Current Organic Chemistry, 2005, 9, 889-898 889

1385-2728/05 $50.00+.00 2005 Bentham Science Publishers Ltd.

Overview of Common Spectroscopic Methods to Determine the
Orientation/Alignment of Membrane Probes and Drugs in Lipidic Bilayers
Slvia C. D. N. Lopes
1
and Miguel A. R. B. Castanho
2
*
1
Centro de Qumica-Fsica Molecular, Instituto Superior Tcnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
2
Centro de Qumica e Bioqumica da Faculdade de Cincias da Universidade de Lisboa, Campo Grande, Ed. C8,
1749-016 Lisboa, Portugal
Abstract: The in-depth location and orientation of membrane probes and drugs inserted in lipidic bilayers are
regarded important key-properties that cannot be overlooked during molecular design and synthesis. Several
spectroscopic phenomena (e.g. excitonic interaction) and molecular recognition (e.g. ligand-receptor interaction)
depend on these properties. However, molecular orientation in lipidic membranes is scarcely addressed. This paper
overviews some of the most important techniques and methodologies used to study orientation of molecules relative
to the surrounding lipidic matrix, namely: FTIR linear dichroism, UV-Vis linear dichroism, Time-resolved
fluorescence anisotropy, NMR, and Surface Plasmon Resonance.
1. INTRODUCTION
Orientation has been a relinquished aspect when studying
membranes, receptor-mediated processes, drugs actions, and
membrane probes, over the years. Nevertheless, biological
membranes are dynamic structures where the orientation and
the molecular ordinance play a basic role in the maintenance
of their own functions. Moreover, molecular orientation (in
biological membranes) is a key-issue to understand immune
responses or cellular communication, for instance
(receptor/ligand interactions, in a broad sense). In medical
technology, for example, orientation of antibodies and
enzymes determines the sensitivity and efficiency of
diagnostic tests [1, 2].
In the membrane catalysis model, Sargent and
Schwyzer proposed that peptides would interact with
membrane lipids in order to adopt the necessary
conformation for docking cell receptors [3]. This way the
molecular mechanism of receptors mediated processes is
based both on receptor and on membrane requirements. In
the case of peptides, the location and orientation of some
specific amino acid residues are crucial factors for both the
interaction with cell receptors, and biological activity (e.g.
tyrosine residue in enkephalin neuropeptides [4]). Many of
these molecular mechanisms of receptors mediated processes
are related with health problems (e.g. pain and neurological
disorders) and one of the main research objectives is to
conceive drugs that are able to eliminate, diminish and/or
relieve the symptoms. Different strategies can be used to
target drugs to its site of action: (1) natural products can be
chemically modified into analogues that are able to maintain
fundamental properties but are better assimilated by the
organism, potentiating its use; (2) new chemical products
can be synthesized so they can mimic natural products and
* Address correspondence to this author at the Centro de Qumica e
Bioqumica da Faculdade de Cincias da Universidade de Lisboa, Campo
Grande, Ed. C8, 1749-016 Lisboa, Portugal. Tel: +351 21 7500931; Fax:
+351 21 7500088; E-mail: castanho@fc.ul.pt
(3) drugs can be associated with a drug carrier system.
However these drugs or analogs must mimic the natural
approach to the natural receptor with favorable orientation
and correct exposure. Therefore orientation must be a
fundamental aspect to be considered in drug designing.
Orientational information on membrane probes is very
scarce. Most times our notion of probes orientation relative
to the membrane surface is guided by chemical intuition
based on what is known for the bilayers structure, mainly the
conformation of the acyl chain of lipids [5, 6]. Nevertheless,
although sometimes chemical intuition proves realistic [7],
others is illusory [8]. Depending on the membrane property
to be reported or the phenomena involved (e.g. excitonic
interaction), probe orientation may be an important feature to
be considered during design and performance tests.
2. INFRARED SPECTROSCOPY (IR)
Although possible, transmission infrared absorption
spectroscopy is not usually used to study molecular
orientation. Technical difficulties and low reproducibility
prevents its ordinary application [9]. Internal reflection
techniques have been therefore long and more widely used
since they circumvent most of these difficulties. Attenuated
total internal reflection-Fourier transform infrared
spectroscopy (ATR-FTIR) methodology is one of the most
popular and it has been used to conclude on the average
secondary structure and orientation of biologically important
molecules (e.g. membrane proteins) inserted in lipid
multibilayers (for reviews see references [10-12]). In a
typical ATR-FTIR experiment, a trapezoidal cell (so-called
internal reflection element (IRE), usually made of
germanium or ZnSe), is covered with lipid multibilayers
containing the molecule under study. These multibilayer
films are obtained by applying an aqueous lipid suspension
onto one side of the ATR crystal and semi-drying it under a
gentle stream of nitrogen. The infrared beam is then focused
into the IRE and the light crosses the plate from one side to
the other, by a series of internal reflections, originating an
evanescent wave (for detailed description see references [10,
890 Current Organic Chemistry, 2005, Vol. 9, No. 9 Lopes and Castanho
11]). This harmonic electromagnetic wave, characterized by
its amplitude, and which oscillates at the same frequency as
the incident radiation, propagates along the IRE surface and
decays exponentially in the direction perpendicular to the
interface. The absorption of the energy of the resulting field
by the lipids and other molecules there inserted (e.g.
proteins) generates the ATR-FTIR spectrum which contains
information about the sample. In macroscopically ordered
deposited membrane on a solid support, as the ones used in
ATR-FTIR experiments, every transition dipolar moments in
the membranes molecules have the same average orientation
relative to the normal of the IRE surface. Therefore if one
changes the orientation of the electric field component, by
means of a polarizer, it is possible to detect orientational
dipole changes. Maximum or minimum light absorption will
be observed, if the dipole transition moment is parallel or
perpendicular to the electric fields component of the incident
light, respectively (Fig. (1)). The difference between the
spectra recorded with parallel and perpendicular
polarizations is denominated dichroic spectrum and it is the
one that possesses orientational information (Fig. (2)). A
larger absorbance for the parallel polarization indicates a
dipole preferentially oriented near the normal of the ATR
plate. However if a larger absorbance is observed for the
perpendicular polarization a dipole oriented parallel to the
ATR plate can be inferred. Nevertheless more detailed
orientational information can be obtained by the calculus of
the dichroic ratio:
R
A//
A
(1)
which is the ratio between the integrated absorption of a
band measured with, a parallel and a perpendicular
polarization of the incident light, respectively. This dichroic
ratio is related to an orientational order parameter (<P
2
>):
R a + b 1+
3 P2
1 P2



