
gw ðCH2 Þ 1315 m 1317, +2 m
gw ðCH2 Þ 1281 wm 1279, –2 wm
gt ðCH2 Þ 1256 wm 1257, +1 w
gt ðCH2 Þ 1202 w 1204, +2 w
gr ðNH2 Þ 1104 w 810, 296 w
nðCNÞ 1084 w 1071, 13 vw
nðCCÞ 1052 m 1040, 12 w
nðCCÞ 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 the
1586–1611 cm1 bands which are from spectra of Gln in aqueous solution (Venyaminov and Kalnin, 1990; Rahmelow
et 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).
Fig. 6. Structure of Asp and Glu.
Table 15
Aspartate side chaina
Assignment {1} Asp–COO Asp–COO Asp–COOH {4} Asp–COO2 H {5} Asp in proteins {6}
in H2O {2} in 2H2O {3}
Band position Band position, Band position Band position, Band position
in cm1 band shift in cm1 in cm1 band shift in cm1 in cm1
nðC ¼ OÞ {7} 1716–1788 (280) 1713–1775, 1732–1762
13 (290)
nas ðCOO Þ {8} 1574–1579 1584–1586, 1578–1595
(290–380) +9 (820)
ns ðCOO Þ {9} 1402 (256) 1404, +2 (450) 1392–1425
dðCOHÞ 1264–1450 955–1058,
309 mw
nðC2OÞ {10} 1120–1250 1270–1322,
(100–200) +88 m
a
{1} According to Pinchas and Laulicht (1971), Chirgadze et al. (1975), Colthup et al. (1975) and Venyaminov and
Kalnin (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 cm1 is for Asp in
2
H2O (Chirgadze et al., 1975; Nara et al., 1994; Wright and Vanderkooi, 1997) and at 1404 cm1 estimated from the
shift observed for sodium acetate (Tackett, 1989). e of the 1404 cm1 band estimated from the comparison with the
nas ðCOO Þ band which has an e of 830 M1 cm1 (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 cm1), hydrogen bonded (1450 cm1, spectra not shown) and free CH3COOH (1264,
1788 cm1) (Pinchas and Laulicht, 1971). Range for the 1120–1250 cm1 band estimated from the band observed for
Asp in H2O at 1250 cm1 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 cm1 (data not
shown). {5} Asp in 2H2O (Chirgadze et al., 1975) (1713 cm1), hydrogen bonded (1058, 1322 cm1) and free
CH3COO2H (955, 1270, 1775 cm1) (Pinchas and Laulicht, 1971). The frequency of the nðC2OÞ band may be 25 cm1
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 cm1) (Fahmy et al., 1993; Sasaki et al., 1994), pike parvalbumin in 2H2O
(1584 cm1) (Nara et al., 1994), calmodulin in 2H2O (1584 cm1) (Fabian et al., 1996), Ca2+ free form of a-lactalbumin
at 1585 cm1 (2H2O) and 1393 cm1 (H2O), Ca2+-loaded form of a-lactalbumin at 1578 and 1592 cm1 (2H2O), and at
1406 and 1425 cm1 (H2O) (Mizuguchi et al., 1997a), Ca2+-loaded form of lysozyme at 1578 and 1595 cm1 (2H2O),
and at 1403 and 1423 cm1 (H2O) (Mizuguchi et al., 1997b). {7} Sensitive to H-bonding: –25 cm1 shift upon formation
of a single H-bond, above 1740 cm1 inverse correlation of nðC¼OÞ and the dielectric constant e (Dioumaev and
Braiman, 1995). For esters, hydrogen bonding not only to the carbonyl oxygen but also to the ether oxygen was found.
Table 15 ( footnote continued )
Both exhibit opposite effects on the nðC¼OÞ band position, with the latter leading to an upshift of 10 cm1 (Maes and
Zeegers-Huyskens, 1983; Maes et al., 1988). While an interaction like this is not observed in aqueous solution, it may
occur 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 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 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 in
aqueous solution. For the monomer a shift of 13 cm1 was found (Pinchas and Laulicht, 1971) but only a shift of
3 cm1 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 cm1 (Fahmy et al., 1993). {8} May shift +60/40 cm1
upon cation chelation (Tackett, 1989; Nara et al., 1994). In extreme cases, a band position as for nðC¼OÞ of the COOH
group 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 band
