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Spectroscopy 24(10) 1
Raman Spectroscopy as a Tool for
Investigating Lipid–Protein Interactions
Raman spectroscopy is a very well-established technique for noninvasive probing of chemical compounds. The fact that Raman scattering is an inherently weak effect has prompted
many new developments in sample signal enhancement and techniques (such as surfaceenhancement Raman spectroscopy [SERS]) as well as improved technical equipment for signal capture (such as improved sensitivity of charge-coupled devices [CCDs]). Combined, these
technological advances have brought Raman spectroscopy into a new era in which hitherto
inaccessible or hardly accessible research areas now are becoming possible to study using
noninvasive vibrational spectroscopy.
Frederic N.R. Petersen and Claus Hélix Nielsen
A
particular area of biophysical research in which
Raman spectroscopy has great potential is the study
of lipid–protein interactions. It is becoming increasingly clear that lipids can modulate membrane-embedded
protein function and vice versa. This modulation can take
both specific and nonspecific forms, but the underlying
framework is defined by the amphiphatic nature of lipids
and proteins. Proteins are embedded in the lipid bilayer by
virtue of their hydrophilic and hydrophobic moieties expressed in the notion of hydrophobic coupling. Of special
interest is the preferential enrichment of aromatic residues
(for example,tryptophan [Trp]) in the interface between the
hydrophilic aqueous phases and the hydrophobic bilayer
core. The reason for this and its relation to hydrophobic
coupling is not clear despite intensive research.
In this article, we will discuss the feasibility of using
Raman spectroscopic markers for the study of lipid–protein complexes in model systems. Specifically, we will briefly
review relevant Raman spectroscopic markers for lipids and
proteins and how both kinds of markers can be used in the
investigation of membrane-embedded proteins in fully hydrated liposomes. We will present data from a model system
consisting of gramicidin incorporated into liposomes of various lipid compositions and discuss the feasibility of using
spectroscopic markers for lipid and Trp residue markers.
The amphiphatic character of membrane-spanning proteins and lipid molecules implies that lipids and proteins
self-assemble to avoid the high energetic cost of exposing
hydrophobic moieties to water (1,2). The interaction — hydrophobic coupling — between lipids and membrane proteins is generally only slightly stronger than the interactions
between lipids (3,4), suggesting an important role for unspecific lipid–protein hydrophobic interactions (3). The number
of available high-resolution structures of integral membrane
spanning proteins is increasing, and there is mounting evidence for regulation of membrane proteins by the host bilayer membrane through the hydrophobic coupling between
membrane and proteins (4,5). This not only raises specific
questions as to how membrane-spanning proteins interact
with the lipid bilayer, but also opens the possibility to design
biomimetic membranes based upon integral membrane proteins incorporated into encapsulated–supported systems for
sensor and separation purposes (6).
One of the specific questions relates to a peculiar feature
of integral membrane proteins: namely, a preference for aromatic (for example, Trp) residues located in the interfacial
region between the hydrophobic core and the hydrophilic
surface of the membrane. This has led to the notion of anchoring residues, in which the anchoring effect is thought
to arise from the amphiphatic nature of interfacial aromatic
residues (7,8). Despite considerable experimental progress
using nuclear magnetic resonance (NMR), electron spin
resonance (ESR), and molecular modeling (9–12) in the investigation of the effect of anchoring aromatic (and polar)
residues, many issues remain unresolved.
Raman spectroscopy has immediate appeal in the study
of lipid–protein interactions because it is a noninvasive and
nondestructive technique, and the excitation wavelength can
be chosen to be below the absorption frequency of water (13).
