vibrational raman optical activity of proteins, nucleic acids, and viruses

Vibrational Raman optical activity of proteins,nucleic acids, and viruses

Ewan W. Blanch, Lutz Hecht, and Laurence D. Barron*

Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK

Accepted 15 October 2002

Abstract

Due to its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in vibrational

Raman scattering from chiral molecules in right- and left-circularly polarized incident light, is a powerful probe of biomolecular

structure in solution. Protein ROA spectra provide information on the secondary and tertiary structures of the polypeptide

backbone, hydration, side-chain conformation, and structural elements present in denatured states. Nucleic acid ROA spectra yield

information on the sugar ring conformation, the base stacking arrangement, and the mutual orientation of the sugar and base rings

around the C–N glycosidic linkage. ROA is able to simultaneously probe the structures of both the protein and the nucleic acid

components of intact viruses. This article gives a brief account of the theory and measurement of ROA and presents the ROA

spectra of a selection of proteins, nucleic acids, and viruses which illustrate the applications of ROA spectroscopy in biomolecular

research.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Raman optical activity; Vibrational spectroscopy; Chirality; Protein conformation; Fold recognition; Conformational disease; Nucleic

acid structure; Virus structure

1. Introduction

X-ray crystallography and high-resolution nuclear

magnetic resonance (NMR) spectroscopy are currently

preeminent in the field of structural biology due to their

ability to reveal the details of biomolecular structure atatomic resolution and will continue to be invaluable.

However, there are limitations to their applicability:

many biomolecules have proven difficult to crystallize

while many others have structures too large to be cur-

rently solved by NMR. Academic and commercial in-

terests in the postgenomic era will demand structural

information about such biomolecules. Although pro-

viding information at atomic resolution, vibrationalspectroscopic techniques will become increasingly valu-

able since they can be routinely applied to a wide range

of biological systems and provide information on

structure, dynamics, local environments and assembly

processes [1–8].

A particularly informative method of vibrational

spectroscopy is vibrational Raman optical activity

(ROA) which refers to a small difference in the intensity

of Raman scattering from chiral molecules in right- andleft-circularly polarized incident light or, equivalently, a

small circularly polarized component in the scattered

light [9,10]. This was first observed in 1973 [11] and has

been developed in our laboratory to the point that it is

now an incisive probe of aqueous solution structures of

peptides, proteins, carbohydrates, nucleic acids, and

intact viruses [12]. Conventional Raman spectroscopy

provides molecular vibrational spectra by means of in-elastic scattering of visible light. During the Stokes

Raman scattering event, the interaction of a molecule

with an incident photon of energy gx (where x is its

angular frequency and g is Dirac�s constant) can leave

the molecule in an excited vibrational state of energy

gxm, with a corresponding loss of photon energy and

hence a shift to lower angular frequency x–xm of the

scattered photon. Therefore, by analyzing the scattered

Methods 29 (2003) 196–209

www.elsevier.com/locate/ymeth

*Corresponding author. Fax: +0141-330-4888.

E-mail address: [email protected] (L.D. Barron).

1046-2023/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

PII: S1046-2023 (02 )00310-9

mail to: [email protected]

light with a visible spectrometer, a complete vibrationalspectrum may be obtained. In this article we provide a

brief survey of the ROA technique, with some typical

applications in biomolecular science illustrated by ex-

amples from recent studies on proteins, nucleic acids,

and viruses.

2. Basic theory

2.1. The ROA observables

The fundamental scattering mechanism responsible

for ROA was discovered by Atkins and Barron [13]. A

more definitive version was developed by Barron and

Buckingham [14], who introduced the following defini-

tion of the dimensionless circular intensity difference(CID),

D ¼ ðIR � ILÞ=ðIR þ ILÞ; ð1Þas an appropriate experimental observable, where IR andIL are the scattered intensities in right- and left-circularly

polarized incident light, respectively. With regard to the

electric dipole–electric dipole molecular polarizability

tensor aab and the electric dipole–magnetic dipole andthe electric dipole–electric quadrupole optical activity

tensors G0ab and Aabc [15,16], the CIDs for forward (0�)

and backward (180�) scattering from an isotropic sam-

ple for incident wavelengths much greater than the

molecular dimensions are

Dð0�Þ ¼4 45aG0 þ bðG0Þ2 � bðAÞ2h i

c 45a2 þ 7bðaÞ2h i ð2Þ

and

Dð180�Þ ¼24 b G0� �2 þ 1

3bðAÞ2

h i

c 45a2 þ 7bðaÞ2h i ð3Þ

where the isotropic invariants are defined as

a ¼ 1

3aaa and G0 ¼ 1

3G0

aa

and the anisotropic invariants as

bðaÞ2 ¼ 1

23aabaab

�� aaaabb

�;

bðG0Þ2 ¼ 1

23aabG0

ab

�� aaaG0

bb

�;

and

bðAÞ2 ¼ 1

2xaabeacdAcdb:

These results apply specifically to Rayleigh (elastic)

scattering. For Raman (inelastic) scattering the samebasic CID expressions apply but with the molecular

property tensors replaced by corresponding vibrationalRaman transition tensors. For the case of a molecule

composed entirely of idealized axially symmetric bonds,

for which bðG0Þ2 ¼ bðAÞ2 and aG0 ¼ 0 [16,17], a simple

bond polarizability theory shows that ROA is generated

exclusively by anisotropic scattering and the CID ex-

pressions reduce to

Dð0�Þ ¼ 0

and

Dð180�Þ ¼ 32bðG0Þ2

c 45a2 þ 7b að Þ2h i :

Unlike conventional Raman scattering intensities, which

are the same in forward and backward directions, ROA

intensity is therefore maximized in backscattering and

zero in forward scattering.

2.2. Enhanced sensitivity of ROA to structure and

dynamics of chiral biomolecules

The normal vibrational modes of biopolymers can be

highly complex as they contain contributions from local

vibrational coordinates in both the backbone and the

side chains. ROA is able to cut through the complexity

of the corresponding vibrational spectra as the largest

ROA signals originate from vibrational coordinates

which sample the most rigid and chiral parts of the

structure. These usually reside within the backbone andgive rise to ROA band patterns characteristic of the

backbone conformation. In proteins these include bands

characteristic of secondary, loop, and turn structures. In

comparison, the parent conventional Raman spectrum

of a protein is dominated by bands arising from the

amino acid side chains which may obscure the peptide

backbone bands. A few distinct ROA signals from the

amino acid side chains, although less prominent thanthose from the peptide backbone, can also provide

useful information. Carbohydrate ROA spectra are

similarly dominated by signals from skeletal vibrations,

principally the constituent sugar rings and the connect-

ing glycosidic links. The ROA spectra of nucleic acids

are dominated by bands characteristic of the stereo-

chemical arrangement of the bases with respect to each

other, to sugar rings, and to signals from the sugar–phosphate backbone.

The time scale of the Raman scattering event

(�3:3� 10�14 s for a vibration with Stokes wavenumber

shift 1000cm�1 excited in the visible) is much shorter

than that of the fastest conformational fluctuations.