_
,

(2)
in which
P2
3cos
2
1
2
(3)
and where a and b depend on the time average square electric
field amplitudes of the evanescent wave in the film at the
IRE/film interface and refers to the angle between the
transition dipolar moment and the normal to the cell. Usually
ATR-FTIR uses a combination of several characteristic
bands to conclude about the orientation and/or second
structure (for examples see references [12-14]). However the
dipole transition moment is not always easily related to the
molecular axis and sometimes is necessary to combine ATR-
FTIR studies with simulation studies in order to relate them
[15]. In those cases it may be convenient to convert the order
parameter obtained for the dipole transition moment to the
one related to the molecular axis so that the overall
molecular orientation can be inferred. This can be achieved
by using:
P2
3cos
2

'
1
2
P2
'
(4)
Where <P
2
> and <P
2
> refer to axis with relative
orientation between them. A more complex expression
can be derived when one deals with -helix proteins, in
which the experimental <P
2
> is a product of three individual
order parameters: (1) of the membrane with respect to the
IRE, (2) of the helix within the membrane plane and (3) of
the dipole orientation of amide I or amide II with respect to
the helix axis (for details see references [11, 16]).
It should be stressed that only one order parameter,
<P
2
>, can be calculated by ATR-FTIR measurements and
that previous knowledge on the sample thickness is needed,
which can be quite complex to achieve [11]. Another
problem is the superimposition of the vibrational bands of
the lipidic matrix with the molecules under study that may
bias or even prevent <P
2
> calculation. To overcome this
limitation Arkin and co-workers [17] have developed an
alternative method to study the structure of -helical
proteins, site-specific infrared dichroism (SSID), which can
be combined with FTIR. SSID uses oriented lipid bilayers
which incorporated, in the original idea, -helical proteins
isotopically labelled at specific residues [17]. Samples are
then analysed using infrared polarised light in a classical
way. The method overcomes the superimposition of bands
Fig. (2). Schematic representation of the internal reflection element
(IRE) used in ATR-FTIR experiments. // and denote the
polarized direction relative to the normal of the IRE surface.
Fig. (1). ATR-FTIR spectra of polyene antibiotic filipin in DLPC
membranes for parallel (A) and perpendicular (B) orientations of
the polarizer at pH 7.4. Dichroic spectrum (C) was obtained as //
minus .
Overview of Common Spectroscopic Methods Current Organic Chemistry, 2005, Vol. 9, No. 9 891
by isotope-label shifting of a specific vibrational mode so
that its dichroism can be directly measured and which can be
used in a large classes of molecules (not only proteins). As
the spectral contribution of the chromophore depends on its
orientation, a three dimensional description of the backbone
of the protein and orientation of a specific group/bond can be
obtained.
Nevertheless, and regardless of some limitations
presented, ATR-FTIR has three major advantages over other
techniques, which are: (1) light scattering problems are
insignificant, (2) sometimes, depending on the molecules to
be studied, it is possible to simultaneously study these
molecules and lipids and (3) small sample volumes (~10l)
are usually needed.
3. UV-VIS LINEAR DICHROISM
In isotropic solution the molecular orientations of
fluorophores are randomly distributed and even if polarized
light is used what is really measured is a spatial average
signal resulting from this distribution. However, in
macroscopically aligned membranes [18] information about
the orientation of the transition dipolar moment can be
obtained through the dependence of linearly polarized light
absorption on the angle formed by light polarization and
sample orientation [18], which is generally called linear
dichroism (LD). Sample preparation is critical in LD
techniques since disordered lipidic matrixes may introduce
artifacts in the orientational distribution of biomolecules
under study. Supported multibilayers can be obtained by
several methods as Langmuir-Blodgett (LB) technique [19,
20], spin-coating [21], vesicle fusion in a quartz substrate
[22], and by semi-drying of an aqueous lipid suspension
under a gentle stream of nitrogen onto one side of the solid
support (quartz slide in UV-Vis LD or IRE in ATR-FTIR
experiments) [11, 18]. To know details regarding sample
properties, namely hydratation, thickness and homogeneity
obtained by the different methods see references [23-26].
In a LD experiment, a quartz slide is covered with sample
and its absorption variation (A