However, Raman spectroscopy of biomembranes in aqueous solution is a technical challenge. The inherent weakness
of Raman scattering and the low concentration of solvated
samples makes the collection of a spectrum with a high
signal-to-noise ratio difficult. But with increased sensitivity and recent developments in instrumentation, Raman
spectroscopy is experiencing a revival in many research
2 Spectroscopy 24(10)
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Oc tober 2009
cence is frequency dependent. Excitation in the near-infrared (NIR) spectrum provides maximum intensity
for a fluorescence-free spectrum and
is therefore usually recommended for
the study of biological samples. Comparing Raman excitations between 406
and 830 nm on tissues, the best defined
lipid features are obtained at 782 and
830 nm (that is, with an excitation in
the very near IR) (22). In fact, 785 nm
seems to be the best suited excitation
wavelength for the identification of
a wide range of biomarkers (23). The
excitation wavelength imposes constraints, such as the power available,
the spot size, the exposure time, grating, and charge-coupled device (CCD)
used. The comparison of mapping at
532 nm versus 785 nm illustrates the
difference between actual collections
at those two frequencies and the actual
superiority of excitation in the NIR for
this type of application (24).
Spectroscopic Markers for Lipids
Figure 1: Lipid conformations and structures: (a) The cis, trans, and gauche conformers of acyl
chains; (b) the structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); and (c) the
structure of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Double bonds in DOPE are
all cis and introduce the same kind of change of direction as does a gauche configuration.
areas, including the study of lipids and
lipid–protein complexes (14–19).
Here, we first discuss the technical
challenges and relevant Raman spectroscopic markers for investigating the
role of lipid–protein interactions in
fully hydrated protein-vesicle samples.
In particular, we present markers relevant for lipid molecular conformations
and markers for the conformation of
anchoring aromatic residues. Then we
present a model system and give a few
examples of how Raman markers appear in spectra of this system.
Raman Spectroscopy on Lipid–
Protein Complexes in Aqueous
Solution
Liposomes present a versatile tool for
investigating lipid–protein interactions where one can fully control the
lipid composition, protein content, and
aqueous phases on both sides of the liposomal membrane. However, the use
of protein-incorporated liposomes in
Raman spectroscopy presents challenges compared to Raman spectroscopy on solid (powder) samples (19).
Longer integration time is needed for
aqueous solution samples and the intensity peaks appear broader (20). Also,
containers used to avoid evaporation
and contamination cause problems like
loss of intensity and interferences, so
windowless, liquid captive cells (21) are
sometimes preferred (20). Raman spectroscopy on biochemical compounds
isolated from living systems (and in
particular, aromatic residues in membrane proteins) is made difficult due to
autofluorescence, which can make the
Raman bands “barely detectable” (20).
A longer excitation wavelength can
alleviate the problem because fluores-
The spectrum of lipids is to a large
extent due to vibrations in the hydrocarbon chains, but vibrations in the
head group region also can be used
as specific lipid fingerprints. Here we
will briefly present the acyl chain lipid
markers (for example, in the CH deformation and the skeletal optical regions)
and lipid head group markers.
Acyl Chain Markers
The CH deformation region includes
the prominent CH bending mode at
1440 cm−1 for CH 2 and 1460 cm−1 for
CH 3 (19,22). It is highly sensitive to
lipid chain architecture and chains
lateral packing (25). It has been used
to detect the main phase transition of
lipid vesicles (26). The other deformation prominent in the spectra is the
CH twist. The CH 2 twisting at 1296
cm−1 is found to decrease in intensity
with increased CC stretching (22). The
two other deformations of methylene
groups, the wagging (out of plane) and
the rocking (in plane), have not been
assigned. For unsaturated chain segments, the =C-H in-plane deformation
is at 1229 cm−1 and the =C-H out-ofplane deformation is at 970 cm−1 (22).
w w w. s p e c t r o s c o p y o n l i n e . c o m
Another region of the spectrum
marked by vibrations in the hydrocarbon chains is the CH stretch region located between 2800 and 3100 cm−1. The
CH bond is highly localized and should
therefore be rather insensitive to chain
configuration or environment (25).
