Journal of Molecular Structure 834–836 (2007) 7–16

www.elsevier.com/locate/molstruc

Raman optical activity: An incisive probe of molecular chirality

and biomolecular structure
a,*
Laurence D. Barron , Fujiang Zhu a, Lutz Hecht a, George E. Tranter b, Neil W. Isaacs a

a
WestCHEM, Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK
b
Biological Chemistry, Division of Biomedical Sciences, Imperial College, London SW7 2AZ, UK

Received 29 August 2006; received in revised form 6 October 2006; accepted 7 October 2006
Available online 8 December 2006

Abstract

The theory, instrumentation and applications of Raman optical activity (ROA), which measures vibrational optical activity by means
of a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarized incident light or,
equivalently, a small circularly polarized component in the scattered light, are briefly reviewed. As well as providing the absolute con-
figuration of small chiral molecules, ab initio simulations of observed ROA spectra provide the three-dimensional structure and confor-
mational distribution. The rich ROA spectra of biomolecules in aqueous solution provide detailed structural information including, in
the case of proteins, the tertiary fold in addition to secondary structure elements. The many structure-sensitive bands in protein ROA
spectra makes them ideal for the application of multivariate analysis methods such as nonlinear mapping to determine structural rela-
tionships between different proteins. ROA studies of unfolded and partially folded proteins provide insight into the residual structure in
denatured proteins and the aberrant behaviour of proteins responsible for misfolding diseases. It is even possible to measure the ROA
spectra of intact viruses, from which information about the fold of the major coat proteins and the structure of the nucleic acid core may
be obtained.
 2006 Elsevier B.V. All rights reserved.

Keywords: Vibrational optical activity; Raman optical activity; Chiral molecules; Absolute configuration; Protein structure; Multivariate analysis;
Structural virology

1. Introduction a powerful spectroscopic technique applicable to a huge

In 1969, Atkins and Barron [1] presented a general the-
ory of the polarization characteristics of Rayleigh and
Raman scattering from chiral molecules which contained
a new interference mechanism between light waves scat-
tered via the polarizability and optical activity property
tensors, leading to the prediction that the scattered light
carries a very small degree of circular polarization and the
scattered intensity is slightly different in right- and left-circu-
larly polarized incident light. This discovery led to the devel-
opment of a new chiroptical phenomenon, now called
Raman optical activity (ROA), which has blossomed into
*
Corresponding author. Tel: +44 141 330 5168; fax: +44 141 330 4888.
E-mail address: laurence@chem.gla.ac.uk (L.D. Barron).
range of chiral samples, from small organic molecules to
intact viruses. ROA theory was refined by Barron and
Buckingham [2], who introduced the dimensionless circular
intensity difference (CID)
D ¼ ðI R  I L Þ=ðI R þ I L Þ ð1Þ
as an appropriate experimental quantity, where IR and IL
are the scattered intensities in right- and left-circularly
polarized incident light, respectively. Recent reviews of
ROA include [3–6].
ROA is a very small effect, with D-values being 103
at best, and conventional vibrational Raman scattering is
intrinsically very weak. This pushed the technology avail-
able at the time of the first attempted observations (scan-
ning Raman spectrometers with single-channel photon

0022-2860/$ - see front matter  2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2006.10.033
8 L.D. Barron et al. / Journal of Molecular Structure 834–836 (2007) 7–16

counting based on photomultiplier detection) to its limit.