) with the measured angle

(, that is the angle formed by the polarization direction
relative to the system director; Fig. (3)), using polarized light
is given by:
(5)
(sin() and the relative refractive index, n, are introduced to
account for experimental artifacts [18]). The term
sin()A

/A
/2
is the dichroic ratio and <P
2
> is obtained
from equation (3) and regards the electronic absorption
transition moment distribution relative to the normal of the
membrane. If the molecular axis and the transition moment
are parallel, the molecular distribution function has the same
<P
2
>.
LD methodology allows the calculation of another order
parameter, known as the fourth rank order parameter (<P
4
>).
<P
4
> is calculated by means of a steady-state fluorescence
spectroscopy having excitation and emission in a 90 angle
geometry, using polarized light [27] (Fig. (4)). If the
absorption and emission dipoles are parallel to the molecular
symmetry axis, <P
4
> can be calculated from:
(6)
( is the angle depicted in Figure 4; m and b depend on both
<P
2
> and <P
4
>, and G, f() and n are correction factors
[18, 27]). The subscripts in I
ij
refer to the position of the
excitation (i) or emission (j) polarizers in the lab frame (v
vertical; h horizontal)).
<P
2
> is known from absorption experiments and <P
4
> can
be obtained from the linear fit to the GI
vh
/(f().I
vv
) vs. sin
2

data, moreover <P

4
> can be calculated from both the slope
and the intercept of GI
vh
/(f().I
vv
) vs. sin
2
. A specific value
of <P
2
> limits the range of possible <P
4
> (eq.7) [27].
P4
min

35 P2
2
10 P2 7
18



_
,
P4
5 P2 + 7
12



_
,
P4
max
(7)
The correction factors involved in <P
2
> and <P
4
>
determination are discussed in detail in reference [27].
However two significant factors should be taken in special
account: (1) the angular dependent propagation of light
passing trough an interface of two isotropic media having
different refraction indices, and (2) the transmission fraction

1
Reprinted from Spectroscopy Int. J., 17, M. A.R.B. Castanho, S. Lopes, M.
Fernandes, Using UV-Vis. Linear dichroism to study the orientation of
molecular probes and biomolecules in lipidic membranes, 377-398,
Copyright (2003), with permission from IOS Press
Fig. (3). Schematic representation of the experimental set-up for
UV-Vis. absorption LD studies in lipidic membranes (not drawn to
scale). The incident beam (A), with polarization B (horizontal in
the lab frame), impinges on the quartz slide and supported bilayers
(only one bilayer is represented for the sake of clarity). The angle
between them () equals the one formed by the system director
(ZZ) and the light polarization (B). variation is accomplished by
rotation of the quartz slide around the axis where A and B
intersect.
1
sin ()A

2
=
3
P
2
cos
2
1 +
1 n
2
P
2
=
A
GI

h
f ' ()I
= m sin
2
+ b
892 Current Organic Chemistry, 2005, Vol. 9, No. 9 Lopes and Castanho
of light passing trough a boundary of two media being
dependent on its polarization state.
Since it is possible to experimentally access two order
parameters, (<P
2
> and <P
4
>) UV-Vis LD methodology
offers, as major advantage, the possibility of an orientational
distribution function (f()) calculation ( is the angle
between the long axis of a molecule and the system director,
which is the normal to the bilayer plane) and the respective
probability density function (f()sin()). f() can be
described as a Legendre polynomial series [27]:
f ( )
1
2
2L+ 1 ( ) PL PL cos ( )
L e v e n