However, the exact frequency seems
to depend upon the methylene position on the chain (27). This region also
has been shown to be sensitive to chain
packing (28), probably due to Fermi
resonance between the CH stretching
and binary combination modes involving CH bending. The result is a broad
feature that is difficult to interpret due
to “a virtual continuum of binary combination states” (29). Despite the complexity of this region, assignments have
been made. The peak at 2860 cm−1 is
assigned to crystal mode interactions
between adjacent hydrocarbon chains
(30). The CH2 symmetric and antisymmetric stretching bands are located at
2840 cm−1 and 2880 cm−1, respectively
(19). The symmetric CH 2 stretching
can be used as an indicator of the number of gauche chain conformers (Steer
et al., 1984--AUTHOR--is this reference
60?). The symmetric and asymmetric CH3 stretching bands are located
slightly higher, around 2930 cm−1 and
2960 cm−1, respectively (19,22,25). The
CH 3 symmetric stretching is modified considerably with polarity (25)
but is insensitive to perturbations of
the terminal methyl group (30). For
unsaturated chain segments, the =CH stretch is located at 3010 cm−1 (19).
The decrease of intensity ratio between
the antisymmetric and the symmetric
stretching modes associated with formation of gauche rotamers or the disruption of lateral packing interactions
between hydrocarbon chains can be
used to detect the main phase transition temperature (31). The CH region
as a whole was used to detect subgel to
gel transition (26), providing evidence
that it can be used to investigate lipid
chain packing.
The skeletal optical region of 1000–
1200 cm −1 is highly sensitive to the
hydrocarbon chain state (32). The CC
stretching bands around 1133 cm −1
and 1064 cm−1 referred to as “all-trans
skeletal vibrations” are associated with
Oc tober 2009
Spectroscopy 24(10) 3
Figure 2: Model system: (a) Gramicidin dimer structure with the two sets of four interfacial
tryptophan residues indicated in bold; (b) Whenever there is a hydrophobic mismatch, the
gramicidin dimer induces a bilayer deformation and there will be an equilibrium distribution
between nonconducting monomers and dimer channels.
the all-trans chain segments (1119 cm−1
and 1079 cm−1, respectively according
to Frank and colleagues [22]). A model
for the contribution of trans chain segment sequences to the intensity at 1130
cm−1 is described in reference 33 and
is thus linked to changes in acyl chain
order-disorder (see also reference 28).
The mode at 1080 cm−1 is probably a
mixture of a C-C stretching and methylene wagging (34). The “gauche band”
or “gauche marker” at 1089 cm−1 is associated with gauche chain segments.
It is superimposed with the O-P-O
symmetric stretch. The gauche band is
found to shift and broaden during the
main phase transition (25). For unsaturated chain segments, the C=C stretch
is around 1660 cm−1 (1654 cm−1 for cis
and 1669 cm−1 for trans) (22) and overlap with protein amide I mode (35).
of acyl chains depends upon environment: it is at 1739 cm−1 for acetic acid
but 1729 cm−1 and 1744 cm−1 for twoacyl chain phospholipids (30). Also,
the characterizing group has distinct
markers: phosphatidylcholine lipids
are identified by stretch vibrations in
the choline group N+(CH3)3, and the
stretching band at 717 cm −1 can be
used as reference intensity, as can the
antisymmetric stretch band at 875 cm−1
(19). Phosphatidylethanolamine lipids
are identified by a minor band at 759
cm−1 associated to the ethanolamine
group (19). Both the C-N symmetric
stretching band and the PO2- band at
1096 cm−1 might have their intensity
affected by cations (such as Ca 2+ and
Mg 2+) linked to the ordering effect of
these ions on lipid headgroups (25).
Other Lipid Regions
Head Group Markers
Lower wavenumber bands than the regions discussed earlier also can be used
for identification of the phospholipids.
The band at 860 cm−1 has been assigned
to the phosphate group common to all
phospholipids, and a band at 1096 cm−1
has been assigned to the phosphodioxy
group PO2- thought to depend upon
the formation of H-bonds (19). The
C=O stretch mode of the ester group
In Raman spectra of vesicle suspensions, a subtractable background appears. Apart from slow fluctuations in
background that can look like apparent
Raman bands (25) and the fluorescence
due to impurities in a glass container,
the main contribution to the background is water, despite the fact that
water is mostly a weak Raman scatterer
in the region investigated. The intense
OH symmetric stretching of water at
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Oc tober 2009
structure, and for example, the peak at
1263 cm−1 depends upon the dihedral
angle ψ (44). Amide II, although less
structure sensitive than amide I and
III, could be used in theory for detecting protein conformation, but in practice it is too weak.