Furthermore the experiment is highly susceptible to arte-
factual signals, and several groups published ROA spec-
tra that were entirely spurious. However, the natural
ROA spectra published by Barron, Bogaard and Buck-
ingham in 1973 [7] were subsequently confirmed by
Hug et al. in 1975 as the first genuine observations [8].
Their success was due to the implementation of a dual
photon counting system synchronized with square-wave
modulation of the incident visible argon-ion laser beam
between carefully prepared right- and left-circular polar-
ization states, together with the measurement of the
depolarized ROA in 90 scattering since the fundamental
theory revealed that this observable, being generated
purely by anisotropic scattering (see 2d below), was the
least susceptible to artefacts. The first reports of genuine
ROA spectra were those of the two enantiomers of 1-
phenylethylamine and 1-phenylethanol, and covered the
small spectral range 300–400 cm1 where a significant
couplet was detected with opposite signs for the two
enantiomers. Fig. 1a shows the original published Raman
and ROA spectra of the (R)-(+)- and (S)-()-enantio-
mers of 1-phenylethanol, the ROA being presented as
the dimensionless CID (1). Also shown (Fig. 2b) are
the Raman and ROA spectra of the same molecules
recorded in backscattering on a more modern instrument
in our Glasgow laboratory, in which a similar couplet
(shaded) appears.
The significance of ROA is that it measures vibrational
optical activity and therefore provides much more stereo-
chemical information than the standard chiroptical tech-
nique of visible and ultraviolet circular dichroism (CD)
which measures electronic optical activity. This is immedi-
Fig. 2. Two equivalent ROA experiments in transparent Stokes vibra-
tional Raman scattering at angular frequency x  xv in incident light of
angular frequency x. (a) ICP ROA measures IR  IL, where IR and IL are
the scattered intensities (shown here as unpolarized) in right- and left-
circularly polarized incident light, respectively. (b) SCP ROA measures
IR  IL, where IR and IL are the intensities of the right- and left-circularly
polarized components, respectively, of the scattered light using incident
light of fixed polarization (shown here as unpolarized). A positive value of
IR  IL corresponds to a small degree of right-circular polarization in the
scattered light. Reproduced from [41] with permission.
ately apparent from the ROA spectra of 1-phenylethanol in
Fig. 1 in which ROA bands are associated with most of the
vibrational Raman bands, the sign and magnitude of each
one reflecting the stereochemistry, including the absolute
chirality, of the part of the skeleton embraced by the corre-
sponding normal mode of vibration. The ROA spectra
consequently report on the conformational details of the
entire structure. In contrast, the ultraviolet CD spectra of
1-phenylethanol will report only on the stereochemical
environment of the chromophore, namely the aromatic
ring. Molecules lacking an electronic chromophore are

Fig. 1. (a) The depolarized ICP Raman and ROA spectra of the two enantiomers of 1-phenylethanol measured in 90 scattering on a scanning Raman
instrument with dual synchronous photon counting (adapted from [7]). The ROA is presented as the dimensionless CID (1). These spectra, recorded in
1972, were the first observations of genuine ROA to be reported. (b) The backscattered ICP Raman and ROA spectra of the two enantiomers of 1-
phenylethanol measured on a more modern instrument. The ROA couplets equivalent to those in (a) are shaded.
 L.D. Barron et al. / Journal of Molecular Structure 834–836 (2007) 7–16 9

2 2
inaccessible to CD but are equally good for ROA. Vibra-  8½45aG0 þ bðG0 Þ  bðAÞ 
tional optical activity spectra may also be recorded using Dð0 Þ ¼ ð2aÞ
2c½45a2 þ 7bðaÞ2 
the complementary technique of vibrational circular 2 2
dichroism (VCD) which measures a difference in absorp-  48½bðG0 Þ þ 13 bðAÞ 
Dð180 Þ ¼ 2
ð2bÞ
tion of left- and right-circularly polarized infrared radia- 2c½45a2 þ 7bðaÞ 
tion [9,10].  12½45aG0 þ 7bðG0 Þ þ bðAÞ 
2 2
It was mentioned above that, as well as the circular Dx ð90 Þ ¼ 2
ð2cÞ
intensity difference, ROA is also manifest as a small cir- c½45a2 þ 7bðaÞ 
cularly polarized component in the scattered beam using  12½bðG0 Þ2  13 bðAÞ2 
Dz ð90 Þ ¼ ð2dÞ
incident light of fixed polarization (including unpolar- 6cbðaÞ
2

