Wednesday 10 July 2013, Strathblane & Cromdale Halls, 16:30-18:30
Poster session B -Biology: structure and function
P.024 An insight into toxin translocation across the gram-negative bacterial membrane
W Arunmanee1, A Solovyova1, C Johnson1, H Ridley1, R Heenan2 and J Lakey1
1
Newcastle University, UK, 2ISIS-STFC, Rutherford Appleton Laboratory, UK
Colicin N (ColN) is a pore-forming bacteriocin produced by gram-negative Escherichia coli which can kill susceptible
E. coli by permeabilising their inner membranes. To reach this cytotoxic site of action, ColN needs to cross the outer
membrane which is known to be a formidable protective barrier due to the tightly packed lipopolysaccharides in the
outer leaflet of this asymmetric bilayer. The translocation requires the interaction of ColN with outer membrane
protein F (OmpF) and the C-terminal domain of the TolA protein located in the periplasm. OmpF has been proposed
to be both a receptor and translocator of ColN whereas the interaction with TolA facilitates the Colicin N import
through the cell envelope.1 In order to investigate ColN translocation across the outer membrane, we are studying
the structure of OmpF/ColN translocation complex and OmpF/TolA complexes using biophysical approaches such
as small-angle neutron scattering (SANS) and isothermal titration calorimetry (ITC). The SANS study of OmpF in
complex with ColN and TolA was carried out by the use of selective deuteration of OmpF, Colicin N, TolA and
detergents, the same strategy used previously by Clifton and coworkers.2 The OmpF/ColN and OmpF/TolA
complexes were formed in the presence of octyl glucoside and sodium dodecyl sulfate, respectively. Furthermore,
the interaction, measured by ITC, of OmpF with truncated and full-length Colicin N in the presence of detergents will
also be presented.
[1]
[2]
Baboolal, T. G., et al. (2008) Structure. 16, 371-379
Clifton, L. A., et al. (2012) J. Biol. Chem. 287, 337-346
P.025 General anaesthesia: membrane interaction & pressure reversal
R Barker1, S Roser2 and P L Chau3
1
Institut Laue-Langevin, France 2University of Bath, UK, 3Institut Pasteur, France
General anaesthetics (GAs) are widely used in medicine, however the precise mechanism of their action is still
poorly understood. The ubiquity of the GA effect and the chemical diversity of the molecules that can lead to it (such
as high pressure nitrogen and the generally unreactive halothanes) seem to suggest that the process must spring
from a structural rather than a specific pharmacological antagonism effect. In a series of ground- breaking
experiments in 1950, Johnson and Flagler were able to show that anaesthesia in tadpoles by 2-5% ethanol was
reversed at a pressure of about 130 atm. [1] This has been demonstrated further in a number of other studies, all of
which identify pressure reversal as a defining feature of general anaesthesia.[2,3]
However, the structural interaction of GAs with the membrane and the mechanism by which reversal occurs is still
poorly understood. Here we present a detailed structural study using neutron reflection from model floating lipid
bilayers to identify both the position of GAs and the resultant effect on the membranes. This is combined with
molecular dynamics (MD) simulations and reflectivity from Langmuir monolayers to investigate the inclusion and
exclusion of GAs through increasing surface pressure. The results offer new insight into the contribution of lipids in
pressure reversal, independent of protein interactions, and present a new opportunity for using MD simulations in
the analysis of neutron reflectivity data.