Therefore the ROA spectrum is a superposition of

‘‘snapshot’’ spectra from all the distinct conformations

present in the sample at equilibrium. ROA intensity isdependent on absolute chirality and therefore yields a

cancellation of contributions from enantiomeric struc-

E.W. Blanch et al. / Methods 29 (2003) 196–209 197

tures which can arise as a mobile structure explores therange of accessible conformations. These factors result

in ROA exhibiting an enhanced sensitivity to the dy-

namic behavior of biomolecular structure. In contrast,

observables that are ‘‘blind’’ to chirality, such as con-

ventional Raman scattering intensities, are generally

additive and therefore less sensitive to conformational

mobility. Ultraviolet circular dichroism (UVCD) also

shows an enhanced sensitivity to the dynamics of chiralstructures. However, the sensitivity is less than that for

ROA due to its dependence on electronic transitions.

3. Instrumentation

Since ROA is maximized in backscattering, a back-

scattering geometry has proven to be essential for theroutine measurement of ROA spectra of biomolecules in

aqueous solution. The optical layout of our current

backscattering ROA instrument is shown in Fig. 1 [18].

A visible argon ion laser beam is weakly focused into the

sample solution which is contained in a small rectan-

gular fused quartz cell. The cone of backscattered light

is reflected off a 45 � mirror, which has a small central

hole drilled to allow passage of the incident laser beam,through an edge filter to remove the Rayleigh line and

into the collection optics of a single grating spectro-

graph. This is a customized version of a fast-imaging

spectrograph containing a highly efficient volume holo-

graphic transmission grating. The detector is a cooled

back-thinned charge-coupled device (CCD) camera with

a quantum efficiency of �80% over the spectral range of

our studies. Operation of the CCD camera in multi-channel mode allows the full spectral range to be mea-

sured in a single acquisition. To measure the small ROA

signals, the spectral acquisition is synchronized with anelectro-optic modulator used to switch the state of po-

larization of the incident laser beam between right- and

left-circular at a suitable rate.

Spectra are displayed in analog-to-digital converter

units as a function of the Stokes Raman wavenumber

shift with respect to the exciting laser line. The green

514.5-nm line of an argon ion laser is used as this pro-

vides a compromise between reduced fluorescence fromsample impurities with increasing wavelength and in-

creased scattering efficiency with decreasing wavelength

due to the Rayleigh k�4 law. Typical laser power at the

sample is �700mW, sample concentrations of proteins,

polypeptides, and nucleic acids are �30–100mg/mL,

and those of intact viruses are �5–30 mg/mL. Under

these conditions ROA spectra such as those presented

here may be obtained in �5–24 h for proteins and nu-cleic acids and �1–4 days for intact viruses. Measure-

ments can also be performed over the temperature range

of �0–60 �C by directing dry air downward over the

sample cell from a device used to cool protein crystals in

X-ray diffraction experiments, to study dynamic be-

havior.

Several novel design features of a new ROA instru-

ment developed by Hug and Hangartner [19] will beespecially valuable for biomolecular studies. It is based

on measuring the circularly polarized component in the

Raman scattered light instead of the Raman intensity

difference in right- and left-circularly polarized incident

light employed in the Glasgow instrument. This allows

‘‘flicker noise’’ arising from dust particles, density fluc-

tuations, etc., to be eliminated because the intensity

differences required to extract the circularly polarizedcomponents of the scattered beam are taken between

two components of the scattered light measured during

Fig. 1. Optical layout of the current backscattering ROA instrument used in Glasgow.

198 E.W. Blanch et al. / Methods 29 (2003) 196–209

the same acquisition period. The flicker noise thereforecancels out, resulting in superior signal-to-noise char-

acteristics. A commercial instrument based on this de-

sign will be available shortly.

4. Studying biomolecules

4.1. Proteins

4.1.1. Folded proteins

Because protein ROA spectra are usually dominated

by bands originating in the peptide backbone they di-

rectly reflect the solution conformation. The enhanced

sensitivity of ROA to dynamic aspects of structure also

makes it a useful new probe of order–disorder transi-

tions and conformational mobility.Vibrations of the peptide backbone in proteins are

usually associated with three main regions of the Raman

spectrum [2,3,20]: the backbone skeletal stretch region

�870–1150cm�1 which arises from CaAC;CaACb and

CaAN stretch coordinates; the extended amide III re-

gion �1230–1340cm�1 which involves mainly the in-

phase combination of the N–H in-plane deformation

with the CaAN stretch along with mixing between theN–H and the CaAH deformations; and the amide I re-

gion �1630–1700cm�1 which originates in the C@O

stretch. The extended amide III region is particularly

important for ROA studies as the coupling between the

NAH and CaAH deformations is sensitive to geometry

and generates a rich and informative band structure [21].

Fig. 2a shows the backscattered Raman and ROA

spectra of the a-helical protein human serum albumin.The main features of the ROA spectrum are in good

agreement with the Protein Data Bank (PDB) X-ray

crystal structure 1ao6, which reports 69.2% a-helix and

1.7% 310-helix with the remainder being made up of

loops and turns. The strong sharp positive band at

�1340cm�1 is assigned to a hydrated form of a-helixwhile the weaker positive band at �1300cm�1 appears

to be associated with a-helix in a more hydrophobicenvironment. We have reported similar bands arising

from elements of a-helix in the ROA spectra of model a-helical polypeptides and globular proteins [12,22,23] and

the coat proteins of intact filamentous bacteriophages

[24]. The relative intensities of these two bands appear to

correlate with the exposure of the polypeptide backbone

to the solvent within the elements of a-helix in each case.

For example, the positive a-helix protein ROA band at�1340cm�1 completely disappears when the protein is

dissolved in D2O [12]. This indicates both that the cor-

responding sequences are exposed to solvent and that

N–H deformations of the peptide backbone make a

significant contribution to the generation of this band

(because corresponding N–D deformations contribute

to normal modes in a spectral region several hundred

wavenumbers lower). The positive ROA band from a-helix at �1300cm�1 retains much of its intensity in D2O.

Conventional Raman bands at similar wavenumbers

have been assigned to a-helix in studies performed on

polypeptides [25] and filamentous bacteriophages [26].

In particular, a recent Raman study of a-helical poly(LL-alanine) [25] has provided definitive assignments of anumber of the normal modes of vibration in the ex-

tended amide III region over the range in which we have

identified ROA bands. These normal modes variously

transform the same as the A, E1, and E2 symmetry

Fig. 2. Backscattered Raman (IR þ IL) and ROA (IR � IL) spectra of

(a) human serum albumin, (b) jack bean concanavalin A, and (c) hen

lysozyme at �pH 5.4 and 20 �C.


species of the point group of a model infinite regularhelix. The ROA bands assigned to a-helix in this region

may be related to a number of these normal modes, with

the ROA intensities and exact wavenumbers being a

function of the perturbations (geometric and/or due to

various types of hydration) to which the particular he-

lical sequence is subjected.