ized). Within the far-from-resonance approximation,

measurement of this circular component, which is now
called scattered circular polarization (SCP) ROA as
(IR  IL)/(IR + IL), where IR and IL denote the intensi-
ties of the right- and left-circularly polarized compo-
nents, respectively, of the scattered light, provides
equivalent information to the CID measurement (1),
which is now called incident circular polarization (ICP)
ROA. These two measurement strategies are illustrated
in Fig. 2.
Until recently, routine ROA studies were held
back by the delicate nature of the measurements.
This situation has now changed with the introduction
of a commercial ROA instrument, the ChiralRAMAN
from BioTools, Inc., which employs the SCP strategy
based on a novel design due to Hug [5,11,12]. In
this article ROA spectra acquired both with our
earlier home-built instruments employing the ICP
strategy and with the ChiralRAMAN instrument are
presented.
2. The ROA observables
ROA measurements can be performed using several dif-
ferent experimental configurations. The scattering angle
can be varied, the most important scattering directions
being forward (0), right-angle (90) and backward
(180). In right-angle scattering (along y with incident
beam along z) two distinct ICP measurements can be made
with a linear polarization analyzer in the scattered beam
with its transmission axis either perpendicular (x) or paral-
lel (z) to the scattering plane (yz) to provide the polarized
and depolarized ROA, respectively. Molecular expressions
for the associated CIDs (1) may be developed semiclassical-
ly by writing down the electric and magnetic field vectors
radiated by the oscillating electric dipole, magnetic dipole
and electric quadrupole moments induced by right- and
left-circularly polarized incident light waves and directly
calculating the circular intensity difference (IR  IL) and
sum (IR + IL) [2,10]. In terms of the electric dipole–electric
dipole molecular polarizability tensor aab and the electric
dipole–magnetic dipole and electric dipole–electric quadru-
pole optical activity tensors G0ab and Aabc, this provides the
following expressions for the CIDs associated with the four
most important scattering geometries for an isotropic col-
lection of chiral molecules [10]:
The various polarizability–polarizability and polarizabili-
ty–optical activity tensor component products have been
averaged over all orientations of the scattering molecule
to generate collections of tensor component products that
are invariant to axis rotations. Specifically,
1 1
a ¼ aaa ¼ ðaxx þ ayy þ azz Þ ð3aÞ
3 3
1 0 1
G ¼ Gaa ¼ ðG0xx þ G0yy þ G0zz Þ
0
ð3bÞ
3 3
are the isotropic invariants of the polarizability tensor and
the electric dipole–magnetic dipole optical activity tensor,
and
1
bðaÞ2 ¼ ð3aab aab  aaa abb Þ ð3cÞ
2
1
0 2
bðG Þ ¼ ð3aab G0ab  aaa G0bb Þ ð3dÞ
2
2 1
bðAÞ ¼ xaab eacd Acdb ð3eÞ
2
are the anisotropic invariants of the polarizability–polariz-
ability and polarizability–optical activity tensor component
products in which the tensor components are referred to
molecule-fixed axes. Common factors in the numerators
and denominators of the CIDs (2) have not been cancelled
so that the relative sum and difference intensities may be
directly compared.
These results apply specifically to Rayleigh (elastic) scat-
tering. For Raman (inelastic) scattering the same basic
CID expressions apply but with the molecular property
tensors replaced by corresponding vibrational Raman tran-
sition tensors between the initial and final vibrational states
nv and mv. Thus aab etc. are replaced by Æmvjaab(Q)jnvæ etc.,
where aab(Q) etc. are effective polarizability and optical
activity operators that depend parametrically on the nor-
mal vibrational coordinates Q so that, within the Placzek
polarizability theory of the Raman effect, the ROA intensi-
ty depends on products such as ðoaab =oQÞ0 ðoG0ab =oQÞ0 and
(oaab/oQ)0eacd(oAcdb/oQ)0 [9,10]. Ab initio calculations of
ROA spectra, which are usually based on the Placzek
approximation, are becoming increasingly successful for
small chiral molecules [13–16]. An early practical applica-
tion was a reliable determination of the absolute configura-
tion of the archetypal small chiral molecule CHFClBr,
which had resisted assignment for over 100 years, from a
comparison of the experimental and ab initio theoretical
10 L.D. Barron et al. / Journal of Molecular Structure 834–836 (2007) 7–16
ROA spectra [17]. Currently, ROA calculations at the den-
sity functional theory (DFT) are implemented in the DAL-
TON and Gaussian 03 software packages, with good
results from the latter exemplified by a study of 1-pheny-
lethanol [18]. There is much ongoing effort by theoreticians
to improve ROA computation to deal with ever-larger
molecules.
For the case of a molecule composed entirely of ideal-
ized axially-symmetric bonds, for which b(G 0 )2 = b(A)2
and aG 0 = 0 [10,19], a simple bond polarizability theory
shows that ROA is generated exclusively by anisotropic
scattering and the forward and backward CID expressions
(2a) and (2b) reduce to [10]
Dð0 Þ ¼ 0 ð4aÞ
0 2
32bðG Þ
Dð180 Þ ¼ 2
ð4bÞ
c½45a2 þ 7bðaÞ 
Unlike conventional Raman scattering intensities, which
are the same in the forward and backward directions,
ROA intensity is therefore maximized in backscattering
and zero in forward scattering. These considerations lead
to the important conclusion that backscattering boosts the
ROA signal relative to the background Raman intensity
and is therefore the best general experimental strategy for
most ROA studies, and is especially valuable for ROA stud-
ies of biomolecules in aqueous solution due to the weak sig-
nals and high backgrounds [20,21].
3. Instrumentation
A backscattering ICP measurement strategy has been
used in our Glasgow laboratory for some years and has
provided a large number of ROA spectra [4,22]. The design
of our Glasgow backscattering ICP ROA instrument is
described in detail elsewhere [23]. Although this ICP
ROA instrument design has served to establish the value
of ROA in stereochemistry and biomolecular science and
will remain useful, a completely new design of ROA instru-
ment with significant advantages accruing from the use of
the SCP strategy has recently been developed by Hug
[5,11,12]. In particular, ‘flicker noise’ arising from dust par-
ticles, density fluctuations, laser power fluctuations, etc. are
