Fang, Yusheng

Huang, Qing

Ke, Zhigang

Liu, Jinghua

Qi, Zeming

Wei, Xiaoli

Yu, Zengliang

Elsevier - Publisher Connector 

Study of Energetic Particle Induced Biological Effect through FTIR and Raman Micro-Spectroscopy 

Tuesday, February 28, 2012 589aBoth isomers appear to interact with lipid bilayers in a similar fashion. Our re-
sults suggest that Ab mediated disruption of ionic homeostasis may occur by
a direct pathway of ion channel formation and may not need to rely on interac-
tions with membrane receptors. Understanding the mechanism of peptide-
membrane interaction and insertion at nanoscale resolution is essential for ther-
apeutic design aiming to control and prevent Ab pore formation. Funded by
NCI Contract HHSN261200800001E (RN) and NIH (National Institute on Ag-
ing AG028709) extramural program (RL).
2995-Pos Board B765
Scanning Probe Microscopy of Serpin Polymers
Pawel A. Osmulski1, Maria Gaczynska1, Przemek Karpowicz1,
Srividya Madabhushi1, Monika Tokmina-Lukaszewska1, Christine Stuart2,
Wesley Jong2, Malgosia Mikolajczyk2, Martha C. Anderson2, Ewa Marszal2.
1UTHSCSA, San Antonio, TX, USA, 2U.S. Food and Drug Administration,
Bethesda, DC, USA.
Serpins belong to a superfamily of structurally homologous proteins with meta-
stable native structures. Their metastability is crucial in performing their func-
tion as regulators of serine and cysteine protease cascades. However, mutated
variants of serpins found in patients with serpinopathies are often prone to po-
lymerization. The most widespread serpinopathy is related to the Z variant of
antitrypsin, which forms toxic polymers in the liver leading to liver degenera-
tion. The understanding of polymerization mechanism holds the key to disease
prevention, diagnostics and cures. Several models of serpin polymerization
have been proposed. Still, the structure of in vivo formed polymers is unknown,
and the relevance of in vitro created oligomers to disease related structures is
far from established. Here we employed atomic force microscopy (AFM) to
compare topography of in vitro and in vivo formed antitrypsin polymers and
oligomers. We established morphometric features of Z monomers and in vitro
formed dimers with different types of linkage. Moreover, we found a remark-
able heterogeneity of unit types and their arrangement in polymer strands iso-
lated from the Z variant mutant mouse liver. Even within the same strand there
were examples of a linear arrangement of monomer units and of compact ar-
rangement of dimers. The in vitro formed wild type oligomer preparations con-
tained structures resembling the native Z variant structures from the liver. Still,
the partition of types of polymers was strikingly distinct, with compact mono-
mer arrangement and polymerized dimers frequenting the in vitro samples. In
addition to AFM, we used scanning tunneling microscopy (STM) to study the
orientation of units in polymers based on the proteins surface charge. Summa-
rizing, SPM imaging provided the unique data on a high heterogeneity of poly-
mers and pointed at certain types of monomer linkages that might help explain
the mechanism of serpin polymerization.Vibrational Spectroscopy
2996-Pos Board B766
Deep-Uvrr Spectroscopy Studies of Amyloidogenic Transthyretin
Fragments
Carol A. Roach, Renee D. JiJi.
University of Missouri-Columbia, Columbia, MO, USA.
The protein transthyretin (TTR) has been implicated as the pathogen in several
amyloid diseases. Normally a transport protein for thyroxine, amyloidosis oc-
curs when the protein aggregates and deposits in organ tissue as b-sheet struc-
tured amyloid fibrils. The formation of amyloid fibril deposits associated with
TTR diseases is poorly understood. In order to study amyloid fibril formation
several amyloidogenic fragments of TTR have been studied in aqueous solu-
tions. It has been suggested that the amyloidogenic fragments TTR(10-20)
and TTR(105-115) contain portions of the protein essential to aggregation
and amyloid fibril formation, however, neither have been studied in the pres-
ence of cell-like lipid membranes. Here, we present the first studies comparing
aqueous and model membrane solution studies of TTR(10-20) and TTR(105-
115) via deep-ultraviolet resonance Raman spectroscopy. Initial results suggest
a change in peptide secondary structure upon interaction with lipid membranes.
2997-Pos Board B767
In Vivo Molecular Labeling of Halogenated Volatile Anesthetics via
Intrinsic Molecular Vibrations using Nonlinear Raman Spectroscopy
Masahiko Kawagishi1, Takayuki Suzuki2, Yu Nagashima1,2, Sumio Terada1,
Kazuhiko Misawa2.
1Tokyo Medical and Dental University, Tokyo, Japan, 2Tokyo University of
Agriculture and Technology, Koganei, Tokyo, Japan.
Halogenated volatile anesthetics are frequently used for inhaled anesthesia in
clinical practice. No appropriate biological method has been available for visu-
alizing their localization in action. Therefore, despite their frequent use, the