Insight into the nature of the hydrophobic and hy-

drated variants of a-helix has been provided by recentelectron spin resonance studies of double spin-labeled

alanine-rich peptides which identified a new, more open

conformation of the a-helix [27,28]. Computer model-

ing indicates that this more open geometry leaves the

hydrogen bonding intact but changes the C@O Nangle, resulting in the splaying of the backbone amide

carbonyls away from the helix axis and into the solu-

tion. Therefore, the more open structure may be thepreferred conformation in aqueous solution as it allows

hydrogen bonding with water molecules. An equilib-

rium would then exist between the canonical form of a-helix, which might be responsible for the positive ROA

band at �1300cm�1, and the more open form, which

may generate that at �1340cm�1, with the former be-

ing favored in a hydrophobic environment and the

latter in a hydrophilic (or hydrogen-bonding) environ-ment.

The amide I ROA couplet, negative at �1640cm�1

and positive at �1665cm�1, is also a characteristic sig-

nature of a-helix and corresponds to the wavenumber

range �1645–1655cm�1 for a-helix bands in conven-

tional Raman spectra [3]. Positive ROA intensity in the

range �870–950cm�1 is a further signature of a-helixwith the detailed band structure in this region appearingto show a dependence on side-chain composition, helix

length, and the presence of irregularities.

Fig. 2b shows the Raman and ROA spectra of the b-sheet protein jack bean concanavalin A which contains,

according to the PDB X-ray crystal structure 2cna,

43.5% b-strand, 1.7% a-helix, and 1.3% 310-helix in a

jelly roll b-barrel with the remainder being hairpin bends

and long loops. The sharp negative band at �1241cm�1

has been assigned to b-structure. Similar bands have

been observed in the ROA spectra of other proteins

containing b-sheet [12,23], which sometimes has another

negative band at �1220cm�1 which appears to be as-

sociated with a distinct variant of b-strand or sheet,

possibly hydrated. As more ROA data have been accu-

mulated on proteins and polypeptides containing b-sheetit has become apparent that the true signature of sometypes of b-sheet in the extended amide III region may be

a couplet, negative at low wavenumber and positive at

high. The negative peak of the couplet in proteins ap-

pears to be constrained either to the region �1220cm�1

or the region �1240cm�1. The positive peak of this

couplet appears to be more variable and appears in the

range �1260–1295cm�1. Bands from loops and turns

may also contribute in this region: in fact the positivecomponent at �1295cm�1 of this b-sheet couplet in

concanavalin A may originate in vibrations of the b-turns in the hairpin bends present in the up-and-down

multistranded b-sheet of this protein. Amide III bands

from b-sheet in conventional Raman spectroscopy are

assigned to the region �1230– 1245cm�1 [3]. The amide

I couplet, negative at �1658cm�1 and positive at �1677

cm�1, is another signature of b-sheet and can be easilydistinguished from the amide I couplet produced by a-helix, which typically occurs �5–20cm�1 lower. This

correlates with b-sheet amide I bands in conventional

Raman spectroscopy which occur in the range

�1665–1680cm�1 [3]. A number of ROA bands associ-

ated with b-sheet may appear in the backbone skeletal

stretch region, as found here for concanavalin A.

However, the details of wavenumber, intensity, andband shape are variable, possibly reflecting differences in

local conformations and amino acid compositions found

within different b-sheets.Proteins containing a significant amount of b-sheet

often display additional ROA signals originating in

loops and turns. Negative ROA bands in the range

�1340–1380cm�1 appear to originate in b-hairpinbends; an example is a band of medium intensity ap-pearing at �1345cm�1 in the spectrum of concanavalin

A. This signature allows ROA to differentiate between

parallel and antiparallel types of b-sheet as only the

latter usually contain hairpin bends while the ends of

strands in the former are usually connected by a-helicalsequences. Many b-sheet proteins also show a strong

positive ROA band at �1314–1325cm�1 similar to the

prominent bands observed in disordered forms ofpoly(LL-lysine) and poly(LL-glutamic acid) [12,29]. These

disordered polypeptides are thought to contain signifi-

cant amounts of the polyproline II (PPII) helix confor-

mation. This signal may therefore be a signature of the

PPII helical elements known from X-ray crystal struc-

tures to occur in some of the longer loops between ele-

ments of secondary structure [30,31]. An example of

such a PPII signal is observed at �1316cm�1 in theROA spectrum of jack bean concanavalin A.

Fig. 2c shows the Raman and ROA spectra of the

a þ b protein hen lysozyme. The fold of this protein is

very different from those of human serum albumin and

concanavalin A mentioned above. This is reflected in the

large differences between their ROA spectra. Hen lyso-

zyme contains 28.7% a-helix, 10.9% 310-helix, and 6.2%

b-sheet according to the PDB X-ray crystal structure1lse, which is consistent with the presence of the positive

ROA bands assigned to hydrophobic and hydrated a-helix at �1299 and 1342cm�1, respectively, and with the

sharp negative band at �1240cm�1 indicative of b-structure. As our database of protein ROA spectra has

expanded it has become apparent that the large positive

peak �1297–1300cm�1 observed for proteins with a


lysozyme-type fold may be boosted by other bands.Proteins with a lysozyme-type fold often contain an

unusually high 310-helix component. It is possible that

the positive band �1299cm�1 contains a contribution

from a 310-helix band, with a positive signal

�1295cm�1. There may also be bands contributing in

this region from turns. A definitive analysis of all ROA

bands in the extended amide III region has not been

possible to date due to the difficulty of deconvolutingthe complex, bisignate spectra.

The amide I couplet, negative at �1641cm�1 and

positive at 1665 cm�1 with a small shoulder at

�1683cm�1, also indicates the presence of a-helix and a

lesser amount of b-sheet. There is a small couplet, neg-

ative at �1426cm�1 and positive at �1462cm�1, from

CH2 and CH3 side-chain deformations. On the low-

wavenumber side of this couplet there is another rela-tively weak couplet, positive at low wavenumber and

negative at high wavenumber, that originates in tryp-

tophan vibrations. In addition, there is a relatively large

positive band �1554cm�1 assigned to the W3-type vi-

brational mode of the indole ring of tryptophan resi-

dues. Miura et al. [32] found that the magnitude of the

torsion angle v2;1 of the tryptophan side chain, which

describes the orientation of the indole ring with respectto the local peptide backbone, can be deduced from the

wavenumber of the corresponding conventional Raman

band. Similarly, the magnitude of the torsion angle can

be determined from the position of the W3-type vibra-

tional mode in the ROA spectrum but with the added

advantage of a possible determination of its sign from

the measured sign of the ROA band and hence the de-

duction of the absolute stereochemistry of the trypto-phan side chain [33]. This information is not normally

available other than from a structure determined at

atomic resolution. Hen lysozyme contains six trypto-

phan residues, four with positive v2;1 values and two

with negative v2;1 values according to the PDB structure

1lse. Partial cancellation of the resulting ROA signals

yields the positive band observed in Fig. 2c [33]. In this

manner the W3 ROA band can also be used as a probeof conformational heterogeneity among tryptophan

residues in disordered protein sequences as the cancel-

lation of signals with opposing signs results in a loss of

ROA intensity. This is similar to the disappearance of

near-UVCD bands from aromatic residues upon the

loss of tertiary structure. Thus these two techniques

provide complementary views of order/disorder transi-

tions as ROA probes the intrinsic skeletal chirality ofthe tryptophan side chain while UVCD probes the

chirality in the immediate vicinity of the aromatic

chromophore.

