nanoscale infrared spectroscopy
Nanoscale infrared spectroscopy of
light harvesting proteins, amyloid
structures and collagen fibres
Andrzej J. Kulik,1 Francesco Simone Ruggeri,1 Wieslaw I. Gruszecki 2 and Giovanni Dietler1
1. Laboratory of Physics of Living Matter, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
2. Department of Biophysics, Maria-Curie Sklodowska University, Lublin, Poland
Introduction
Several analytical methods have been
developed that apply scanning probe
microscopy to extract information about
local physical or chemical properties of
biological macromolecules. Most of these
are very application specific, tedious
or difficult to use, which limits their
popularity. Kelvin probe microscopy
and Piezoelectric probe microscopy
are among the most commonly used
now. The mapping of local mechanical
properties is quite popular in biology,
although there are reproducibility
problems related to methodology and
the non-reproducibility of commercially
available cantilevers. Chemical force
microscopy [1-3] measures tip-sample
friction, or pull-off force, and is
influenced heavily by the chemical
properties of the sample. This method is
very specific but does not fully deliver
quantitative analytical information.
nano Infrared analysis
A recently developed infrared
spectroscopy technique by Dazzi [4]
and its subsequently commercialized
version, the nanoIR system from Anasys
Instruments, appears to offer an easy-touse solution for local chemical analysis
with the spatial resolution of an atomic
force microscope (AFM).
The nanoIR technique uses
thermomechanical detection. A pulsed,
tunable laser beam is passed through a
ZnSe prism and undergoes total internal
reflection from the upper face of the
crystal (Figure 1). An evanescent wave
interacts with the sample, mounted
as a thin layer on the ZnSe prism. If a
given wavelength is absorbed by the
sample, its temperature rises and its
height subsequently changes through
thermal expansion. This ‘kicks’ the AFM
cantilever in contact with the sample
surface. The cantilever resonates at
several Eigen frequencies. One mode is
chosen and its amplitude is proportional
to IR absorption within the specimen.
The user may change the wavelength
(or wavenumber) by tuning the laser.
It is possible to record IR spectra at one
location of the sample, or alternatively
map IR absorption at a single
wavenumber. Additionally, wide band
excitation with a short pulse excites
simultaneously many Eigen frequencies
of the cantilever. This ability gives the
technique a strong advantage over other
analytical methods in that it measures
several cantilever resonant frequencies
which can be related to the local stiffness
of the specimen [5].
In this article we review the
application of the nanoIR technique
to the study of several biologically
important macromolecules.
materials and methods
LHCII was isolated from spinach
chloroplasts as described in [6]
Samples were deposited at the surface
of the attenuated total internal
reflection element prism made of ZnSe
monocrystals and dried overnight.
Then, they were scanned in contact
mode by a nano-IR system from
Anasys Instruments with a rate line
within 0.02-0.1 Hz. We used a silicon
(AppNano) cantilever with a nominal
radius of 10 nm and a nominal spring
elastic constant of 0.5 N/m. All images
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have a resolution within 256x256 and
1024x1024 pixels for line. Spectra
were collected with a sampling of
1-2 cm-1, within the range 1200-1800
cm-1. Measurements were done within
5-100% of the instrument maximum
laser power. All measurements were
performed at room temperature.
FIGURE 1
Schematic of the
nanoIR system.
See text for explanation.
(Courtesy of Anasys
Instruments.)
results
photosynthetic pigmentprotein complex
As a first application of the system, we
performed a study on the molecular
organization of the major plant
photosynthetic pigment-protein,
the light-harvesting complex of
photosystem II (LHCII), in the
environment of chloroplast lipid
membranes [6]. This complex is the most
abundant membrane protein in the
biosphere and is estimated to comprise
half of the chlorophyll pool on Earth.
Its main physiological function is to
collect light quanta and to transfer
excitation energy towards the reaction
centres where electric charge separation
11
nanoscale infrared spectroscopy
takes place. The lipid membranes
of the photosynthetic apparatus of
plants are exceptionally enriched with
proteins which occupy more than 70%
of the membrane surface. Owing to
this fact the structural aspects of the
LHCII presence in the lipid phase are
expected to be important determinants
of the molecular architecture of the
chloroplasts membranes.