mechanism of action of these drugs has not been fully investigated. We mea-sured coherent anti-Stokes Raman scattering (CARS) spectra of sevoflurane
and isoflurane, two of the most representative volatile anesthetics, and deter-
mined the low-frequency vibrational modes without nonresonant background
disturbance.Molecular dynamics calculations predict that these modes are asso-
ciated with multiple halogen atoms. Because halogen atoms rarely appear in bi-
ological compounds, the entire spectral landscape of these modes is expected to
be a good marker for investigating the spatial localization of these drugs within
the intracellular environment. Using live squid giant axons, we could detect the
unique CARS spectra of sevoflurane for the first time in a biological setting.
2998-Pos Board B768
Cell Surface Protein Detection using Surface-Enhanced Raman Scattering
(SERS) Gold Nanoparticles
Christina M. MacLaughlin, Shell Ip, Nisa Mullaithilaga, Edward Parker,
Chen Wang, Gilbert C. Walker.
University of Toronto, Toronto, ON, Canada.
Surface enhanced Raman scattering (SERS) Au nanoparticles have been used
as novel cell surface receptor labels for the identification of markers of interest
in chronic lymphocytic leukemia (CLL) and lung cancer. Biocompatibility of
the particles was improved using multiple coating and particle protection strat-
egies. Each of these strategies facilitated different methods for the inclusion of
Raman active reporter molecules, as well as for different types of targeting
moieties. Characterization of the SERS nanoparticles was undertaken including
quantification of the number of antibodies bound to the surface. Long-term sta-
bility of both the nanoparticle Raman signal intensity and monodispersity was
assessed under standard storage conditions, as well as conditions suitable for in
vitro biological experiment. The SERS labeling platform has been demon-
strated as being compatible with traditional pathology protocols including
flow cytometry, and stains such as giemsa. SERS detection using these particles
has been adapted to models for both adherent and circulating malignancies, in
addition to patient cell samples in the example of CLL. The narrow vibrational
spectra of SERS particles used in this study greatly increase the multiplexed
labeling potential over traditional fluorescence-based technologies. Preliminary
multiplexed labeling of CLL has also been demonstrated.
2999-Pos Board B769
Micro-Raman of Cancer Cells: Toward Label-Free Sorting of Circulating
Tumor Cells from Whole Blood
Gregory L. Futia, Allison G. Caster, Omid Masihzadeh, Kian Behbakht,
Emily A. Gibson.
University of Colorado, Denver, Aurora, CO, USA.
Even in the early stages of cancer, circulating tumor cells (CTCs) can travel
from a primary tumor site to other organs through the blood and lymphatic sys-
tem. Detecting and isolating CTCs from the blood has great potential for early
cancer detection and studies of metastasis. However, they are notoriously rare
(a few CTCs per mL of blood) and difficult to distinguish from epithelial non-
tumor cells and leukocytes. Attempts to analyze CTC genetic or protein
changes in response to treatment have been hampered by the difficulty in
isolating intact clonogenic cells. Toward the goal of developing a rapid,
non-invasive tool for detecting and isolating live CTCs from the blood, we
measured the Raman spectra of live, unlabeled single cells. Using principal
components analysis (PCA), we can distinguish WBCs from cancer cells,
and tumorigenic ovarian cancer cells (A2780, OVCAR2, CaOV3) from non-
tumorigenic ovarian cancer cells (OV429). Adherent cell lines and suspended
(laser trapped) cells from cancer patient fluid samples were measured. A micro-
fluidic platform with pressure control has been implemented to transport such
cells single-file through the Raman laser focus. Custom microfluidics are in de-
velopment and will sort the live, unmarked cancer cells into separate reservoirs
for cell culture. Each Raman spectrum requires ~1-2 minutes, so we are concur-
rently developing a faster, coherent anti-Stokes Raman scattering (CARS) mi-
croscope for higher throughput analysis. Rapid detection and sorting of live
CTCs will give prognostic information and allow for observation of the effect
of targeted cancer treatments via a minimally invasive blood test and possibly
well before the availability of response data.
3000-Pos Board B770
Study of Energetic Particle Induced Biological Effect through FTIR and
Raman Micro-Spectroscopy
Qing Huang1, Jinghua Liu1, Xiaoli Wei1, Yusheng Fang2, Zhigang Ke1,
Zeming Qi3, Zengliang Yu1.
1Hefei Institute of Physical Science, Chinese Academy of Sciences, Hefei,
China, 2School of Nuclear Science and Technology, University of Science
and Technology of China, Hefei, China, 3National Synchrotron Radiation
Laboratory, University of Science and Technology of China, Hefei, China.