Since ROA spectra contain bands characteristic of

loops and turns and bands indicative of secondary

structure, they provide information about the overall

three-dimensional solution structure of a protein. It

is possible to deduce motif types of supersecondaryelements and even the domain fold class from ROA

spectra. We are developing a pattern recognition pro-

gram, based on principal component analysis (PCA), to

identify protein folds from ROA spectral band patterns

[34]. From the ROA data, the PCA program calculates a

set of subspectra that serve as basis functions. The al-

gebraic combination of these subspectra, weighted with

appropriate expansion coefficients, can be used to re-construct the original set of experimental ROA spectra.

Our current set contains over 70 entries comprising

ROA spectra of several polypeptides in model confor-

mations, many proteins with well-defined folds known

from X-ray crystallographic or solution NMR mea-

surements, and several viruses with coat protein folds

known from X-ray crystallographic or fiber diffraction

data.

4.1.2. Unfolded proteins

As yet it has not been possible to obtain useful ROA

spectra of fully unfolded denatured states of proteins

which have well-defined tertiary folds in the native state.

This is because the intense Raman bands from chemical

denaturants typically used at high concentration pre-

clude ROA measurements, while thermally unfoldedproteins often give rise to intense Rayleigh light scat-

tering due to aggregation. We have, however, reported

interesting results for partially unfolded denatured

protein states associated with molten globules and re-

duced proteins and for proteins which are unfolded in

their native biologically active states [35].

Molten globules are partially unfolded denatured

protein states, stable at equilibrium, with well-definedsecondary structure but lacking the specific tertiary in-

teractions characteristic of the native state [36,37]. A

much studied molten globule is that supported by a-lactalbumins at low pH and called the A-state. Native

bovine a-lactalbumin has the same fold and X-ray crystal

structure very similar to that of hen lysozyme and this is

apparent from its ROA spectrum [22] shown in Fig. 3a,

which is similar to that of hen lysozyme depicted in Fig.2c. Nevertheless, differences in detail the spectra are

apparent. This highlights the sensitivity of ROA to small

differences in structure in proteins with the same fold.

The Raman and ROA spectra of A-state bovine a-lact-albumin are shown in Fig. 3b. The sensitivity of ROA to

the complexity of order in molten globule states is indi-

cated by the large differences observed here between the

ROA spectra of the native and A-states of bovine a-lactalbumin as opposed to the small differences between

the corresponding parent Raman spectra. Much of the

ROA band structure in the extended amide III region of

the A-state spectrum has disappeared and is replaced by

a large couplet consisting of a single negative band ob-

served at �1236cm�1 and two distinct positive bands at

�1297 and 1312cm�1. The negative �1236cm�1 signal


may originate in residues clustering around an ‘‘average’’

of the conformations corresponding to the two negative

signals in the native spectrum, observed at �1222 and1246cm�1 in Fig. 3a, assigned to b-structure. The posi-

tive band at �1297cm�1 may arise from the same a-he-lical sequences in a hydrophobic environment, and

possibly 310-helical sequences, as those responsible for a

similar band in the spectrum of the native state. The

positive �1340cm�1 band assigned to a-helix in a hy-

drated state that dominates the ROA spectrum of the

native state is completely lost in the A-state, with a newpositive band appearing at �1312cm�1 that may origi-

nate in reduced hydration in some a-helical sequences. Inthe amide I region the positive signal of the couplet has

shifted by �10cm�1 to higher wavenumber compared to

that for the native state. The signal originating from the

W3 vibrational mode of tryptophan residues at

�1551cm�1 has almost completely disappeared in the

ROA spectrum of the A-state and been replaced by a

weak positive band at �1560cm�1 indicating confor-

mational heterogeneity among these side chains associ-ated with a loss of the characteristic tertiary interactions

found in the native state.

The Raman and ROA spectra of reduced hen lyso-

zyme are shown in Fig. 3c [35]. This sample was prepared

by reducing all the disulfide bonds and keeping the

sample at low pH to prevent their reoxidation. There are

significant changes of the ROA spectrum of reduced ly-

sozyme compared with that of the native state shown inFig. 2c. The bands in the backbone skeletal stretch region

are suppressed, along with differences in detail, indicat-

ing the loss of much of the fixed structure. Most of the

ROA band patterns in the extended amide III region

have disappeared and have been replaced by a large

broad couplet with some hints of weak band structure,

similar to that observed in the ROA spectrum of A-state

a-lactalbumin, suggesting that the reduced protein sup-ports a number of conformations with a range of local

residue /;w angles clustering in the same regions of the

Ramachandran surface as those in the native state. The

disappearance of the positive band at �1340cm�1 im-

plies that none of the significant amount of hydrated a-helix found in the native state persists in the reduced

form. The positive signal of the amide I couplet has also

shifted by �9cm�1 to higher wavenumber and has be-come quite sharp, indicating the presence of a significant

amount of b-structure. The couplet from �1426–

1462cm�1 in the spectrum of the native state originating

in aliphatic side chains and the band at �1554cm�1 as-

signed to tryptophan side chains are all greatly dimin-

ished, presumably due to conformational heterogeneity.

Although of similar appearance, the ROA spectra of

different denatured proteins display differences in detail,possibly reflecting the different residue compositions and

their different /;w propensities.

Although human lysozyme has a stability similar to

that of hen lysozyme with regard to temperature and

low pH, its thermal denaturation behavior is subtly

different. Below �pH 3.0 the thermal denaturation of

human lysozyme is not a two-state process. Unlike hen

lysozyme, it supports a partially folded molten globule-like state at elevated temperatures [38]. Incubation at

57 �C and pH 2.0, under which conditions the partially

folded state is the most highly populated, induces the

formation of amyloid fibrils while incubation at 70 �Cand pH 2.0, under which conditions the fully denatured

state is the most highly populated, leads to the forma-

tion of amorphous aggregates [39].

Fig. 3. Backscattered Raman and ROA spectra of (a) native bovine a-lactalbumin at �pH 4.6 and 20 �C, (b) A-state bovine a-lactalbumin at

�pH 1.9 and 2 �C, and (c) reduced hen lysozyme at �pH 2.4 and 20 �C.


The Raman and ROA spectra of the native andpartially folded prefibrillar–amyloidogenic intermediate

states of human lysozyme are shown in Fig. 4a and b,

respectively [40]. The structure and ROA spectrum of

the native state of human lysozyme are similar to those

of hen lysozyme. However, large changes are apparent

in the ROA spectrum of the prefibrillar intermediate.