eliminated since the intensity difference measurements
required to extract the circularly polarized components of
the scattered beam are taken between two orthogonal com-
ponents of the scattered light measured during the same
acquisition period. The flicker noise consequently cancels
out, resulting in greatly superior signal-to-noise character-
istics. The basic design is illustrated in Fig. 3. The incident
visible laser beam at 532 nm from a frequency-doubled Nd/
YAG laser, the initial linear polarization of which is
‘scrambled’ by a fast rotation of the azimuth, is deflected
into the sample cell using a very small prism. The cone of
backscattered light is collimated onto a liquid crystal
retarder set to convert right- and left-circular polarization
states into linear polarization states with azimuths perpen-
Fig. 3. Simplified optical design of the SCP BioTools ChiralRAMAN
backscattering ROA instrument. Reproduced from [41] with permission.
dicular and parallel, respectively, to the plane of the instru-
ment, followed by an edge filter to remove the intense
Rayleigh line. A beam-splitting cube then diverts the per-
pendicular component at 90 to the propagation direction
of the parallel component, which passes through undivert-
ed. In this way, the right- and left-circularly polarized com-
ponents of the backscattered light are separated and
collected into the ends of two fiber optics. Each fiber optic
converts the cross section from circular at the input end to
a linear configuration at the output end that matches the
entrance slit of a fast imaging spectrograph, thereby
enabling separate Raman spectra for the right- and left-cir-
cularly polarized components of the scattered light to be
dispersed simultaneously one above the other on the chip
of a backthinned CCD detector. Subtraction then provides
the required SCP ROA spectrum corresponding to tiny cir-
cularly polarized components in the Raman bands. Small
differences in the two detection channels are compensated
by interconverting their function through the switching of
the liquid crystal retarder from the k/4 to the k/4 state.
A commercial instrument based on the Hug design that
also incorporates a sophisticated artefact suppression pro-
tocol, based on a ‘virtual enantiomers’ approach which
greatly facilitates the routine acquisition of reliable ROA
spectra [12], has recently become available (the Chi-
ralRAMAN from BioTools, Inc.). The first production
model of the ChiralRAMAN instrument has been opti-
mized in our laboratory for protein samples. It provides
high-quality protein ROA spectra in 2–5 h, around five
times faster than our home-built ICP ROA instruments,
using a sample volume of 30 ll and 500 mW of focused
laser power at the sample, and extends protein ROA data
acquisition routinely to the low-wavenumber region
250–600 cm1. For neat liquid samples of small chiral
 L.D. Barron et al. / Journal of Molecular Structure 834–836 (2007) 7–16
molecules, it provides high-quality ROA spectra in just a
few minutes.
4. Some applications of ROA
4.1. Small chiral molecules
A large number of small chiral organic molecules were
studied in the early days of ROA, either as neat liquids
or concentrated solutions. Stereochemical information
was deduced by comparing ROA band patterns with those
of related molecules of known structure [24], or by the
application of simple model theories [10]. However, ab ini-
tio ROA computations are now the method of choice for
obtaining structural information from experimental ROA
spectra. We refer to the results of Zuber and Hug [25] on
galaxolide, for which ROA provided the absolute configu-
rations at two independent chiral centres, for an example of
the impressive results that can now be achieved on quite
large molecules. Because ROA spectra may be easily
acquired down to 100 cm1 and below, interesting low-
wavenumber modes of vibration such as methyl torsions
[10,26] may be studied as well as low-wavenumber skeletal
modes that bring about conformational transitions [27].
Another application is the measurement of enantiomeric
excess in samples containing unequal amounts of mirror-
image enantiomers and which, in favourable circumstanc-
es, may be determined to an accuracy of 0.1% [28].
4.2. Biomolecules
Work in our Glasgow laboratory has shown that appli-
cations of ROA in the realm of biomolecular science are
highly promising. ROA is more incisive than conventional
vibrational spectroscopy, infrared or Raman, in the study
of biomolecules on account of its exquisite sensitivity to
chirality. We have demonstrated that ROA spectra may
be measured routinely over a wide spectral range on the
central molecules of life, namely proteins, carbohydrates,
nucleic acids and viruses; all in aqueous solution to reflect
their natural biological environment [22,29].
The normal modes of vibration of biomolecules can be
highly complex, with contributions from vibrational coor-
dinates within both the backbone and the side chains.
ROA is able to cut through the complexity of the corre-
sponding vibrational spectra since the largest signals are
often associated with vibrational coordinates which sample
the most rigid and chiral parts of the structure. These are
usually within the backbone and often give rise to ROA
band patterns characteristic of the backbone conforma-
tion. Proteins are particularly favourable in this respect
since signals from the peptide backbone usually dominate
the ROA spectrum, unlike the parent conventional Raman
spectrum in which the many bands from the amino acid
side chains often obscure the peptide backbone bands. Car-
bohydrate ROA spectra are similarly dominated by signals
from skeletal vibrations, in this case centred on the constit-
11
uent sugar rings and the connecting glycosidic links.
Although the parent Raman spectra of nucleic acids are
dominated by bands from the intrinsic base vibrations,
their ROA spectra tend to be dominated by bands charac-
teristic of the stereochemical dispositions of the bases with
respect to each other and to the sugar rings, together with
signals from the sugar–phosphate backbone.
4.2.1. Small biomolecules and ab initio computations
Ab initio ROA computations are starting to make an
impact on studies of the aqueous solution conformations
of smaller biomolecules. For example, a recent analysis
of the conformational space of zwitterionic L-alanine
revealed that shapes of Raman and ROA bands are to a