Energetic particles exist ubiquitously in nature and may cause varied biological
effects which have been found useful applications such as radiotherapy of
590a Tuesday, February 28, 2012human cancers and mutation breeding of crops and microbes by means of
ion-beam technology. However, the underlying mechanism for the energetic
particle induced biological effects is still elusive because the interacting system
involves radiolysis of different biomolecules such as DNA, proteins, sugar and
lipids, and moreover, there are cross-correlations among these varied sub-
stances due to their interactions and signal transductions in living cells.
A new trend of research is therefore to investigate the interaction between
energetic particles and organisms with consideration of the entire cellar
micro-environment and the entangled processes occurring in a whole cell,
and to scrutinize the changes of cellular structure and compositions on the
micro-scale and also monitor the dynamic and kinetic processes for the inter-
actions. In this context, it is very useful and powful to apply micro-spectral im-
aging technology such as FTIR and Raman confocal microscopy because it can
provide not only good time and spatial resolution, but also non-invasive mea-
surement. In this work, we utilized the high spatial resolution FTIR and Raman
microscopy to study the cellular changes of some model microbes and cells by
mapping and monitoring the fingerprint bands of the cellular components such
as lipids, carbohydrates, polyunsaturated fatty acids, proteins and nucleic acids
under the irradiation of energetic particles. Correspondingly, the cell activity,
the intracellular content of ROS, the level of MDA and GSH, the activity of
CAT and SOD were measured to explain the biological effect induced by
energetic particles. This work is supported by NSFC (No. 10975152, No.
11175204), CAS Innovative Project (KJX2-YW-N34-1) and Hundred Talents
Program of CAS, China.
3001-Pos Board B771
Simulating the Amide I IR Signal of a Peptide in Solution using a Classical
Implicit Water Approximation
William R. Welch.
University of Wyoming, Laramie, WY, USA.
Behavior of the amide I IR signal of peptides has been the object of much study
because it can provide valuable information about the environments and con-
formations of peptides in solution. Numerous models have been proposed to
predict the amide I band shape, the most successful of which may have limited
utility in studying dynamic processes due to high computational cost. Here
a new, simple method is employed to predict the effects that the aqueous envi-
ronment and side chains have on the shape of the amide I IR signal for a poly-
peptide in water. Using GEPOL to generate a solute cavity, apparent charges at
the solvent accessible surface are generated considering a conductor like re-
sponse from the solvent to partial charges on atoms (AMBER, or CHARMM).
Potentials on the atoms in amide groups resulting from the surface charges and
from the side groups are then used along with an electrostatic potential map to
predict how the amide I signals are perturbed from gas phase signals. Effects of
cavity size and atomic partial charges on the integrity of predicted band shapes
are examined. The method gives results comparable to much more computa-
tionally intensive methods.
3002-Pos Board B772
Measuring Moments of Protein Conformation Distributions using Infra-
red Spectroscopy
Curtis W. Meuse.
NIST, Rockville, MD, USA.
Infrared spectroscopy is often used to characterize the concentration and sec-
ondary structures of proteins in a variety of static and dynamics samples.
Our work develops new methods to compare the structure, dynamics and func-
tion of nearly identical protein samples, in order to help characterize bio-similar
protein therapeutics. We have developed a method to describe protein confor-
mational variations around the average molecular values. By comparing the
moments of the protein structural distributions and amide hydrogen/deuterium
exchange methods, we explore the relationships between protein stability and
dynamics. Examples include lysozyme and albumin in solution, cytochrome
c interacting with lipid membranes of varying net-negative surface charge den-
sity, and bacteriorhodopsin during its photocycle.
3003-Pos Board B773
Isotope-Edited FT-IR Spectra of Isotopmers of Helical Hexamers of
Alpha-Aminoisobutyric Acid
Matthew A. Kubasik, Steven F. Hannigan.
Fairfield University, Fairfield, CT, USA.
Isotope-edited FT-infrared spectra of the Amide I region of hexamers of alpha-
aminoisobutyric acid (Z-Aib6-OtBu) have been collected in order to explore the
effects of 13C=O enrichment on the FT-IR spectra in the conformational con-
text of 310 helices. Oligomers of Aib are known to adopt predominantly 310 he-
lical structures, even at short peptide lengths. The Amide I band is sensitive tothe details of peptide secondary structure, but the competency of this band to