The most significant of these is the loss of the positive

�1345cm�1 band assigned to hydrated a-helix and theappearance of a new positive band at �1325cm�1 as-

signed to PPII helix. This suggests that hydrated a-helix

has undergone a conformational change to PPII struc-ture. The disappearance of the positive �1550cm�1

band assigned to tryptophan vibrations indicates that

major conformational changes have occurred among the

five tryptophan residues, four of which lie within the a-helical domain. Thus the ROA data suggest that the a-domain destabilizes and undergoes a conformational

change in the prefibrillar intermediate and that PPII

helix may be a critical conformational element involvedin amyloid fibril formation [40]. There is no sign of an

increase in b-sheet content in the intermediate. The

ROA spectrum of hen lysozyme, which has a much

lower propensity to form amyloid fibrils than the human

variant [41], is virtually native-like under the same

conditions of low pH and elevated temperature [40].

ROA has proven useful in several recent studies of

natively unfolded proteins of biological and physiolog-ical significance. The ROA spectra of bovine milk

caseins [42], several wheat prolamins [35], and the hu-

man recombinant synuclein and tau brain proteins,

some of which are associated with neurodegenerative

disease [42], were all found to be dominated by a strong

positive band at �1316–1322cm�1 assigned to PPII

helix. The ROA spectrum of c -synuclein shown in Fig.

4c displays an example at �1321cm�1. The absence of awell-defined amide I ROA couplet in this spectrum in-

dicates the lack of secondary structure. Although the

other natively unfolded proteins studied so far have

ROA spectra that are similar overall, there are differ-

ences of detail which reflect differences in residue com-

position and minor differences in structural elements.

The studies on the caseins, synucleins, and tau suggested

that these proteins may be more realistically classified asbeing ‘‘rheomorphic,’’ meaning flowing shape [43], ra-

ther than ‘‘random coil.’’ The rheomorphic state is dis-

tinct from the molten globule state which is a more

compact entity containing a hydrophobic core and a

significant amount of secondary structure [36,37].

The conformational plasticity supported by mobile

regions within native proteins, partially denatured pro-

tein states, and natively unfolded proteins underliesmany of the conformational (protein misfolding) dis-

eases [44,45], many of which involve amyloid fibril for-

mation. As it is extended, flexible, and hydrated, the

PPII helical structure identified in some of these systems

imparts a plastic open character to the structure and

may be implicated in the formation of regular fibrils in

the amyloid diseases [40,42]. This is because the elimi-

nation of water molecules between extended polypeptidechains with fully hydrated N–H and C@O groups to

form b-sheet hydrogen bonds is a highly favorable

process entropically [46]. As the PPII- and b-sheet re-

gions are adjacent on the Ramachandran surface it is

expected that elements of PPII helix would readily un-

dergo this type of aggregation with each other and with

the edges of preexisting b-sheet to form the cross-b

Fig. 4. Backscattered Raman and ROA spectra of native human ly-

sozyme at �pH 5.4 and 20 �C, (b) the prefibrillar intermediate of hu-

man lysozyme at �pH 2.0 and 57 �C, and (c) human c-synuclein at

�pH 7.0 and 20 �C.


structures typical of amyloid fibrils. True random coil isexpected to lead to amorphous aggregation rather than

fibril formation and this is what is generally observed.

However, although the presence of significant amounts

of PPII structure may be necessary for the formation of

regular fibrils, other factors might be important too

because, of the milk and brain proteins studied by ROA,

only a-synuclein and tau are fibrillogenic and associated

with disease. It appears necessary to consider propertiesof the constituent residues such as mean hydrophobicity

and charge also [42].

PPII helix is not readily amenable to traditional

methods of structure determination due to its inherent

flexibility and the lack of intrachain hydrogen bonding,

which has hindered its recognition in globular proteins.

For instance, the PPII conformation is indistinguishable

from an irregular backbone structure for free peptides insolution by 1H NMR spectroscopy [47]. PPII helix may

be recognized in polypeptides by UVCD [47–49] and

vibrational circular dichroism [7,50,51] and in proteins

from the deconvolution of UVCD spectra [52]. In con-

trast, ROA provides a clear and characteristic signature

of PPII helix in both proteins and polypeptides.

4.2. Nucleic acids

The study of the structure and function of nucleic

acids remains a central problem in molecular biology.

Although ROA studies of nucleic acids are at an early

stage, the results obtained so far are promising as ROA

appears to be sensitive to three different sources of chi-

rality: the chiral base-stacking arrangement of intrinsi-

cally achiral base rings, the chiral disposition of the baseand sugar rings with respect to the C–N glycosidic link,

and the inherent chirality associated with the asym-

metric centres of the sugar rings. Model studies on

pyrimidine nucleosides [53] and polyribonucleotides [54–

56] have provided a basis for the interpretation of ROA

spectra of DNA and RNA.

The Raman and ROA spectra of calf thymus DNA

and of phenylalanine-specific transfer RNA (tRNAPhe)in the presence and absence of Mg2þ ions are shown in

Fig. 5a–c, respectively [57]. ROA bands in the region

�900–1150cm�1 originate in vibrations of the sugar

rings and phosphate backbone. The region �1200–

1550cm�1 is dominated by normal modes in which the

vibrational coordinates of the base and sugar rings are

mixed. ROA band patterns in this sugar–base region

appear to reflect the mutual orientation of the two ringsand possibly the sugar ring conformation. The region

�1550–1750cm�1 contains ROA bands characteristic of

the types of bases involved and the particular stacking

arrangements. Although the ROA spectra of the DNA

and the two RNAs are similar there are many differences

in detail. The main differences originate in the DNA

taking up a B-type double helix in which the sugar

puckers are mainly C20-endo and the RNAs taking up

A-type double helical segments where the sugar puckers

are mainly C30-endo. There are smaller differences be-

tween the two RNA spectra and these are most apparent

in the sugar–phosphate region �900– 1150cm�1. It is

known that Mg2þ ions are necessary to hold RNAs in

their specific tertiary folds. For tRNAPhe in the presenceof Mg2þ this is a compact L-shaped form, whereas in the

absence of Mg2þ the tRNAPhe adopts an open cloverleaf

structure [58]. The ROA spectrum of the Mg2þ-freetRNAPhe shows a strong negative, positive, negative

triplet at �992, �1048, and �1091cm�1 which is very

Fig. 5. Backscattered Raman and ROA spectra of (a) calf thymus

DNA, (b) Mg2þ-bound tRNAPhe, and (c) Mg2þ-free tRNAPhe at �pH

6.8 and 20 �C.


similar to that found in A-type polyribonucleotides andhas been assigned to the C30-endo sugar pucker. This

signature is weaker and more complex in the ROA

spectrum of the Mg2þ-bound sample, suggesting a wider

range of sugar puckers. Switching the sugar pucker

conformations from C30-endo to C20-endo would elon-

gate the sugar–phosphate backbone and may assist the

formation of the loops and turns that characterize the

tertiary structure of the folded form.