large extent determined by rotation of NHþ 3 , COO and
CH3 groups and hence that it is essential to take into
account dynamic factors for successful simulations [30].
And by transferring molecular property tensors from
smaller fragments to the whole structure, together with
optimization in normal coordinates (rather than in Carte-
sian or internal valence), the influence of side chains on
Raman and ROA spectra of poly(L-proline) was simulated
by averaging different proline ring conformations [31]. This
is the largest system for which ROA calculations have been
performed to date.
Carbohydrates in aqueous solution are highly favour-
able samples for ROA studies, giving rich and informative
band structures over a wide range of the vibrational spec-
trum [32]. Monosaccharide ROA spectra contain informa-
tion on the ring conformation, the relative disposition of
hydroxyl groups around the ring, the absolute configura-
tion and axial or equatorial orientation of groups attached
to the anomeric carbon and the exocyclic CH2OH confor-
mation; disaccharide ROA spectra contain information on
the type and conformation of the glycosidic link; and oligo-
and polysaccharide ROA spectra can distinguish disor-
dered structure from extended order such as helical. The
long timescale of NMR means that it only senses structure
averaged over interconverting conformers and requires reli-
able molecular dynamics simulations; whereas the short
timescale of the Raman scattering process means that
ROA provides ‘short-time snapshot’ spectra of the individ-
ual conformers present in solution. This advantage of ROA
was recently illustrated in a determination of conforma-
tional populations of some simple sugars in aqueous solu-
tion, for comparison with conformations observed in the
gas phase, in which, using the Gaussian 03 program, basis
sets of computed ROA spectra were used to construct
weighted sums which approximately reproduced the
observed ROA spectra [33].
4.2.2. Proteins
Although individual protein ROA bands may be
assigned to elements of secondary structure such as a-helix
and b-sheet, the presence of clear bands originating in the
loops and turns connecting the secondary structure ele-
ments leads to overall ROA band patterns characteristic
12 L.D. Barron et al. / Journal of Molecular Structure 834–836 (2007) 7–16