(8)
(P
L
(cos) are Legendre polynomials; <P
L
> is the
ensemble-average of P
L
(cos) and is referred to as the L
th
rank order parameter). However, and since only <P
2
> and
<P
4
> can be known from experiment, the quest for
f()sin() resumes to two steps: (1) <P
2
> and <P
4
>
determination (previously explained), and (2) finding an
approximated function for f()sin() from <P
2
> and <P
4
>
only. The most common realistic approximation combines
the application of the maximum entropy method with the
Lagrange multipliers method [28] in which the resulting
distribution is the broadest possible from all the universe of
distributions having that particular (<P
2
>, <P
4
>) pair. (for
details see ref [29] and Fig. (5)). Table I summarizes the
different types of distributions obtained for particular (<P
2
>,
<P
4
>) pairs [30].
UV-Vis. LD methodologies have been applied in a large
number of studies involving different classes of molecules
such as membrane probes [7, 8], polyene macrolide
antibiotics [31, 32], multifunctional peptides [33, 34] and
DNA-ligand systems [35].
UV-Vis. LD techniques present numerous advantages
such as: (1) can be easily implemented, from both the
instrumental and methodological points of view, (2)
instrumental adaptation, sample preparation and data
analysis are simple, and <P
2
> and <P
4
> can be obtained for
strongly absorbing and emitting molecules, enabling a quite
straightforward estimate of the orientational density
probability function. Moreover for samples having a small
absorption values, <P
2
> obtained from other LD techniques
(e.g. ATR-FTIR LD) can be used, as long as the same system
director is considered.
Recently fluorescence imaging of two-photon LD have
been applied to study molecular orientation in cell
membranes [36] which represents a new insight and a
promising tool in orientational studies.
4. TIME-RESOLVED FLUORESCENCE ANISO-
TROPY DECAYS
Fluorescence emission anisotropy measurements can be
used to obtain information on size, shape and location of
biomolecules and/or rigidity of various molecular
environments (e.g. phospholipids) [37]. Large unilamellar
vesicles (obtained by the extrusion method [38]) with
different phospholipids composition are the most common
model system of membranes used in these studies.
Anisotropy measurements are based in a principle of

2
Reprinted from Spectroscopy Int. J., 17, M. A.R.B. Castanho, S. Lopes, M.
Fernandes, Using UV-Vis. Linear dichroism to study the orientation of
molecular probes and biomolecules in lipidic membranes, 377-398,
Copyright (2003), with permission from IOS Press
3
Reprinted from Biophys J., 68, M. A. Bos and J. M. Kleijn, Determination
of the orientation distribution of adsorbed fluorophores using TIRF. I.
Theory, 2566-2572, Copyright (1995), with permission from Byophysical
Society
Fig. (4). Schematic representation of the experimental set-up for
fluorescence LD studies in lipidic membranes (not drawn to scale).
The excitation beam (A), with polarization horizontal (H) or
vertical (V; perpendicular to the figure plane) in the lab frame,
impinges on the quartz slide and supported bilayers (only one is
represented for the sake of clarity). Fluorescence light collection
(B) is carried out in a 90 angle geometry The angle between them
() equals the one formed by the system director (ZZ) and the light
polarization (B). Rotation of the quartz plate causes a variation in
angle .
2
Fig. (5). Interrelation between the order parameters <P
2
> and
<P
4
>. The physical boundaries of <P
2
> and <P
4
> are indicated by
solid curves. The insets show the shape of the distribution function
f() for between 0 and 90 as calculated following the
maximum-entropy method.
3
Overview of Common Spectroscopic Methods Current Organic Chemistry, 2005, Vol. 9, No. 9 893
photoselectivity; fluorophores preferentially absorb photons
whose electric vectors are aligned parallel to its transition
moment [37], which in turn has a defined orientation relative
to the molecular axis. In general fluorescence emission
anisotropy, r, can de defined as
r
I// I
I // +2I
(9)
where I
//
and I

are the intensities of the polarization

components parallel and perpendicular to the polarization of
the excitation radiation, respectively. However the
anisotropy in its functional form is:
r
Ivv GIvh
I vv +2GIvh
; G
I hv
I hh
(10)
(where G is a correcting factor that accounts for the different
efficiency of the detecting system for parallel and
perpendicular polarized light and the two subscripts in I are
used to indicate the orientation of the excitation and
emission polarizers, respectively; v-vertical, h-horizontal).
Table 1. Summary of the different types of distributions obtained for different <P
2
> and <P
4
> as discussed in reference [30]
<P2> <P4> Distribution
0 0 Total disorder (random)
3cos
2
c 1
2
<P4>MIN Total order fixed orientation along
c arccos
2
3
P2 +
1
3



_
,

1
2
(conic surface distribution)
1
2
cos0 1+ cos0 ( ) ( )
1
8
cos0 1+ cos0 ( ) 7cos
2
0 3 ( )
Analogous to a particle in a conic box:
homogeneous distribution inside the cone
of surface at angle 0.
f ( )
1
2 1+ cos0 ( ) ( )
if 0<<0
( ) 0 f if >0
0 unknown The molecular position is considered
dependent on its interaction with a Maier-
Saupe (Gaussian) angular potencial.
When only <P2> is known, f() is
obtained from
f ( )
exp 2P2 cos ( )
sen ( ) exp 2P2 cos ( ) ( )
d
0

(Equation 18 in reference [18] with 4=0.)