position indicating a lower pKa (Wright and Vanderkooi, 1997). {9} Band position in 2H2O estimated from the shift
observed for sodium acetate (Tackett, 1989). May shift +60/90 cm1 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 2H2O
estimated from the comparison with the nas ðCOO Þ band which has an e of 830 M1 cm1 (Chirgadze et al., 1975) using
spectra of sodium acetate (Tackett, 1989; Wright and Vanderkooi, 1997). 20 cm1 shift upon 13C labelling (Fahmy
et al., 1993). {10} For esters it was found that a hydrogen bond to the carbonyl oxygen leads to an upshift of 21 cm1
and a hydrogen bond to the ether oxygen to a downshift of 17 cm1 (Maes and Zeegers-Huyskens, 1983).
Table 16
Glutamate side chaina
Assignment {1} Glu–COO Glu–COO Glu–COOH {4} Glu–COO2H {5} Glu in proteins {6}
in H2O {2} in 2H2O {3}
nðC¼OÞ {7} 1712–1788 (220) 1706–1775,
13 (280)
nas ðCOO Þ {8} 1556–1560 1567–1568, 1553–1575
(450–470) +9 (830)
dðCH2 Þ 1452 sh 1451 w
dðCH2 Þ 1440 s 1417 m
ns ðCOO Þ {9} 1404 (316) 1406, +2 (450) 1397–1424
gw ðCH2 Þ, 1388 w
dðCHÞ, nðCCÞ
gw ðCH2 Þ 1359 w
gt ðCH2 Þ, gw ðCH2 Þ 1343 sh
gt ðCH2 Þ 1323 m
gw ðCH2 Þ, dðCHÞ 1313 w
dðCHÞ, dðNHÞ 1283 w
gw ðCH2 Þ 1292 w
dðCOHÞ 1264–1450 955–1058, 309 mw
nðNCÞ, dðCHÞ 1260 mw
nðC2OÞ {10} 1120–1253 1270–1322, +88 m
(100–200) ms
Table 16 (continued)
Assignment {1} Glu–COO Glu–COO Glu–COOH {4} Glu–COO2H {5} Glu in proteins {6}
in H2O {2} in 2H2O {3}
gt ðCH2 Þ, dðCHÞ 1225 sh
gw ðCH3 Þ 1212 w
gt ðCH2 Þ, dðCHÞ 1187 mw 1181 w
gt ðCH2 Þ, dðCHÞ 1130 m
nðNCÞ, nðCCÞ 1118 m
nðCCÞ 1074 vw 1083 w
nðCCÞ 1040 vw 1060 w
nðCCÞ 1018 vw
1028 w
nðCCÞ 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 of
Table 1. {2} Glu in H2O (Venyaminov and Kalnin, 1990; Rahmelow et al., 1998) (1560 and 1404 cm1) and poly-
l-glutamate (Sengupta and Krimm, 1984). {3} Band position and extinction coefficient of the 1567 cm1 band are for
Glu in 2H2O (Chirgadze et al., 1975; Nara et al., 1994; Wright and Vanderkooi, 1997). Band position at 1406 cm1
estimated from the shift observed for CH3COO in H2O and 2H2O (Tackett, 1989), e of this band estimated from the
comparison with the nas ðCOO Þ band which has an e of 830 M1 cm1 (Chirgadze et al., 1975) using spectra of sodium
acetate (Tackett, 1989; Wright and Vanderkooi, 1997). Band shifts upon H2O/2H2O exchange as observed for
CH3COO (Tackett, 1989). {4} Band positions are for poly-l-glutamate (Sengupta and Krimm, 1984), Glu in H2O
(Venyaminov and Kalnin, 1990) (1712 and 1250 cm1), hydrogen bonded (1450 cm1, spectra not shown) and free
CH3COOH (1264, 1788 cm1) (Pinchas and Laulicht, 1971). Range for the 1120–1253 cm1 band estimated from the
band observed for Glu in H2O at 1250 cm1 or poly-l-Glu at 1253 cm1 and from the shift upon hydrogen bonding
observed for hydrogen bonded and free CH3COOH (Pinchas and Laulicht, 1971). {5} Glu in 2H2O (Chirgadze et al.,
1975) (1706 cm1), hydrogen bonded (1058, 1322 cm1) and free CH3COO2H (955, 1270, 1775 cm1). The frequency of
the nðC2OÞ band may be 25 cm1 lower for Glu 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} 1553–1575 cm1 band: pike parvalbumin with bidentate Ca2+ coordination (1553 cm1) or
unidentate Mn2+ coordination (1575 cm1) (Nara et al., 1994), Ca2+ loaded calmodulin (1555 cm1) (Fabian et al.,
1996) in 2H2O. 1397–1424 cm1 band: Asp or Glu of pike parvalbumin in 2H2O with bound Ca2+, Mn2+ or Mg2+. {7}
Sensitive to H-bonding: –25 cm1 shift upon formation of a single H-bond, above 1740 cm1 inverse correlation of
nðC¼OÞ and the dielectric constant e (Dioumaev and Braiman, 1995). For esters, hydrogen bonding not only to the