of the three-dimensional structure, or fold, of the protein.
Unlike the parent Raman band patterns, the ROA band
patterns for some fold types can therefore be quite distinct.
To illustrate this, Fig. 4 displays the backscattered SCP
ROA spectra of three proteins with different folds, namely
human serum albumin (SCOP class: all a; fold: Serum-
albumin-like), human immunoglobulin G (all b; Immuno-
globulin-like) and b-casein (natively unfolded). The corre-
sponding MOLSCRIPT diagrams [34] of the first two are
displayed for convenience. The ROA band pattern for
human serum albumin is very similar to that for poly(L-ly-
sine) in a model a-helical conformation [35], reflecting the
large amount of extended a-helix contained within its fold.
The ROA band pattern for human immunoglobulin G has
similarities with that for poly(L-lysine) in a model b-sheet
conformation [36], which accords with the large amount
of antiparallel b-sheet within each of its 12 b-sandwich fold
domains based on the Greek key motif. The ROA spec-
trum of b-casein is similar to that of disordered polypep-
tides, which are thought to contain large amounts of
poly(L-proline)II (PPII) helix [37].
Vibrations of the backbone in polypeptides and proteins
are usually associated with three main regions of the
Raman spectrum [38]. These are the backbone skeletal
stretch region 870–1150 cm1 originating in mainly
CaAC, CaACb and CAN stretch coordinates; the amide
III region 1230–1310 cm1 often assigned mostly to the
in-phase combination of the in-plane NAH deformation
with the CAN stretch; and the amide I region 1630–
1700 cm1 which arises mostly from the C@O stretch.
However, it is now recognized that the amide III region
involves much more mixing between the NAH and CaAH
deformations than previously thought, and should be
extended to at least 1340 cm1 [39,40]. This extended amide
III region is particularly important for ROA studies
because the coupling between NAH and CaAH deforma-
tions is very sensitive to geometry and generates a rich
and informative ROA band structure. Side-chain vibra-
tions also generate many characteristic Raman bands
[38]: although these are often less prominent in ROA spec-
tra due to some conformational freedom which can sup-
press the ROA intensities, a few side-chain vibrations,
especially those associated with tryptophan and phenylala-
nine, do generate useful ROA signals [41].
The large number of resolved structure-sensitive bands
in protein ROA spectra makes them highly suitable for
the application of multivariate analysis (pattern recogni-
tion) techniques to extract structural information. We
have shown that useful structural relationships among
proteins may be obtained by analyzing their ROA spectra

Fig. 4. Backscattered SCP Raman (IR + IL) and ROA (IR  IL) spectra of: (a) human serum albumin, (b) human immunoglobulin G and (c) bovine
ribonuclease A, all in aqueous solution and recorded on the ChiralRAMAN instrument. The MOLSCRIPT diagrams for (a) and (b) correspond to PDB
structures 1ao6 and 1hzh, respectively.
 L.D. Barron et al. / Journal of Molecular Structure 834–836 (2007) 7–16 13

using the well-known method of principal component 4 all α 1344

1662
analysis (PCA) [29,36,41]. More recently, we have found 2 1125 1302

that the application of the more advanced multivariate 0

analysis method called nonlinear mapping (NLM) yields -2

1092 1632

even better results [42]. Multivariate analysis techniques 4 all β 1311 1674
1288 1318
start by considering each digitized spectrum to be a vec- 2
0
tor from the origin to a point in a multidimensional
-2
space, with the axes representing the digitized wavenum- 1242 1356 1654
4 all disordered/irregular 1318
bers. Points close to or distant from each other in this
2 1677
multidimensional space are then taken to represent simi-
0
lar or dissimilar spectra, respectively. Like PCA, NLM -2 1263
seeks to create a lower dimensional space in which the 4 αβ
relative positions of the points approximately preserve 2
the relationships between the spectra, thereby providing 0
a more easily comprehended representation. The advan- -2
tage of NLM over PCA is that it aims to best represent 4 mainly α
the relationships between all spectra rather than just 2
describing the gross overall variance (which can lead to 0
poor representation of some sample members), so that -2