distinguish between alpha- and 310 helical secondary structure remains an
open question. The 310 helix is shorter than an alpha-helix of the same number
of residues and exhibits an i to iþ3 hydrogen bonding pattern, instead of an i to i
þ4 pattern of the alpha-helix. These differences bring amide oscillators of a 310
helix slightly closer in space and in shorter periodicity of hydrogen-bonding
partnership as compared to an alpha-helix. We have collected infrared spectra
of isotopomers of hexamers of Aib (e.g., Z-Aib-Aib-Aib-Aib-Aib-Aib-OtBu,
Z-Aib*-Aib-Aib*-Aib-Aib-Aib-OtBu, and Z-Aib*-Aib*-Aib-Aib-Aib-Aib-
OtBu, Aib* = 13C enrichment at *C=O) in dichloromethane (a nonpolar aprotic
solvent) and methanol (a polar, protic solvent) to examine the effects of carbon-
13 enrichment on the spectra. Differences among the spectra of peptide iso-
topomers enriched with one or two 13C labels will be examined, detailing the
effects of coupling between pairs of 13C-labeled Amide I oscillators.
3004-Pos Board B774
Under Pressure: Measuring Desolvation of Model a-Helical Peptides
Teraya Donaldson, Alice Smith-Gicklhorn, Sean M. Decatur.
Oberlin College, Oberlin, OH, USA.
Model helical peptides have been shown to unfold with temperature, but are
stabilized with increased pressure 1-3. In these peptides, as pressure increases,
the solvent environment becomes ordered allowing for an increase of the heli-
cal content of the peptide. Our work investigates hydration of the peptide back-
bone using both temperature and pressure as modifying conditions. The
synthetic peptide sequence, (AAAAK)3-AAAAY is well-characterized and
ideal for the study of helix properties3. To measure the coupling of pressure
and temperature, Fourier transform infrared spectroscopy (FTIR) monitors
the perturbations in the secondary structure via the amide I’ band. FTIR is a sen-
sitive technique for detecting changes in hydrogen bonding and has been used
for the estimation of amide photon exchanging with the solvent. Pressure on the
peptide is applied using a manually manipulated Diamond Anvil Cell (DAC).
Isotopically labeled residues within the peptide have been exploited for probing
local interactions due to the shift of the heavier masses of the labeled residues to
a lower frequency compared to the global 12C amide I band around 1633 cm1.
Our goal is to compare the location of isotopically edited alanines and the abil-
ity to remain desolvated with increasing pressure and temperature. With this
technique, it was found that alanines proximal to the lysines were protected
from solvent hydrogen interactions due to side chain shielding in the model
sequence.
(1) Barber-Armstrong, W.; Donaldson, T.; Wijesooriya, H.; Silva, R. A.;
Decatur, S. M. J Am Chem Soc 2004, 126, 2339.
(2) Gnanakaran, S.; Hochstrasser, R. M.; Garcia, A. E. Proc Natl Acad Sci
U S A 2004, 101, 9229.
(3) Paschek, D.; Gnanakaran, S.; Garcia, A. E. Proc Natl Acad Sci U S A 2005,
102, 6765.
3005-Pos Board B775
Dependence of Plant Cell Wall Composition and Structure on Cellulose
Synthase-Like Knock Out Mutant
Andreia M. Smith-Moritz1, Jeemeng Lao1, Joshua L. Heazlewood1,
Pamela C. Ronald1,2, Miguel E. Vega-Sanchez1.
1Joint BioEnergy Institution , Lawrence Berkeley National Labs, Berkeley,
CA, USA, 2University of California, Davis,Dept of Plant Pathology, Davis,
CA, USA.
Plant cell walls are a complex mixture of polysaccharides, proteins and the phe-
nolic polymer lignin that have been recently targeted as possible sources of fer-
mentable sugars for the production of biofuels. The development of a biomass-
based biofuels industry is partly dependent on genetic engineering and breeding
next generation crops containing, among other traits, easily extractable cell
wall sugars. Thus, a better understanding of how plants synthesize, deposit
and modify their cell walls is necessary for the selection of traits important
for biofuel crop improvement. The identification of plants with altered cell
wall composition or structure can prove useful in the discovery of novel genes
involved in the biosynthesis and modification of the cell wall.
The CELLULOSE SYNTHASE-LIKE 6 (CSL6) gene has been recently shown
to mediate the biosynthesis of mixed-linkage glucan (MLG), a cell wall poly-
saccharide that is thought to be necessary for cell wall expansion in the primary
cell wall of young seedlings. A detailed analysis of a loss-of-function MLG rice
mutant has been recently conducted revealing surprising results. Though the
mutant showed a 99% reduction of MLG content, the rice clsf6 knock out mu-
tant showed only a slight decrease in growth compared to wildtype. The cell
wall properties of both mutant and wild type were determined via biochemical
and various spectroscopic (Fourier Transform Mid-Infrared spectroscopy)


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Study of Energetic Particle Induced Biological Effect through FTIR and Raman Micro-Spectroscopy

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