4.3. Viruses

Knowledge of the structure of viruses at the molecular

level is essential for enterprises such as structure-guided

antiviral drug design [59]. However, the application of

key structural biology techniques such as X-ray crystal-

lography or fiber diffraction is often hampered by prac-tical difficulties. Conventional Raman is valuable in

studies of intact viruses at the molecular level as it is able

to simultaneously probe both the protein and the nucleic

acid constituents [3,6]. The additional incisiveness of

ROA further enhances the value of Raman spectroscopy

in structural virology.

The first virus ROA spectra were reported for fila-

mentous bacteriophages [24]. The Raman and ROAspectra of Pf1 [24], fd, and PH75 [33] are shown in Figs.

6a–c, respectively, as examples. At high concentrations

(above �10–20mg/mL depending on virus and detailed

experimental conditions) filamentous bacteriophages

form liquid crystals which give rise to artifacts which

prevent the acquisition of reliable ROA data. Hence the

viral concentrations were kept below the liquid crystal-

forming thresholds which consequently resulted in adecrease of signal-to-noise levels. This was partly offset

by an apparent boost in viral protein ROA CID values,

possibly due to decreased conformational mobility

within closely packed environments. The water back-

ground has been subtracted from each of the corre-

sponding Raman spectra shown in Fig. 6 to aid

presentation.

Filamentous bacteriophages are long flexible rodsmade up of a loop of single-stranded DNA surrounded

by a shell consisting of a helical array of several thou-

sand identical copies of a major coat protein containing

�50 amino acids. There are also a few copies of minor

coat proteins. X-ray fiber diffraction has shown that the

major coat protein subunits have the fold of an extended

a-helix and that they overlap each other so that the

exterior half of each protein is exposed to water whilethe interior half is protected by neighboring coat pro-

teins [60]. The ROA spectra are dominated by bands

assigned to a-helix in the backbone skeletal stretch,

amide I, and extended amide III regions which have

been discussed already. A couplet, negative at �10955cm�1 and positive at �1125 5cm�1, also seems to be

associated with well-defined a-helix. Further, the rela-

tive intensities of the ROA bands at � 1300 and

1342cm�1 assigned to hydrophobic and hydrated a-he-lix, respectively, correlate well with the hydration of the

peptide backbone in each coat protein [24]. The interior,

protected region of the coat protein of Pf1 contains a

continuous sequence of 20 hydrophobic residues whilethe exterior, exposed region contains a 20-residue se-

quence of mixed hydrophobic and hydrophilic residues.

In Fig. 6a the maximum intensities of the corresponding

ROA bands at �1299 and 1343 cm�1 are virtually

identical. For bacteriophage fd the sequence of pure

hydrophobic helix in the interior region of the coat

protein is shorter and there is a longer sequence of

Fig. 6. Backscattered Raman and ROA spectra of the filamentous

bacteriophages (a) Pf1, (b) fd, and (c) pH 7.5 at �pH 7.6 and 20 �C.


mixed hydrophobic and hydrophilic residues. In thecorresponding ROA spectrum in Fig. 6b the hydrated a-helix band is now relatively stronger than the band

originating in hydrophobic a-helix.An obvious difference between the three bacterio-

phage spectra is the behavior of the band associated

with the W3-type vibrational mode of tryptophan resi-

dues. The major coat protein of Pf1 does not contain

any tryptophan residues and there is no ROA signal�1550cm�1. Bacteriophage fd contains a single trypto-

phan residue in the major coat protein which generates

the positive ROA band at �1559cm�1 while PH75,

which has unusual thermophilic properties [61], shows a

negative band at �1550cm�1 originating from the single

tryptophan in its coat protein. This suggests that the

absolute stereochemistry of the indole ring of the tryp-

tophan residue relative to the local peptide backbone infd is quasienantiomeric to that in PH75. From the

wavenumbers and signs of the W3 ROA bands it was

deduced that v2;1 ¼ þ120 � for fd and )93� for PH75

[33].

Fig. 7a–c show, respectively, the Raman and ROA

spectra of tobacco mosaic virus (TMV) [62], a tob-

amovirus, and the potexviruses potato virus X (PVX)

and narcissus mosaic virus (NMV) [34]. These are helicalplant viruses containing a single strand of positive-sense

RNA encapsidated within a rigid rod-shaped particle in

the case of TMV or a flexuous filamentous particle in the

case of the potexviruses, made up of multiple copies of a

single-coat protein. The structure of the coat protein

subunits of TMV has been determined by X-ray fiber

diffraction [63,64] to be based on a four-helix bundle

motif which contains both water-exposed residues andresidues at the hydrophobic interfaces between a-helices.The ROA band pattern in the extended amide III region

of the TMV spectrum is characteristic of a helix bundle,

with a positive band �1295cm�1 assigned to a-helix in a

hydrophobic environment and a slightly stronger band

�1342cm�1 assigned to hydrated a-helix. The amide I

couplet and bands in the backbone skeletal stretch re-

gion are also characteristic of a-helix.Little is known about the conformations of the coat

protein subunits of PVX and NMV from other tech-

niques but the similarities of their ROA spectra to the

ROA spectrum of TMV reveal they are both based on

helix bundle folds. All three ROA spectra also contain a

positive band at �1316cm�1 assigned to elements of

PPII helix in long loops and a small negative band at

�1220cm�1 from segments of b-strand, possibly hy-drated. Although the three ROA spectra are similar, the

differences in detail illustrate the sensitivity of ROA to

different structural parameters and types of subunit

packing within different virus capsids.

In addition to filamentous and rod-shaped viruses,

ROA has also been applied to icosahedral viruses. Sa-

tellite tobacco mosaic virus (STMV) is a small T ¼ 1

icosahedral virus with a single strand of RNA inside a

capsid composed of 60 identical copies of a coat protein

with 159 amino acids. The X-ray crystal structure of

STMV [65] reveals the fold of the coat protein to be ajelly roll b-barrel. Although it is not shown here, the

ROA spectrum of STMV is very similar to that of

proteins with a jelly roll fold such as concanavalin A,

shown in Fig. 2b. MS2 is a small T ¼ 3 icosahedral virus

with a single strand of RNA contained inside a capsid

made up of 180 identical copies of a coat protein con-

taining 129 amino acids. The X-ray crystal structure of

MS2 [66] reveals that the coat protein subunits fold into

Fig. 7. Backscattered Raman and ROA spectra of the helical plant

viruses (a) TMV, (b) PVX, and (c) NMV at �pH 7.4 and 20 �C.


a 5-stranded antiparallel b-sheet with an additionalshort hairpin at the N terminus and two a-helices. Thea-helices are responsible for interactions with a second

subunit to form a dimer containing 10 adjacent anti-

parallel b-strands. This fold is quite distinct from those

of the jelly roll type of STMV and most other icosahe-

dral viruses, and the ROA spectrum of the MS2 capsid

was found to reflect this.