characterisation of spectral and hence structural similari- 4 maily β

ties is optimized. A two-dimensional (2D) NLM plot for 2

a set of 80 polypeptide, protein and virus ROA spectra 0

in aqueous solution over the range 702–1773 cm1 shows -2

excellent clustering corresponding to different types of
structure (as determined mostly by X-ray or NMR for
proteins with well-defined native folds, and UVCD
and/or NMR for polypeptides and natively unfolded
proteins) [42]. Specifically, clusters corresponding to the
following structural classes are observed: all a, mainly
a, ab, mainly b, all b, mainly disordered/irregular and
all disordered/irregular. We have also found that map-
ping into three dimensions has the advantage of separat-
ing distinct clusters that are otherwise superimposed and
therefore indistinguishable in the 2D NLM plot [42]. The
average standardized ROA spectra of the polypeptides
and proteins falling within each structure class in the
2D NLM plot are presented in Fig. 5. Each has several
distinct features that are characteristic of the type of
structure. The peak wavenumbers of some of the more
prominent ROA bands in the average spectra of the all
a, all b and all disordered/irregular classes have been
marked in the top three spectra in Fig. 5. Although these
marked protein ROA bands have been noted previously
[35,36,41], their appearance in the average spectra strong-
ly reinforces the validity of the earlier assignments. The
average all disordered/irregular ROA spectrum is in fact
very similar to that characteristic of PPII helix [43]
which, as mentioned above, is thought to be a major
conformational element in disordered polypeptides.
Intact glycoproteins also provide excellent ROA spectra
with clear bands originating in both the polypeptide and
carbohydrate components from which information about
the structure of both components may be deduced
[32,44]. This should be especially valuable in view of the
central importance of glycoproteins in biochemistry and
the fact that they are difficult to study using the conven-
tional techniques of structural biology.
4 mainly disordered/irregular
2
0
-2
800 1000 1200 1400 1600
-1
wavenumber /cm
Fig. 5. Averages of the standardized backscattered ROA spectra (ICP and
SCP) in aqueous solution for seven main structure classes. Adapted from
[42]. The vertical axis represents arbitrary intensities standardized to put
the spectra on an equal footing, as described in [42].
4.2.3. Viruses
Knowledge of virus structure at the molecular level is
essential for understanding their modus operandi. Remark-
ably, ROA spectra may be obtained for most types of
intact virus in aqueous solution, including filamentous,
helical, rod-shaped and icosahedral [45]. To illustrate the
enormous potential of ROA in structural virology, we dis-
play in Fig. 6 a set of spectra measured on cowpea mosaic
virus [45]. This virus has a nucleic acid genome consisting
of two different RNA molecules (RNA-1 and RNA-2)
which are separately encapsidated in identical icosahedral
protein shells, the structure of which is known from X-
ray crystallography. Fig. 6a illustrates how the icosahedral
capsid is constructed from 60 copies of an asymmetric unit
made up of three different protein domains A, B and C
each of which, as illustrated by the MOLSCRIPT diagrams
in Fig. 6b, has a similar structure rich in b-sheet within the
same ‘jelly-roll b-sandwich’ fold. Virus preparations can be
separated into empty protein capsids, capsids containing
RNA-1 and capsids containing RNA-2. The top panel in
Fig. 6c shows the Raman and ROA spectra of the empty
protein capsid, the band patterns being characteristic of
14 L.D. Barron et al. / Journal of Molecular Structure 834–836 (2007) 7–16

Fig. 6. (a) The icosahedral capsid of cowpea mosaic virus. (b) The asymmetric unit comprising three different protein domains, each having the same jelly-
roll b-sandwich fold represented as MOLSCRIPT diagrams (from PDB structure 1ny7). (c) The backscattered ICP Raman (IR + IL) and ROA (IR  IL)
spectra measured in aqueous solution of the empty protein capsid (top pair), the intact capsid containing RNA-2 (middle pair), and the difference spectra
obtained by subtracting the top from the middle spectra to reveal the spectra of the viral RNA-2 (bottom pair). Adapted from [45].