A nonamide mode at 1395 cm−1 referred to as the amide S mode has been
shown to arise from Cα(C)H bending
with contribution from CN stretch and
NH in plane bending. It is enhanced by
coupling to the amide III mode via CH
bending and CC stretching. When the
Cα-H bond is rotated, there is uncoupling of the two modes and disappearance of the amide S band. The Cα-H
bond is oriented cis to the carbonyl
group in α-helices and trans in loop or
β-sheet structures. The intensity of the
amide S band can thereby be linearly
related to the nonhelical content of the
protein. It has been used for the structure analysis of proteins (42).
Raman Markers for Trp
Figure 3: Raman spectra of fully hydrated vesicles with (a) DMPC, (b) DMPC and gramicidin
(100:4), (c) DOPE:DMPC (3:1), and (d) DOPE:DMPC (3:1) and gramicidin (100:4).
3400 cm−1 and asymmetric stretching
at 3455 cm−1 appears as a shoulder in
the CH stretch region. The bending at
1640 cm−1 varies, depending upon the
extent of hydrogen bonding (36,37).
Apart from the intramolecular bands,
there are intermolecular libration bands
from water (38), which form a broad
envelope from 300 to 1000 cm−1 (37,39)
including rocking at 440 cm−1, twisting
at 550 cm−1 or 650 cm−1, and wagging
at 730 cm−1. All three are found to decrease in wavenumber with increasing
temperature (37). In addition, there are
weak unassigned bands between 1000
cm−1 and 2600 cm−1 at 1300 cm−1, 1555
cm−1, 2118 cm−1, and 2390 cm−1 (36,39).
Protein Spectroscopic Markers
The bands can be divided into two
groups: the main-chain bands (amide
bands) related to the protein backbone and the bands related to the side
chains, where we will focus on the Trp
side chains.
The amide I and amide III bands are
classical markers for the conformation
of proteins (40). The α-helix, β-sheet,
and random coil secondary structural
motifs have different peak wavenumbers and band shapes, and the composition of the protein in terms of these
secondary structures can be calculated
from these bands (41,42). However,
using the amide I band for structural
analysis might be difficult, necessitating a combination of experiments with
classical and quantum methods for
advanced interpretation. The amide
I mode usually is described as CO
stretching mode with some mixture
of CCN deformation (43). The exact
wavenumber decreases with hydrogen bonding to the carbonyl group.
The amide III is a CN stretch and NH
in plane bending mode with C(C)H3
symmetric bending and CO in plane
bending contributions between 1200
and 1300 cm−1. The exact wavenumbers depend upon environment and
Apart from the main chain bands, protein spectra also have bands specific to
the amino acid side chains (for example,
NH stretch at 3300 cm−1, CH stretch at
2800–3000 cm−1, and CH bending at
1450 cm−1). Aromatic groups are rich
in electrons and therefore prominent
in spectra, and Trp has several bands
that have been reported to be suitable
as conformational markers (45). The
assignments have been confirmed by
computer simulation (46).
The peak around 1542 cm−1 can be
used as a conformation marker for the
orientation of the Trp residue because
the exact wavenumber (υW3) of the
peak is determined by the angle χ2,1
(47):
uW 3 = 1542 + 6.7 cos 3 c 2.1 +1
(1)
The intensity ratio of the doublet at
1340 cm−1 and 1360 cm−1 I1360/I1340 is
a marker (denoted W7) of the Trp’s environment hydrophobicity: the stronger
the hydrophobicity, the higher the ratio.
The participation of Trp in H-bonding
w w w. s p e c t r o s c o p y o n l i n e . c o m
as a hydrogen donor can be monitored
using the markers at 877 cm −1 (denoted W17), 1490 cm−1 (denoted W4),
and 1430 cm−1 (denoted W6) (45,47).
For W17 ,a scale has been proposed in
which 883 cm−1 corresponds to a nonH-bonded state, 877 cm−1 to a medium
strength H-bonding, and 871 cm−1 to
strong H-bonding (47).
Model System
Spectra of living systems and macromolecules are highly complex superimpositions of bands differing only
slightly from one another as they originate from identical chemical groups.