[1]
[2]
[3]
Johnson, F.H. et al.., Science, 112, 91-92 (1950)
Trudell, J.R. et al., Biochimica et Biophysica Acta, 291, 328-334 (1973)
Halliday, D.J.X. et al., British Journal of Pharmacology, 67, 229-237 (1979)
ICNS 2013 International Conference on Neutron Scattering
P.026 Study of bovine serum albumin dynamics in the vicinity of dynamical transition by inelastic neutron
scattering
J P Embs1, S Lushnikov2 and A Svanidze2
1
Paul Scherrer Institut, Switzerland, 2A.F. Ioffe Physical Technical Institute, Russia
The great interest to the dynamical transition in hydrated biopolymers is connected with the fact that it has the
features typical for glass transitions in amorphous systems, and, at the same time, strong changes in dynamics at
temperature decrease below dynamical transition affect the protein biological activity and functioning [1]. Despite
numerous studies the nature of the dynamical transition is still up for debate. The purpose of our work was to study
the low-frequency part of vibrational spectrum of model protein, bovine serum albumin (BSA) in the vicinity of the
dynamical transition temperature by inelastic neutron scattering. Particularly, the temperature behavior of the boson
peak and the contribution of relaxational motions have been analyzed. The lyophilized powder of BSA provided by
Sigma has been used for the neutron scattering experiments without future treatment. The neutron scattering
measurements have been performed at the direct geometry cold neutron time-of-flight spectrometer FOCUS (SINQ,
Paul Scherrer Institut) using 4.3 Å neutrons. The temperature was varied from 100 K up to 340 K. As a result, the
inelastic incoherent dynamic structure factors and the density of states have been obtained for BSA in broad
temperature range. The analysis of these data gave us the possibility to reveal anomalies in the temperature
evolution of the boson peak and the relaxation dynamics around 240 K.
[1]
W. Doster, Biochim. Biophys. Acta 1804 (2010) 3
P.027 Clustered and arrested states of highly concentrated lysozyme systems
P D Godfrin1, Y Liu2 and N J Wagner1
1
University of Delaware, USA, 2NIST Center for Neutron Research, USA
Studying the solution structure of highly concentrated proteins interacting with both a short-range attraction and
long-range repulsion is directly applicable to our understanding of their properties in highly crowded environments,
such as within the human body or in biopharmaceutical therapeutics for subcutaneous injection. We use lysozyme
as a model protein and treat it within the framework of colloid science to extract the effective interactions and phase
behavior using liquid state theories. Using a multi-faceted approach, including small angle neutron scattering
(SANS), neutron spin echo (NSE), rheology, and simulations, the formation of viscous and arrested states are
shown to correspond to distinct solution structures, including dynamic clusters and percolated networks. In order to
further reveal the structure of a fluid system with these two competing potential features, we have also performed
Monte Carlo (MC) simulations of a generic system of particles interacting via an isotropic potential with short-range
attraction and long-range repulsion. By using a corresponding states argument, a master phase diagram has been
shown to exist for these systems with competing interactions. Specifically, the predicted binodal of a reference
system interacting with only the hard core and attractive potentials, and not the long range repulsions, has been
shown to predict the transition from a monomer dominating state to a clustered state. This simulational and
theoretical work provides a deeper understanding of the states realized in highly concentrated lysozyme systems
investigated using both SANS and NSE techniques.
ICNS 2013 International Conference on Neutron Scattering
P.028 Structure of α-synuclein and glucocerebrosidase at the lipid membrane interface
F Heinrich1, C M Pfefferkorn2, T L Yap2, Z Jiang2, E Sidransky3 and J C Lee2
1
Carnegie Mellon University, USA, 2National Heart, Lung, and Blood Institute, National Institute of Health,
USA, 3National Human Genome Research Institute, National Institute of Health, USA
While most proteins adopt folded structures linked to specific cellular functions, some polypeptides are highly
unstructured, remaining dynamic and undergo large structural rearrangements that are dependent on the
environment. Understanding how environmental factors affect α-synuclein conformation is of great importance
because its misfolding is intimately connected to Parkinson's disease etiology. The role of membranes is of
particular interest because membranes not only affect the protein-folding landscape, but are also ubiquitous in vivo.
Neutron reflectometry was used to characterize membrane-induced conformational changes in α-synuclein,
revealing the polypeptide structural envelope and changes in bilayer structure. Moreover, we investigated the
structure of the complex of α-synuclein and glucocerebrosidase, a lysosomal enzyme deficient in Gaucher disease,
to understand why mutations in glucocerebrosidase are a risk factor for parkinsonism. Additional information on
residue-specific environments was gained via steady state and time-resolved fluorescence measurements of single
fluorophore-containing variants. Beyond the biomedical significance of these findings, we will discuss the
methodical developments in neutron reflectometry that were critical for this work.