Determination of the structures of the nucleic acidcomponent of intact viruses has proven difficult in even

the best-resolved X-ray crystal structures. Several of the

ROA spectra of viruses recorded in our laboratory have

displayed bands attributed to nucleic acid. However,

these are usually weak because of either the low nucleic

acid content of the virus or its flexible nature. Recently,

though, we have recorded the first nucleic acid ROA

spectra from an intact virus [67].Cowpea mosaic virus (CPMV), the type member of

the comovirus genus, has a bipartite genome, meaning

that two RNA molecules (called RNA-1 and RNA-2)

are separately encapsidated. CPMV particles can be

separated into four distinct isolates by ultracentrifuga-

tion with a CsCl gradient [68]. The top component (T-

CPMV) consists of empty protein capsids, the middle

component (M-CPMV) contains capsids with a mole-cule of RNA-2 bound, the bottom-upper component

(BU-CPMV) contains capsids with RNA-1 bound, and

the bottom-lower component (BL-CPMV) contains

capsids with RNA-2 bound plus Csþ ions permeating

the interior of the particles. The Raman and ROA

spectra of T-CPMV and M-CPMV are shown in Figs.

8a and b, respectively. CPMV has an icosahedral capsid

of T ¼ 3 symmetry, with two protein chains formingalarge (L) coat protein and a third protein chain

forming a small (S) coat protein. Despite the low se-

quence homology between these three protein chains,

X-ray crystallography shows that they exhibit very

similar eight-stranded jelly roll b-barrel folds [68,69].

The details of the T-CPMV ROA spectrum are con-

sistent with those of a jelly roll b-barrel fold (see the

ROA spectrum of concanavalin A in Fig. 2b). There areseveral large changes in the ROA spectrum of M-

CPMV due to new bands originating from the RNA-2

molecule, which constitutes �24% of the particle by

mass. This is clearer in Fig. 8c which shows the Raman

and ROA spectra of the bound RNA-2 molecule ob-

tained by the subtraction (M-CPMV)-(T-CPMV).

Bands corresponding to conformational changes in-

duced in the protein structure by nucleic acid bindingmay also be present. Comparison of this ROA spec-

trum with those of the nucleic acids shown in Fig. 5

indicates that RNA-2 has an A-type helical conforma-

tion. A similar analysis has also shown that the RNA-1

molecule in the BU-CPMV particle has the same con-

formation. The RNA is not observed in the X-ray

crystal structure of CPMV [69].

5. Concluding remarks

The sensitivity of ROA to molecular chirality makes

it a valuable new tool for investigating biomolecular

structure and behavior in solution which may now be

applied routinely to a wide range of proteins, nucleic

acids, and viruses. It is already providing informationcomplementary to that obtained from high-resolution

techniques such as X-ray crystallography and NMR

spectroscopy. Particularly promising applications for

ROA spectroscopy include high-throughput protein fold

Fig. 8. Backscattered Raman and ROA spectra of the icosahedral vi-

ruses (a) T-CPMV and (b) M-CPMV at �pH 7.0 and 20 �C, and (c) the

difference (b–a) (M-CPMV)-(T-CPMV).


recognition in structural proteomics, the protein mis-folding diseases, and structural virology. With the ex-

pected availability of a commercial instrument in the

near future, we hope that this brief review will encour-

age wide use of ROA in biomolecular science.

Acknowledgment

L.D.B. and L.H. thank the BBSRC for a research

grant.

References

[1] P.R. Carey, Biochemical Applications of Raman and Resonance

Raman Spectroscopies, Academic Press, New York, 1982.

[2] M. Diem, Modern Vibrational Spectroscopy, Wiley, New York,

1993.

[3] T. Miura, G.J. Thomas Jr., in: B.B. Biswas, S. Roy (Eds.),

Subcellular Biochemistry Proteins: Structure Function and Engi-

neering, 24, Plenum Press, New York, 1995, pp. 55–99.

[4] T.A. Keiderling, in: G.D. Fasman (Ed.), Circular Dichroism and

the Conformational Analysis of Biomolecules, Plenum Press, New

York, 1996, pp. 555–598.

[5] P.L. Polavarapu, Vibrational Spectra: Principles and Applications

with Emphasis on Optical Activity, Elsevier, Amsterdam, 1998.

[6] G.J. Thomas Jr., Annu. Rev. Biophys. Biomol. Struct. 28 (1999)

1–27.

[7] T.A. Keiderling, in: N. Berova, K. Nakanishi, R.W. Woody

(Eds.), Circular Dichroism: Principles and Applications, second

ed., Wiley, New York, 2000, pp. 621–666.

[8] B.R. Singh (Ed.), Infrared Analysis of Peptides and Proteins:

Principles and Applications, in: ACS Symposium Series, vol. 750,

American Chemical Society, Washington DC, 2000.

[9] L.A. Nafie, T.B. Freedman, in: N. Berova, K. Nakanishi, R.W.

Woody (Eds.), Circular Dichroism Principles and Applications,

second ed., Wiley, New York, 2000, pp. 97–131.

[10] L.D. Barron, L. Hecht, in: N. Berova, K. Nakanishi, R.W.

Woody (Eds.), Circular Dichroism. Principles and Applications,

second ed., Wiley, New York, 2000, pp. 667–701.

[11] L.D. Barron, M.P. Bogaard, A.D. Buckingham, J. Am. Chem.

Soc. 95 (1973) 603–605.

[12] L.D. Barron, L. Hecht, E.W. Blanch, A.F. Bell, Prog. Biophys.

Mol. Biol. 73 (2000) 1–49.

[13] P.W. Atkins, L.D. Barron, Mol. Phys. 16 (1969) 453–466.

[14] L.D. Barron, A.D. Buckingham, Mol. Phys. 20 (1971) 1111–1119.

[15] A.D. Buckingham, Adv. Chem. Phys. 12 (1967) 107–142.

[16] L.D. Barron, Molecular Light Scattering and Optical Activity,

Cambridge University Press, Cambridge, 1982.

[17] L.D. Barron, A.D. Buckingham, J. Am. Chem. Soc. 96 (1974)

4769–4773.

[18] L. Hecht, L.D. Barron, E.W. Blanch, A.F. Bell, L.A. Day, J.

Raman Spectrosc. 30 (1999) 815–825.

[19] W. Hug, G. Hangartner, J. Raman Spectrosc. 30 (1999) 841–852.

[20] A.T. Tu, Adv. Spectrosc. 13 (1986) 47–112.

[21] S.J. Ford, Z.Q. Wen, L. Hecht, L.D. Barron, Biopolymers 34

(1994) 303–313.

[22] E.W. Blanch, L.A. Morozova-Roche, L. Hecht, W. Noppe, L.D.

Barron, Biopolymers 57 (2000) 235–248.

[23] L.D. Barron, E.W. Blanch, A.F. Bell, C.D. Syme, L. Hecht, L.A.

Day, It is my book, in: Hicks, J.M. (Ed.), Chirality: Physical

Chemistry. ACS Symposium Series, vol. 810, American Chemical

Society, Washington DC, 2002, pp. 34–49.

[24] E.W. Blanch, A.F. Bell, L. Hecht, L.A. Day, L.D. Barron, J. Mol.

Biol. 290 (1999) 1–7, doi:10.1006/jmbi.1999.2871.