Often, overlapping bands make the
interpretation of spectra difficult. A
model system such as lipid vesicles
with incorporated model proteins and
peptides has fewer components than a
living cell, making the interpretation of
Raman spectra feasible.
A well-suited model system in which
to investigate the role of interfacial
aromatic residues for the hydrophobic
lipid–protein interactions is gramicidin incorporated into lipid bilayers. In
lipid environments, the 15-amino-acid
gramicidin sequence adopts a β6.3–helical fold with four interfacial Trp residues (48) (Figure 2). Two monomers can
join by hydrogen bonding and form an
antiparallel dimer. This arrangement
results in a transmembrane protein
with a hydrophobic length l of about 22
Å spanning the bilayer (49,50). Highresolution structures of gramicidin in
the β6.3–helical structure favored in
lipid environments are available (51),
and Raman bands for gramicidin in
the crystalline state have been assigned
(52,53).
Whenever gramicidin dimers are
incorporated in bilayers having a hydrophobic thickness d0 larger than the
hydrophobic length of the gramicidin
dimer channel, there will be a hydrophobic mismatch, and this mismatch
leads to local protein-induced bilayer
deformation (that is, lipid monolayer
bending and compression) (3,54,55)
(Figure 2).
Changes in acyl chain conformation, whether induced by monomers or
dimers, should in principle be visible
as changes in the lipid Raman mark-
Oc tober 2009
ers discussed earlier and exemplified
in the relative porportion of gauche
versus trans chain segments. The Trp
“interfacial anchoring” effect seems
to be related to lipid species because
molecular dynamics (MD) simulations suggest that the Trp residues of
gramicidin can interact preferentially
with the phosphatidylethanolamine
(PE) lipid headgroups rather than with
phosphatidylcholine lipid headgroups
(10). Thus, Raman markers for Trp rotameric states and H-bonding will in
principle provide information about
how this anchoring is coupled to lipid
head group specifcity and hydrophobic
mismatch.
Spectroscopy 24(10) 5
3b and 3d, the spectra were smoothed
using adjacent averaging. Subtraction,
Gaussian peak fitting, and smoothing
routines were performed using Origin
Pro 8 analysis software (OriginLab,
Northampton, state?).
Results
We investigated the gramicidin-induced changes in acyl chain conformation through the bands sensitive to
lipid chain conformations. Then we
analyzed the role of lipid headgroups
(bilayer composition) on the change in
conformation of the bilayer lipids. Finally, we illustrated how the Trp bands
for gramicidin are affected by lipid bilayer composition.
Materials and Methods
The saturated dimyristoylphosphatidyl
choline (DMPC) and the unsaturated
dioleoylphosphatidy-ethanolamine
(DOPE) (Avanti Lipids, Alabaster, Alabama). are both in the liquid state at
room temperature. DMPC and DOPE:
DMPC (3:1 mol:mol) vesicles were prepared using standard protocols (56) to
a final aqueous concentration of 10
mg/mL. Gramicidin (Sigma Aldrich,
St. Louis, Missouri) in powder form
was dissolved in ethanol. The final
gramicidin:lipid ratio in DMPC and
DOPE:DMPC (3:1) vesicles was 4:100
(mol:mol).
The measurements were made using
an inVia Raman microscope with 50×
objective (Renishaw plc, Old Town,
UK) with a laser wavelength of 785
nm. A maximal effect of 100 mW and
a grating with 1200 lines/mm was used.
A quantity of 200 μL of lipid suspension was placed on a glass cover slip in
the microscope stage. The microscope
objective was lowered until it came in
contact with the drop and subsequently
raised several mm without breaking
contact with the drop. An extended
mode scan of 1–5 min was used at maximum power. To improve the signal-tonoise ratio, up to 10 scans were accumulated, depending on the sample. The
spectra were normalized to the band
at 717 cm−1. The background was approximated by fast Fourier averaging of
the spectra where the prominent bands
had been removed and subtracted from
the spectra. For the inserts in Figures
Gramicidin Induces Gauche
Conformational Acyl Chain
States
The perturbation of bilayer lipids on
gramicidin binding was investigated
for a DMPC bilayer and a DOPE:
DMPC (3:1) bilayer by comparison of
the Raman intensity of the bands dependent upon lipid chains conformation and packing in the absence and in
the presence of gramicidin. The Raman
spectra of DMPC vesicle suspensions in
the absence and presence of gramicidin
are given in Figures 3a and 3b, respectively. The Raman spectra of DOPE:
DMPC (3:1) mixture vesicles suspensions in the absence and presence of
gramicidin are given in Figures 3c and
3d, respectively.