With the application of the nanoIR
technique, we studied lipid multibilayers containing incorporated
LHCII. We found that LHCII proteins
self-organized into oligomeric structures
in the plane of the membrane. Moreover
the protein formed pillar-like transmembrane structures stabilized
by hydrogen bonds between the
peripheral (polar) protein fragments
of the complexes anchored in the
adjacent lipid bilayers. Formation of
these type of complexes was found to
be strongly dependent on the protein
phosphorylation state (which is light
intensity-controlled in vivo). Such a
finding led to several specific conclusions
on the role of molecular organization of
LHCII in response of the photosynthetic
apparatus of plants to stress conditions
associated with strong light.
Figure 2
Light-harvesting complex II. Model of the
lipoprotein multibilayer composed of lipids
and integrated LHCII. The model is based on
the crystallographic structure of LHCII (coordinates downloaded from the PDB-database,
PDB ID:2bhw. The horizontal dimension of the
LHCII trimer is close to 10 nm [8]. For more
details of the model, see [6].
a
Figure 3
Light-harvesting complex II. (a) Surface
topography, (b) mechanical stiffness map and
(c) IR absorption pattern of the lipoprotein
multibilayer, recorded simultaneously on a 1x1
µm area of the sample, the thickness of which
was approximately 1.2 µm. Data contributing
to the IR-absorption map were acquired at a
wavenumber of 1650 cm-1.
Scan resolution: X = 512 points, Y = 256 points;
scan rate: 0.4 Hz, 8 co-averages.
amyloid structures
One of greatest benefits of nanoIR is that
the nanoscale chemical analysis can be
performed with very small quantities
of biological samples. Purification
and high throughput production of
proteins is extremely expensive and
time consuming; for this reason, the
reduction of sample quantity required
to perform routine FTIR measurements
and secondary structure analysis are
problems of primary importance which
nanoIR can solve.
This is the case of proteins aggregating
into amyloid structures, which are
involved in neurodegenerative disorders.
Amyloids are insoluble proteins that
form fibrillar aggregates. During
aggregation, initially monomeric
proteins undergo internal structural
rearrangement forming amyloid
fibrils with a universal cross b-sheet
quaternary structure. This structure
is independent on the monomeric
initial one and it is the fingerprint of
amyloid fibrils. FTIR is a key method
for studying their structural conversion
during amyloid formation. Nevertheless,
during fibrillation, several coexisting
amyloidogenic species are formed. So,
conventional FTIR is only able to give
average spectra of the heterogeneous
solution. Sub-micrometer chemical
characterization of amyloidogenic
structures as oligomers and fibrils is
central to understanding how proteins
misfold and aggregate.
In this work, the nanoIR system
was initially used to analyze proteins
deposited as patterned microdroplets
12
b
c
Compositional Analysis May 2014 | MicroscopyandAnalysis
nanoscale infrared spectroscopy
d
a
b
c
Figure 4
NanoIR imaging and spectra acquisition of
lysozyme microdroplets containing amyloid
structures.
(a) AFM height channel of a monomeric droplet.
(b) IR absorption of monomers at 1620 cm-1.
(c) IR absorption of monomers at 1530 cm-1.
(d) Spectra of monomers and fibrils.
by means of a microfluidic system [7]
(Figure 4). The instrument allowed
us to distinguish spectra of lysozyme
droplets (Figures 4 a,b,c) containing
amyloids from ones containing native
globular proteins. In particular, the
shift in the amide I band allowed us to
identify an a-to-b secondary structure
transition which is associated with
amyloid formation (Figure 4d). No other
technique had been available to give
chemical analysis at this sub-micrometer
scale.
The same approach was used to study
a-synuclein macromolecular aggregates.