3 < cos
2
> 1
2
P4
MAX

5 < P2 > +7
12
When <P4>=<P4>MAX, a double
function is obtained along the parallel and
perpendicular orientations relative to the
lipidic bilayer plan. f() is described
by
f ( )
exp 4P4 cos ( )
sen ( ) exp 4P4 cos ( ) ( )
d
0

(Equation 18 in reference [18] with 2=0.)

894 Current Organic Chemistry, 2005, Vol. 9, No. 9 Lopes and Castanho
The fluorescence anisotropy decay (anisotropy is followed in
a nanosecond time scale, after pulse excitation of the
fluorophores), r(t), depends both on the molecular
orientational order and dynamics during the fluorophores
excited state. At t=0, r(t), is only dependent on intrinsic
spectroscopic characteristics of the fluorophore. Maximal
value for anisotropy is r(t=0)=r
0
=0.4 in macroscopically
random distributions of molecular orientation (i.e.
macroscopically isotropic systems). r
0
will decrease with
time, trough Brownian rotation, and will tend to zero in an
unrestricted isotropic distribution. However, fluorophores
that follow orientational distributions that are restricted (as
most molecules inserted in lipidic membranes) lead to
limiting anisotropies, r

(r(t) in the limit t ), different

from zero (i.e. depolarisation is not complete, Fig. (6)). r
0
and r

are related to the second rank order parameter (<P
2
>
r
)
can be obtained by the analysis of the decay trough the
following relation:
r r0 P2
r
2
(11)
Nevertheless this relation is only valid if: (1) the
transition moments are parallel to the symmetry axes of the
molecule, and (2) if the orientational distribution function is
the same in the excited and ground state. A more general
equation would be [39]:
r r0 P2
r
P2
r
*
(12)
(* denotes an excited state parameter).
No information can be obtained on the orientation of the
director axis relative to the lipidic matrix, since they are
unoriented, but both equations hold true regardless of the
orientation of the director axis relative to the membrane
surface [18]. One of the major disadvantages of this method
is that the molecular orientation relative to the membrane
cannot be known; nevertheless options can be restrained (for
more detailed discussion see reference [18]) and attempts
have been made to circumvent the limitation [40].
The r(t) decay from r
0
to r

is not only dependent on

<P
2
>
r
but also dependent on the fourth rank order
parameter, <P
4
>
r
, and the rotational diffusion rate of the
fluorophore around an axis perpendicular to the transition
dipole, D

. Zannoni et al. [41] attained a quantitative model

for r(t), assuming that D

is the same in the whole bilayer.

Van der Meer et al. [42] worked out simplified approximated
equations (see equations 13 to 20), consisting of the sum of
three exponentials plus a constant in which <P
2
>
r
, <P
4
>
r
and D

can be obtained as fitting parameters in unoriented

vesicles (e.g. [43]). A more detailed knowledge about
fluorophore orientation can be obtained through an
orientational distribution function which can be derived,
using the same approximation used in UV-Vis LD
methodology.
r(t) r0 gi exp(t i )
i1
3