All of the regions sensitive to lipid
chains conformation are modified upon
binding of gramicidin in both bilayers.
In the CH bend region, the intensity at
1440 cm−1 is more than halved in the
DMPC–gramicdin bilayer compared to
DMPC alone. In the CC stretch region,
the intensity at 1130 cm−1 related to the
number of trans segments in the chains
is almost halved upon gramicdin incorporation. The ratio I1089/I1128 quantifying the number of gauche bonds
(relative to trans bonds) increases by
40% in DMPC with gramicidin. In the
CH2 twist region, the intensity at 1296
cm−1, which decreases with the number
of trans chain segments, is decreased
fourfold in DMPC upon gramicidin incorporation. In the CH stretch region,
6 Spectroscopy 24(10)
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Oc tober 2009
the amplitude of the peak at 2850 cm−1
is increased by 30% in DMPC when
gramicidin is present.
The quality of the spectra in DOPE:
DMPC (3:1) alone and with gramicidin
incorporated are not as good as the
DMPC and DMPC–gramicidin spectra. The skeletal region is difficult to
analyze, and it is therefore not possible
to quantify any changes in gauche acyl
chain conformations. However, the CH
stretch at 2850 cm−1 is reduced about
15% in DOPE:DMPC (3:1) upon gramicdin incorporation, in contrast to the
observation for DMPC–gramicidin
versus DMPC.
Tryptophan Rotameric States
and H-Bonding Depend on Lipid
Composition
The conformational state of Trp residues of gramicidin as a function of the
host lipid bilayer was investigated by
comparing the Raman Trp markers in
the spectra shown in Figure 3.
The W3 conformational marker
is centered at 1554 cm −1 in DMPC–
gramicidin, corresponding to a |χ2,1|
of around 100°°(Figure 3b). It has a
bimodal distribution in DOPE:DMPC
(3:1)–gramicidin, with approximately
two-thirds of the intensity around
1558 cm−1 corresponding to a |χ 2,1| of
around 120°, and one-third of the intensity around 1545 cm−1 corresponding to
a |χ 2,1| of around 80° (Figure 3d).
For W17 in DMPC–gramicidin,
Gaussian fitting reveals a trimodal
distribution with most of the intensity
centered on 876 cm−1 and two lesser
peaks at 882 cm−1 and 889 cm−1, indicating that on average, most tryptophan
residues ngage in medium strength
H-bonding, whereas a smaller population tend to be non-H-bonded (the
band at 883 cm−1) (see insert in Figure3b.) In DOPE:DMPC (3:1)–gramicidin, the spectral region also has a
trimodal distribution, with most of
the intensity centered on 876 cm−1 and
two lesser peaks at 864 cm−1 and 886
cm−1. This corresponds to a mediumto-strong H-bonding (stronger and
broader band at 877 cm−1) but with a
possible contribution from nonbonded
interactions (see insert in Figure 3d).
The W7 environmental hydropho-
bicity marker I1360/I1340 is difficult
to anlyze in the spectra because there
are additional lipid bands in that region
(Figures 3a and 3c). DMPC I1360/I1340
appears to be about unity, indicating
low hydrophobicity. In DOPE:DMPC
(3:1)–gramicidin I1360/I1340 may be
<1, but the quality of the spectra is not
sufficiently good to conclude further
on this marker.
Discussion
Lipid Conformation
There is an increase in the number of
gauche conformations when gramicidin
is incorporated in DMPC. The increase
in the trans-to-gauche ratio has the
same magnitude as the disordering observed during the main phase transition
or during a large increase in temperature in the fluid phase (57). An increase
in lipid chain disorder likely reflects a
compression of the lipids associated
with gramicidin channels to compensate for the hydrophobic mismatch.