The protein was deposited as droplets
on the ZnSe substrate in two forms:
monomeric and fibrillar (Figure 5). A
first macro-observation showed that
monomers formed a uniform layer on
the surface, while fibrils were strongly
organized (Figure 5 a-d). The monomeric
layer is chemically and mechanically
homogeneous, in contrast to the tubular
fibrillar macro-aggregate (Figure 5
b,c,e,f). This difference was confirmed
by the spectra acquisition of the two
samples which are quite different and
show a random-coil to b-sheet transition,
typical of the formation of amyloids
from unordered proteins. Spectra
modification during amyloid formation
is in very good agreement with the
previous ones for lysozyme.
g
collagen fibres
Finally, as last example of the versatility
of the nanoIR technique, other
measurements were realized on collagen
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FIGURE 5
NanoIR imaging and spectra
acquisition of a-synuclein
film of monomers and of a
supramolecular aggregate.
(a) AFM height channel.
(b) IR absorption at 1620 cm-1.
(c) Mechanical response of the
cantilever at 1620 cm-1 for a film of
monomers.
(d) AFM height channel.
(e) IR absorption at 1620 cm-1.
(f) Mechanical response of the
cantilever at 1620 cm-1 for a
supramolecular aggregate.
(g) Spectra of the monomeric film
and the supramolecular rod.
13
nanoscale infrared spectroscopy
biography
a
Andrzej J. Kulik has
worked as scientist at
EPFL in Switzerland since
1981. He has developed
several new methods
for non-destructive
material testing,
quantitative acoustic microscopy, nanoscale
measurements applied to nanoscale objects,
AFM-based nanomanipulation and optical
tweezers-based 3D nanomanipulation.
Recently, he has been using a nanoIR
system to study biomolecules. He is the
co-author of over 110 papers, seven book
chapters, and has two patents with more
than 4000 citations.
b
c
abstract
d
fibres. The system allowed us to acquire
the spectral chemical composition of a
single collagen fibril with a diameter of
about 100 nm (Figure 6).
conclusions
In conclusion, we have demonstrated
that the nanoIR technique is stable
and easy-to-use, giving novel and
meaningful results from studies of
biologically important macromolecules
that were previously not available.
References:
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14
FIGURE 6
NanoIR imaging and
spectra acquisition of a
bundle of collagen fibrils.
(a) AFM height channel.
(b) IR absorption at 1655
cm-1.
(c) Mechanical response of
the cantilever as a function
of sample stiffness at 1655
cm-1.
(d) Spectrum of typical
collagen fibril with a
diameter of about 100 nm.
(Sample courtesy of
L. Kreplak, Dalhousie
University, Canada.)
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By combining atomic force microscopy with
photo-thermal induced resonance detection
of infrared spectra (nanoIR), extraordinary
high nanoscale spatial resolution can
be obtained. This method is very useful
to study inhomogeneous samples by
functional imaging at a single wavenumber.
This allows the detection of the distribution
of chemical properties of the constituents
of the sample. As well, the method provides
meaningful infrared spectra of very small
quantities of sample (e.g. proteins). Studies
of light harvesting complex II, amyloid and
collagen samples are presented.
acknowledgements
We would like to thank the Swiss National
Foundation for Science for funding,
Tuomas Knowles’ lab in Cambridge, who
worked with us on lysozyme, and Lashuel
Hilal’s lab for alpha-synuclein.
Corresponding author details
Dr Andrzej J. Kulik,
Laboratory of Physics of Living Matter,
Ecole Polytechnique Fédérale de Lausanne,
BSP/Cubotron,
CH-1015 Lausanne, Switzerland
Tel: +41 21 693 3359
Email: [email protected]
Microscopy and Analysis 28(4):11-15 (EU),
May 2014
©2014 John Wiley & Sons, Ltd
T. P. J., Dietler, G. Nanoscale spatially
resolved infrared spectra from single
microdroplets. Lab on a Chip 14(7), 13151319, 2014.
8. Barros, T., Royant, A., Standfuss, J.,
Dreuw, A., Kühlbrandt, W. Crystal
structure of plant light-harvesting
complex shows the active, energytransmitting state. The EMBO Journal
28(3):298-306, 2009.
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