+ g4




_
,


(13)
g1
1
5
+
2 P2
r
7
+
18 P4
r
35
P2
r
2
(14)
g2
2
5
+
2 P2
r
7

24 P4
r
35
(15)
g3
2
5

4 P2
r
7
+
6 P4
r
35
(16)
g4 P2
r
2
(17)
1 g1 6D
1
5
+
P2
r
7

12 P4
r
35



_
,




1
]
1
(18)
2 g2 12D
1
5
+
P2
r
14
+
8 P4
r
35



_
,




1
]
1
(19)
3 g3 12D
1
5

P2
r
7

2 P4
r
35



_
,




1
]
1
(20)
Unusual time-resolved fluorescence anisotropy decays,
in which an initial decay is followed by a rise, are sometimes
observed (e.g. [32, 40, 44]). In general this phenomenon has
been interpreted as the result of microheterogeneity of
environments for the fluorophores, which may be a result of
a variation of local lifetime and/or local fluorescence
anisotropy.
5. RAMAN SPECTROSCOPY
Raman spectroscopy is related to changes in
polarizability associated with molecular vibrations and is
usually performed with green, red or near-infrared lasers
[45]. These changes give rise to scattered radiation which
can be collected in a specific direction, usually named
analyzer direction [45]. The wavelengths, which are usually
below the first electronic transitions of most molecules (as
assumed by the scattering theory), and intensities of the
scattered light can be used to identify functional groups in a
molecule. Because the intensity of the Raman signal is
inversely proportional to the fourth power of the excitation
wavelength, it is advantageous to use as short wavelengths as
possible. Polarized Raman spectroscopy has been used to
conclude on the orientation of diverse molecules such as,
single wall carbon nano tubes [46], protein [47, 48] or DNA
residues [49]. Nevertheless the most popular Raman methods
Fig. (6). Time-resolved fluorescence anisotropy decay of polyene
antibiotic filipin in aqueous solution (A) and inserted in large
unilamellar vesicles of DPPC (B), at room temperature.
B
A
Overview of Common Spectroscopic Methods Current Organic Chemistry, 2005, Vol. 9, No. 9 895
have been the ones that used the so-called resonance Raman
effect. In fact, if the wavelength of the exciting laser is
within the range of the electronic spectrum of a molecule, the
intensity of some Raman-active vibrations increases by a
factor of 10
2
-10
4
(Resonance-Enhanced Raman Scattering,
[50, 51]). Surface-enhanced Raman spectroscopy (SERS)
has been used to study orientation of several molecules in
contact with lipid monolayers deposited on planar supports
[52]. Surface-enhanced Raman spectroscopy needs the
presence of a thin silver layer (15-20 nm thick) deposited on
the planar support before the transfer of the planar mono- or
bilayer (through the Langmuir-Blodgett technique [19])[50-
52]. These conditions makes it possible to enhance the
Raman signal of deposited molecules (by six orders of
magnitude), which otherwise could not be detected. Because
of the short range of SERS, the observed Raman spectrum is
mainly due to the monolayer in contact with silver. The
SERS effect can be increased, if the laser wavelength used is
in the absorption band of the molecule under study (surface-
enhanced resonance Raman scattering, SERRS). The
scattered light is then collected in a direction perpendicular
to the surface of the sample and orientation can be conclude
from the analysis of characteristic bands. Moreover,
Resonance Raman spectroscopy is also a major probe of the
chemistry of fullerenes, polydiacetylenes and many other
molecules which strongly absorb visible radiation [50-52]. A
variation to this technique is the deep ultra-violet resonance
Raman (UVRR) spectroscopy. UVRR uses a UV laser source
and is a potentially powerful tool because at a wavelength
below 250nm fluorescence emission do not interfere the
UVRR spectrum and it can provide similar information
obtained from IR without the same level of interference from
water [53]. However one of the most powerful recent
methods to study orientation is the Raman linear intensity
difference (RLID) method. This method was first described
by Takeuchi and co-workers [54] to study orientation of an
indole ring in a filamentous virus, where the authors derived
an equation relating the RLID to the orientation of the
ultraviolet resonance Raman (UVRR) chromophore. In RLID
the molecules under study are aligned by hydrodynamic
shear force in a flow cell composed of an outer rotating
cylinder and an inner stationary rod (flow orientation) (for
more experimental details see reference [54]). The difference
between the two resonance Raman spectral intensities,
obtained by the UV laser, with parallel and perpendicular
polarizations with respect to the direction of the alignment,
gives information about chromophores orientation. The
reduced RLID, , is defined as [54]:

3 I// I ( )
I// +2I

3(5cos
4
+ 6cos
2
3)
2(cos
4
+ 3)
(21)
(where is the angle of inclination of the transition moment
under study with respect to the flow orientation). However
this equation only olds true if all the molecules have perfect
uniaxial alignment; if not (as in most of the cases), a
correction for the fraction of randomly oriented particles as
to be taken into account ([54]; equation (22)):