the jelly-roll b-sandwich fold of the individual protein
domains. The middle panel shows the spectra of the capsid
containing RNA-2, with bands from the nucleic acid now
evident in addition to those from the protein. The bottom
panel shows the spectra obtained by subtracting the top
spectrum from the middle spectrum. The difference ROA
spectrum looks very similar to those of synthetic and nat-
ural RNA molecules and is therefore taken as originating
mainly in the viral RNA: the details reflect the single-
stranded A-type helical conformation of the RNA-2 pack-
aged in the core. Hence new information about both the
protein and nucleic acid constituents of an intact virus
may be deduced from ROA data.
4.2.4. Dynamics and behaviour
Since the time scale of the Raman scattering event is
much shorter than that of the fastest conformational fluc-
tuations, an ROA spectrum is a superposition of ‘snapshot’
spectra from all the distinct conformations present in the
sample at equilibrium. ROA intensity depends on absolute
chirality with respect to the arrangement of bonds, so there
tends to be a cancellation of contributions from structures
with ‘opposite’ chirality which can arise as a mobile struc-
ture explores the range of accessible conformations about
single bonds. These factors result in ROA exhibiting an
enhanced sensitivity to the dynamic behaviour of biomo-
lecular structure. In contrast, observables that are ‘blind’
 L.D. Barron et al. / Journal of Molecular Structure 834–836 (2007) 7–16
to chirality, such as conventional Raman band intensities, References
are generally additive and consequently less sensitive to
conformational mobility. [1]
This enhanced sensitivity of ROA to dynamic aspects of [2]
[3]
biomolecular structure has proved valuable in monitoring [4]
the partially structured intermediate states accessed by pro-
teins as they are induced to unfold under the influence of
heat and/or reduced pH [46], and in detecting glass-like [5]
transitions in nucleic acids [47]. These special attributes
of ROA are also providing a new perspective on the impor- [6]
tant problem of protein misfolding and disease. For exam-
ple, ROA has revealed that PPII helix could be an [7]
important intermediate structure in the formation of amy-
[8]
loid fibrils [48], and that the prion protein is able to support
unusually flat b-sheet structures [36]. [9]
5. Future directions
[10]
Application of two-dimensional spectroscopic correla-
[11]
tion methods, based on linear relationships between spectral [12]
data obtained under a perturbing influence such as change of [13]
temperature or pH, has the potential to greatly increase the [14]
amount of information that may be extracted from ROA [15]
spectra. Spectral resolution is enhanced by spreading bands [16]
[17]
over a second dimension, which can provide unambiguous
band assignments and order of spectral changes. A prelimin- [18]
ary study of the a-helix to b-sheet transition of poly(L-lysine)
as a function of temperature has demonstrated the value of [19]
[20]
two-dimensional ROA measurements [49].
[21]
Experimental and theoretical studies of resonance ROA [22]
have been reported [50]. Although resonance ROA is still
in its infancy, it has the potential to boost the intensity [23]
by several orders of magnitude thereby enabling much
more dilute samples to be studied. Resonance and pre-res- [24]
[25]
onance ultraviolet ROA measurements on biomolecules, [26]
using ultraviolet lasers or perhaps even synchrotron beams [27]
for the light source, could be especially valuable. The pos-
sibility of a huge increase in sensitivity via surface-en- [28]
hanced ROA is also attractive, and a report claiming the
[29]
first observation, in adenine, has recently appeared [51].
Because adenine is not chiral, the authors suggest that [30]
the observed ROA signals result from surface-induced chi-
rality. However, equal numbers of mirror-image chiral sur- [31]
face domains are expected on silver nanoparticles in the [32]
bulk sample, resulting in zero ROA, so the reported obser- [33]
vations almost certainly originate in experimental artefacts.
A sufficient body of experimental data has now been [34]
accumulated and analysed to demonstrate that ROA can [35]
provide new and incisive information complementary to
[36]
that obtained from other physical methods. With the recent
availability of a commercial instrument, ROA should find [37]
many applications in chemical and biomolecular science.
[38]
Acknowledgements
[39]
Our Glasgow ROA work has been supported by EPSRC
and BBSRC.
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