carbonyl 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 cm1 (Maes and Zeegers–Huyskens, 1983; Maes et al., 1988). While an
interaction like this is not observed in aqueous solution, it may occur 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 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 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 in aqueous solution. For the monomer a shift of 13 cm1 was found
(Pinchas and Laulicht, 1971) but only a shift of 3 cm1 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 cm1 (Fahmy et al.,
1993). {8} May shift +60/40 cm1 upon cation chelation (Tackett, 1989; Nara et al., 1994). In extreme cases, a band
position as for nðC¼OÞ of the COOH group is possible (Deacon and Phillips, 1980). In aqueous solution is the band
Table 16 ( footnote continued)
position 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). {9} Band position
estimated from the shift observed for CH3COO in H2O and 2H2O (Tackett, 1989). May shift +60/90 cm1 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 2H2O estimated from the comparison with the nas ðCOO Þ band which has an e of
830 M1 cm1 (Chirgadze et al., 1975) using spectra of sodium acetate (Tackett, 1989; Wright and Vanderkooi, 1997).
20 cm1 shift upon 13C labelling (Fahmy et al., 1993). {10} For esters it was found that a hydrogen bond to the
carbonyl oxygen leads to an upshift of 21 cm1 and a hydrogen bond to the ether oxygen to a downshift of 17 cm1
(Maes and Zeegers-Huyskens, 1983).
Acknowledgements
The author gratefully acknowledges continuous support by W. Mäntele. Current work is

supported by grants Ba 1887/1-1 and 2-1 of the Deutsche Forschungsgemeinschaft.
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The Infrared Absorption of Amino Acid Side Chains

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

The infrared absorption of amino acid side chains

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

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

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

The infrared spectrum of a protein provides a wealth of information on structure and

2. Infrared absorption of amino acid side chains

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

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

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

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

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

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

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

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

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

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

ds ðCH3 Þ 1375 1378 1391

2.2. Aliphatic side chain groups

Fig. 1. Structure of aliphatic amino acids.

2.3. Aromatic side chains: Tyr, Phe, Trp

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

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

2.6. Side chains with amide groups: Asn, Gln

2.7. Side chains with carboxyl groups: Asp, Glu

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

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

nas ðCN3 Hþ5Þ 1672–1673 (420–490) 1608 (460) 1688–1695, 1595–1605

das ðNHþ3Þ 1626–1629 (60–130) 1201, 428

Fig. 5. Structure of Asn and Gln.

Fig. 6. Structure of Asp and Glu.

Table 15 ( footnote continued )

Table 16 ( footnote continued)

The author gratefully acknowledges continuous support by W. Mäntele. Current work is

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das ðNHþ3Þ 1626–1629 (60–130) 1201, 428