15 f (5cos
4
+6cos
2
3)
10 f (cos
4
+3)
(22)
More details can be obtained in references [54-56].
One of the major advantages of Raman spectroscopy is
that it can provide similar information to the one obtained,
for example from infra-red spectroscopy, without the same
level of interference from water absorption which is ideal for
studying biological systems. However, Raman spectroscopy
is not more widely used for orientation determination
because: (1) when visible wavelength lasers are used as the
excitation source (because they are reliable and cheap)
fluorescence is also excited and this swamps the Raman
scattered signal making the measurements virtually
impossible, and (2) due to the high cost of lasers and optics
for UV spectral region suitable for Raman spectroscopy.
6. SURFACE PLASMON RESONANCE
Surface plasmon resonance (SPR) has been one of the
most used optical techniques for studying surface and
interfacial phenomena in pharmaceutical and analytical
chemistry research [57] structural properties (e.g. metals-
electrolyte surfaces and lipid bilayers, [58]), and, more
recently, for the study of membrane biochemistry and
biophysics [59, 60]. The SPR technique exploits the fact that
light, at certain resonance conditions, excites a wave, which
results from collective oscillations of conducting electrons
(plasmons) in a thin metallic film, providing a source of an
evanescent electromagnetic field, which can probe the
optical properties of systems in direct contact with the
plasmon-generating medium [61, 62]. The resonance is
achieved by varying the incident light wavelength at a fixed
angle (at or above the critical angle) or by changing the angle
(at a fixed wavelength) [62]. The amplitude of the plasmon
electromagnetic wave is maximal at the interface between
the plasmon-generating and the emergent medium (air, water
or lipid film in contact with water) [62]. In practice SPR uses
a polarized laser wave to excite plasmons on a thin silver (or
gold) film deposited onto the front of a glass prism. A Teflon
sheet, with a central pin-hole (which is coupled to the
metallic film), allows the spreading of small amounts of the
molecules/system under study, which in some controlled
conditions may spontaneously orientate on the solid support.
The most common solid supports are made of silver or gold
and can have highly variable surfaces, including
carboxymethylated surfaces, dextran free flat surfaces and
chelated nickel surfaces (for binding His-tagged ligands).
SPR makes use of the evanescent wave, which decays
exponentially with the penetration distance, to sense the
optical properties of the metal/system interface without any
interference from the properties of the bulk volume [57, 62].
The refractive index near a sensor surface, for instance, can
be used to conclude on receptor-ligand interactions and
probing mechanisms of drug/lipid membrane interactions
[60]. However in the most common SPR methodology used,
the plasmons are generated only by perpendicular polarized
component of the excitation light relative to the film surface
and more detailed conclusions about orientation of the
systems is prevented. With the purpose of having more
detailed information about anisotropic systems Salamon and
co-workers developed a new variant of this technique, in the
late nineteens, which they called coupled plasmon-
waveguide resonance (CPWR) spectroscopy [63]. CPWR
couples SPR and waveguide modes being more effective for
characterize anisotropic biological systems (as membranes)
and events which occur there.
896 Current Organic Chemistry, 2005, Vol. 9, No. 9 Lopes and Castanho
In CPWR the thin metal film is coated with a thicker
silica layer (which is not present in SPR). As consequence
the molecules that are attached at the outer surface of the
silica, and under the appropriate experimental conditions,
can be excited by light polarized perpendicular (p) or parallel
(s) to the Ag-SiO
2
plane. By measuring resonances with both
p and s excitation CPWR allows the study of the anisotropic
optical properties of biomembranes systems (e.g. drug-lipid
or protein-lipid interactions). The degree of molecular order
is characterized by the refractive-index anisotropy (A
n
) or by
the absorption anisotropy (A
k
). A
n
is related to the mean
polarizabilities along the p and s polarizations. A
k
is related
to the degree of order of molecular segments containing
chromophore groups that absorb at the excitation wavelength
[64]. A
k
is proportional to <P
2
>:
Ak
3
2
3 cos
2
1 ( ) 3 P2
(23)
(where is the tilt angle of the molecular principal axis
relative to the surface normal).
As in LD studies if the molecules are macroscopically
aligned, and the orientation of the transition dipoles with
respect to the molecular axes are known, the anisotropy
provides overall information about molecules orientation.
The major advantages of CPWR are: (1) the detection
method allows the measures to be performed in either time-
resolved or steady-state modes, (2) its high sensitivity, (3)
high simplicity and (4) is fast, which are crucial factors
mainly when a small quantity of the sample is available.
7. NMR
NMR has met important advancements in the domain of
membrane-inserted peptide orientation recently [65-67].
Such advancements have been thoroughly reviewed by
Bechinger et al. in 2004 [68], including peculiar sample
spinning experimental set-ups, which will not be covered in
this paper. Only the basics of solid-state NMR applied to
static samples and oriented samples are within the scope of
this review. Readers are referred to references [69] and [68]
for additional information on motional averaging, rotational
dynamics and dipolar couplings, for instance, and overview
of peptides where the methodologies have been applied.
Likewise, segmental orientation of flexible molecules (e.g.
[70]) will not be addressed.
Considering the amide groups in peptides labeled with
15
N in such dilution that interactions between different labels
are negligible and that
1
H and
15
N nuclei are decoupled, a
broad (~160 ppm) powder-pattern is observed in solid state
NMR (Fig. (7D), [68]). However under adequate orthogonal
coordinates (the principal axis system) the diagonal
elements of a 33 matrix tensor (
11
,
22
,
33
; the
terminology and approach of references [69] and [68] are
adopted) suffice to describe the system. The tensor results
from the arrangement of the nuclei and bonds in the
molecular frame and can be converted into any other
coordinate systems. Namely, it is possible to relate the
orientation of the tensor within the molecule with the
magnetic field direction of the NMR spectrometer
(laboratory macroscopic frame). The component of the
chemical shift tensor projected in the magnetic field
direction (ZZ component) is related to the experimental
NMR chemical shift value. When expressed in terms of
Euler angles and (Fig. (7E), [68]) and the elements of
the chemical shift tensor
11
,
22
and
33
, the measurable
zz
is:
zz 11 sin
2
cos
2
+ 22 sin
2
sin
2
+ 33 cos
2
(24)
A possible graphical depiction of the chemical shift is an
ellipsoid (Fig. (7E), [68]) where the length of the three main
axes are 1/ ii
(i=1, 2, 3). The intersect of the magnetic

4
Reprinted from Biochim. Biophys. Acta Biomemb., 1666, Bechinger, B.;
Aisenbrey, C.; Bertani, P., The alignment, structure and dynamics of
membrane-associated polypeptides by solid-state NMR spectroscopy,
Copyright (2004), with permission from Elsevier
Fig. (7). Proton-decoupled
15
N solid-state NMR spectra of the in-
plane oriented model peptide LK15 in C20-PC with alignments of
the membrane normal parallel (A) and perpendicular (B) to the
magnetic field direction. (C) Simulated powder spectra under
conditions of fast rotational diffusion around the membrane normal.
(D) Static simulated powder spectrum. (E) The
15
N chemical shift
tensor is represented as an ellipsoid. (F) Transmembrane-inserted
helical peptide is represented as a cylinder and the tilt and rotational
pitch angles and are indicated. The approximated alignment of
the static tensor elements is shown within the helix. Fast rotation
around the bilayer normal results in averaged tensor elements
//
and