[25] S.-H. Lee, S. Krimm, J. Raman Spectrosc. 29 (1998) 73–80.

[26] M. Tsuboi, M. Suzuki, S.A. Overman, G.J. Thomas Jr.,

Biochemistry 39 (2000) 2677–2684.

[27] P. Hanson, D.J. Anderson, G. Martinez, G. Millhauser, F.

Formaggio, M. Crisma, C. Toniolo, C. Vita, Mol. Phys. 95 (1998)

957–966.

[28] K.A. Bolin, G.L. Millhauser, Accs. Chem. Res. 32 (1999) 1027–

1033.

[29] G. Wilson, L. Hecht, L.D. Barron, J. Chem. Soc. Faraday Trans.

92 (1996) 1503–1510.

[30] A.A. Adzhubei, M.J.E. Sternberg, J. Mol. Biol. 229 (1993) 472–

493, doi:10.1006/jmbi.1993.1047.

[31] B.T. Stapley, T.P. Creamer, Prot. Sci. 8 (1999) 587–595.

[32] T. Miura, H. Takeuchi, I. Harada, J. Raman Spectrosc. 20 (1989)

667–671.

[33] E.W. Blanch, L. Hecht, L.A. Day, D.M. Pederson, L.D. Barron,

J. Am. Chem. Soc. 123 (2001) 4863–4864.

[34] E.W. Blanch, D.J. Robinson, L. Hecht, C.D. Syme, K. Nielsen,

L.D. Barron, J. Gen. Virol. 83 (2002) 241–246.

[35] L.D. Barron, E.W. Blanch, L. Hecht, Adv. Prot. Chem. 62 (2002)

51–90.

[36] O.B. Ptitsyn, Adv. Prot. Chem. 47 (1995) 83–229.

[37] M. Arai, K. Kuwajima, Adv. Prot. Chem. 53 (2000) 209–282.

[38] P. Haezebrouck, M. Joniau, H. vanDael, S.D. Hooke, N.D.

Woodruff, C.M. Dobson, J. Mol. Biol. 246 (1995) 382–387,

doi:10.1006/jmbi.1994.0093.

[39] L.A. Morozova-Roche, J. Zurdo, A. Spencer, W. Noppe, V.

Receveur, D.B. Archer, M. Joniau, C.M. Dobson, J. Struct. Biol.

130 (2000) 339–351, doi:10.1006/jsbi.2000.4264.

[40] E.W. Blanch, L.A. Morozova-Roche, D.A.E. Cochran, A.J. Doig,

L. Hecht, L.D. Barron, J. Mol. Biol. 301 (2000) 553–563,

doi:10.1006/jmbi.2000.3981.

[41] M.R.H. Krebs, D.K. Wilkins, E.W. Chung, M.C. Pitkeathly,

A.K. Chamberlain, J. Zurdo, C.V. Robinson, C.M. Dobson, J.

Mol. Biol. 300 (2000) 541–549, doi:10.1006/jmbi.2000.3862.

[42] C.D. Syme, E.W. Blanch, C. Holt, R. Jakes, M. Goedert, L.

Hecht, L.D. Barron, Eur. J. Biochem. 269 (2002) 148–156.

[43] C. Holt, L. Sawyer, J. Chem. Soc. Faraday Trans. 89 (1993) 2683–

2692.

[44] R.W. Carrell, D.A. Lomas, Lancet 350 (1997) 134–138.

[45] C.M. Dobson, R.J. Ellis, A.R. Fersht (Eds.), Philos. Trans. R.

Soc. Lond. B, 356, 2001, pp. 127–227.

[46] Y.C. Sekharudu, M. Sundaralingham, in: E. Westhof (Ed.), Water

and Biological Macromolecules, CRC Press, Boca Raton, FL,

1993, pp. 148–162.

[47] G. Siligardi, A.F. Drake, Biopolymers 37 (1995) 281–292.

[48] M.L. Tiffany, S. Krimm, Biopolymers 6 (1968) 1379–1382.

[49] R.W. Woody, Adv. Biophys. Chem. 2 (1992) 37–79.

[50] R.K. Dukor, T.A. Keiderling, Biopolymers 31 (1991) 1747–

1761.

[51] T.A. Keiderling, R.A.G.D. Silva, G. Yoder, R.K. Dukor, Bioorg.

Med. Chem. 7 (1999) 133–141.

[52] N. Sreerama, R.W. Woody, Biochemistry 33 (1994) 10022–10025.

[53] A.F. Bell, L. Hecht, L.D. Barron, J. Chem. Soc. Faraday Trans.

93 (1997) 553–562.

[54] A.F. Bell, L. Hecht, L.D. Barron, J. Am. Chem. Soc. 119 (1997)

6006–6013.

[55] A.F. Bell, L. Hecht, L.D. Barron, Biospectroscopy 4 (1998) 107–

111.

[56] A.F. Bell, L. Hecht, L.D. Barron, J. Raman Spectrosc. 30 (1999)

651–656.

[57] A.F. Bell, L. Hecht, L.D. Barron, J. Am. Chem. Soc. 120 (1998)

5820–5821.

[58] W. Saenger, Principles of Nucleic Acid Structure, Springer, New

York, 1984.


[59] W. Chiu, R.M. Burnett, R.L. Garcia, Eds, Structural Virology of

Viruses, Oxford University Press, New York, 1997.

[60] D.A. Marvin, Curr. Opin. Struct. Biol. 8 (1998) 150–158.

[61] D.M. Pederson, L.C. Welsh, D.A. Marvin, M. Sampson, R.N.

Perham, M. Yu, M.R. Slater, J. Mol. Biol. 309 (2001) 401–421,

doi:10.1006/jmbi.2001.4685.

[62] E.W. Blanch, D.J. Robinson, L. Hecht, L.D. Barron, J. Gen.

Virol. 82 (2001) 1499–1502.

[63] A. Klug, Philos. Trans. R. Soc. Lond. B 354 (1999) 531–535.

[64] G. Stubbs, Philos. Trans. R. Soc. Lond. B 354 (1999) 551–

557.

[65] S.B. Larson, J. Day, A. Greenwood, A. McPherson, J. Mol. Biol.

277 (1998) 37–59, doi:10.1006/jmbi.1997.1570.

[66] K. Valeg�aard, L. Liljas, K. Fridborg, T. Unge, Nature 345 (1990)

36–41.

[67] E.W. Blanch, L. Hecht, C.D. Syme, V. Volpetti, G.P. Lomonoss-

off, L.D. Barron, J. Gen. Virol. 83 (2002) 2593–2600.

[68] G.P. Lomonossoff, J.E. Johnson, Prog. Biophys. Mol. Biol. 55

(1991) 107–137.

[69] T. Lin, Z. Chen, R. Usha, C.V. Stauffacher, J.-B. Dai, T. Schmidt,

J.E. Johnson, Virology 265 (1999) 20–34, doi:10.1006/

viro.1999.0038.


vibrational raman optical activity of proteins, nucleic acids, and viruses

Documents