Because each gramicidin monomer
will have about 10 annular lipids (that
is, 20 for a gramicidin dimer channel),
a gramicidin:lipid ratio of 4:100 implies
that 40% of all the lipids are in direct
contact with a monomer and that only
a few (<3) lipids are “traversed” when
going from one gramicidin monomer–
dimer to the other. Thus, a considerable
portion of lipids can be compressed but
at the same time motionally restricted.
This is consistent with observations
using spin-labeled lipids, where addition of gramicidin results in a decreased
motionally averaging (58).
Trp Conformation and
Environment
The W3 marker seems most promising
in lipid–protein analysis here, showing re-orientation of the Trp residues,
whereas the W17 and W7 markers are
more difficult to use due to lipid bands
in these spectral regions. While W7
cannot be anlyzed using the spectra
obtained, W17 suggests that there is
a small increase in H-bonding when
going from a DMPC bilayer to a DOPE:
DMPC (3:1) bilayer. A reason for this
could be that the DOPE lipids can engage in carbonyl-Trp H-bonding (and
more cation-α interactions [10]) with
the Trp indole as the PE moity can be
closer to the indole than the PC moiety. The closer packing will thus tend
to make tryptophan residues more
accessible to lipid H-bonding but less
accessible to water H-bonding. This
is also consistent with the observation that gramicidin Trp becomes less
accessible to water and is deuterated
more slowly when going from DMPC
to DPPC (that is, with an increase in
hydrophobic mismatch (45)). Although
the choline group stretch vibration at
875 cm−1 precludes an exact H-bonding
estimate based upon W17, we note that
the lower molar content of choline in
DOPE–DMPC in its own right would
dimish the 875 cm−1 band, but we observe an increase in that region when
going from DMPC to DOPE:DMPC
(3:1), suggesting that H-bonding is increased. The conformational changes
also could be due to gramicidin tilt adaptation to mismatch (59) because this
could cause reorientation of the Trp
and change their H-bonding capability.
However, the orientation of gramicidin
Trp residues does not change when
comparing DLPC with DMPC (that
is, when the hydrophobic mismatch is
decreased [45]).
Outlook
We have used a relatively simple way to
measure Raman scattering on vesicles:
measuring during prolonged times on
concentrated suspensions. An improvement leading to better spectra would be
to use optical trapping on a single large
vesicle using a Raman microscope (15).
Another way to improve the approach
would be to combine Raman spectroscopy with “Raman-complementary” IR
spectroscopy. In IR, it should be possible to identify the well-defined wagging bands that correspond to different conformations (gauche end, kink,
double gauche) (34). The methylene
and methyl stretching modes can confirm the change in the relative number
of trans and gauche conformations on
peptide binding obtained by Raman
spectroscopy (60). Future work also
should include a temperature study to
determine what the intensity is in the
all-trans configuration (57). Finally, IR
phosphate bands can be related to H-
w w w. s p e c t r o s c o p y o n l i n e . c o m
bonding and thereby report on headgroup conformation (25,61).
Raman spectroscopy of vesicle suspensions shows that gramicidin affects
and is affected by the bilayer lipids.
Gramicidin incorporation increases
the number of gauche bonds in the lipid
acyl chains, indicating an increase in
acyl chain compression. Raman spectroscopic markers for the Trp residues
of gramicidin (in particular W3) show
that Trp rotametric states (and perhaps
H-bonding) are influenced by the lipid
composition of the host bilayer.
Acknowledgments
The authors would like to thank
Erik Skibsted at Novo Nordisk A/S
for providing access to their Raman
equipment. This work was supported
by a grant from the Danish National
Research Foundation to QUP. Claus
Hélix Nielsen also received additional
support through MEMBAQ, a Specific
Targeted Research Project (STREP), by
the European Commission under the
Sixth Framework Programme (NMP4CT-2006-033234) and from WATERMEMBRANE, a project supported by
The Danish National Advanced Technology Foundation (023-2007-1).
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