.
4
Overview of Common Spectroscopic Methods Current Organic Chemistry, 2005, Vol. 9, No. 9 897
field vector with the ellipsoid corresponds to 1/ zz
with
zz
being the apparent chemical shift at a given orientation of the
molecule [68]. To convert the NMR (label) information into
structural molecular information, it is mandatory to know the
positioning of the tensor elements within the molecule. The
proton-decoupled
15
N chemical shift tensors in the peptidic
bond show modest variation with respect to the secondary
structure of the peptide chain ([68] and references therein).
Using molecular models Bechinger and co-workers [68]
have shown that the
33
tensor is oriented within a few
degrees along an -helix long axis (Fig. (7F), [68]). Figure
(8) shows simulations of
15
N solid-state NMR obtained from
a model for -helical peptide reconstituted into oriented
membranes with the membrane normal parallel to the
magnetic field direction [68]. To create the simulated
spectra, Bechinger and co-workers [68] have considered all
backbone atoms of the 18-residue helices were labeled with
15
N. The measured
15
N chemical shift is a function of both
the tilt angle () and the rotational pitch angle (, Fig. (7F),
[68]).
8. FINAL REMARKS - OTHER TECHNIQUES
Other techniques have contributed to enlighten molecular
orientation in lipidic matrices, although not as extensively as
the ones mentioned above. Two of them are worth
remarking.
Electron spin resonance has long been used with spin
labeled molecules inserted in oriented membranes [70]. At
first the technique was used to gather information on the
alignment of supported multibilayers and membranes but
was later used to conclude on the orientation of membrane
proteins (e.g. [71]). Recent developments include the
magnetical alignment of phospholipidic bicelles [72], which
have been used for instance to study the structural
orientation and dynamics of a stearic acid, in combination
with NMR spectroscopy [70]. However, the intrinsic
complexity of the technique associated to a scarce diversity
of spin-labels hinders its generalized application.
Fluorescence resonance energy transfer (FRET)
phenomena depend on the relative orientation of donor and
acceptor molecules. Nevertheless, it is not usually used for
orientation determination because this dependence is very
weak [73]. Moreover, orientation relative to the lipidic
surface is difficult to ascertain and the results may be biased
by the uncertainty in the donor-acceptor distance and
refractive index.
In the future, single molecule orientation will probably be
a hot topic. So far only a few experiments in very controlled
conditions have succeeded. Recently fluorescence imaging
of two-photon LD have been applied to study molecular
orientation in cell membranes [36], which represents a new
insight and a promising tool in orientational studies.
ACKNOWLEDGEMENTS
The authors acknowledge Fundao para a Cincia e
Tecnologia (Portugal) for funding and grant SFRH / BD /
6497 / 2001 to S. Lopes. Reproduction permission is
acknowledged to IOS Press (Figures 3 and 4), Biophysical
Society (Figure 5), Elsevier (Figures 7 and 8) and respective
articles authors.
ABBREVIATIONS LIST
ATR-FTIR = attenuated total internal reflection-Fourier
transform infrared spectroscopy
CPWR = coupled plasmon-waveguide resonance
DLPC = dilauroylphosphatidylcholine
DPPC = dipalmitoylphosphatidylcholine

5
Reprinted from Biochim. Biophys. Acta Biomemb., 1666, Bechinger, B.;
Aisenbrey, C.; Bertani, P., The alignment, structure and dynamics of
membrane-associated polypeptides by solid-state NMR spectroscopy,
Copyright (2004), with permission from Elsevier
Fig. (8). Simulations of proton-decoupled
15
N chemical shift
spectra of helices oriented with their long axes at the angles
indicated. The sum of the spectra from the individual amide sites is
shown a single site label will, therefore, be found within the
anisotropic chemical shift dispersion indicated. During the
simulations, the static main tensor elements were 223, 75 and 61
ppm with an alignment of the tensor elements within the molecular
frame.
5
898 Current Organic Chemistry, 2005, Vol. 9, No. 9 Lopes and Castanho
FRET = fluorescence resonance energy transfer
IRE = internal reflection element
LD = linear dichroism
LUV = large unilamellar vesicle
NMR = nuclear magnetic resonance
RLID = Raman linear intensity difference
SERRS = surface-enhanced resonance Raman
scattering
SERS = surface-enhanced resonance Raman
spectroscopy
SPR = surface plasmon resonance
SSID = site-specific infrared dichroism
UVRR = ultra-violet resonance Raman
UV-Vis = utra-violet-visible
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