P.029 Conformational differences in membrane bound retroviral gag proteins
F Heinrich1, M Barros1, S Datta2, A Rein2, M Lösche1 and H Nanda1
1
Carnegie Mellon University, USA, 2National Cancer Institute, USA
The structural protein termed Gag is an essential component for the assembly of new retroviral particles in infected
host cells. Cryo-EM of immaure virions indicated Gag molecules laterally pack on the plasma membrane in long
extended conformational states. All Gag proteins contain structured domains that are separated by linker regions.
These linkers can be flexible, as in the Human Immunodeficiency Virus (HIV), or rigid, as in the Murine Leukemia
Virus (MLV). Previous work by our group showed that HIV-1 Gag can undergo large conformational depending on its
biochemical environment. In particular transitioning from a compact to extended conformation required
simultaneous binding with both the lipid membrane and single stranded DNA segments [J. Mol. Biol. (2011) 406
pp.205-214]. In contrast to HIV-Gag, current evidence from small angel X-ray scattering suggests MLV Gag is
constantly extended in solution [J. Virol. (2011) 85 pp.12733-12741]. The properties of these linkers may indicate
different mechanisms for controlling molecular reorganization that leads to proper assembly and membrane
budding. However, the structure of MLV Gag on the membrane in intermediate stages of viral assembly is not
known. Using reflectivity, we determined the dimensions of MLV Gag bound to the membrane. We compared wildtype MLV Gag to a linker deletion mutant termed dp12. Comparative analysis of the SPR data between these two
constructs confirmed similar membrane binding affinity. However, both neutron and x-ray reflectivity measurements
showed significant structural differences. As a result we propose a tentative model for the different mechanisms of
HIV-Gag and MLV-Gag membrane assembly.
ICNS 2013 International Conference on Neutron Scattering
P.030 A molecular control mechanism for PTEN membrane signaling
F Heinrich1, H Nanda1, S Shenoy1, A H Ross2, A Gericke3 and M Lösche1
1
Carnegie Mellon University, USA, 2University of Massachusetts Medical School, Department of Biochemistry and
Molecular Pharmacology, USA, 3Worcester Polytechnic Institute, Department of Chemistry and Biochemistry, USA
PTEN, an antagonist to PI3K in cell signaling, performs its phosphatase activity on PI(3,4,5)P 3 at the plasma
membrane-cytoplasm boundary. It is the second-most frequently mutated protein in human tumors, but how PTEN
binds to membranes, is activated, and acts as a phosphatase remains unknown. Neutron reflection studies of the
PTEN-membrane complex reveal the structural envelope of the protein and show the enzyme penetrating the
headgroup region only marginally. The truncated core domain PTEN structure (PDB 1D5R) fits the membraneproximal region of the envelope remarkably well while the membrane-distal region can be attributed to PTEN’s
unstructured C-terminal tail. All-atom MD simulations of PTEN on PC/PS membranes support this model by
reconstructing the experimentally determined neutron scattering length distributions almost perfectly, and show the
protein-membrane complex and the dynamics of protein-lipid interactions in great detail. Comparative simulations
of PTEN in solution show the C-terminal tail in vastly different organizations in solution and on the membrane,
suggesting a mechanism for the regulation of PTEN membrane association by the C-terminus, and quantify subtle
changes of the structure upon membrane binding. Together, these results provide a reference structure for a
critically important cell signaling enzyme on a fluid membrane.
P.031 Synthesis of deuterated lipids, phospholipids and heterocycles: applications in SANS, diffraction and
neutron reflectometry
P Holden, T Darwish, E Luks, G Moraes, N Yepuri, A Le Brun and M James
ANSTO, Australia
The expansion of the range of molecules deuterated by Australia’s National Deuteration Facility through custom
chemical synthesis or biodeuteration has led to exciting diverse neutron scattering studies previously hampered by
the lack of relevant labelled molecules. The deuteration of oleic acid (gram quantities) and derivative lipids (glycerol
monooleate) and phospholipids has enabled reflectometry and diffraction studies on biomembrane and antioxidant phenomena (e.g. Phloretin) as well as a SANS study of surfactant interactions in lyotropic liquid crystals (in
the form of cubosomes and hexosomes). Phospholipids produced include head (d-9) or tail (d-66) deuterated
DOPC; DPPC; DMPC; and 1,2-diphytanoyl-sn-glycero-3-PC (d78). Deuterated DOPE is significant as it is the major
phospholipid component in bacterial (E. coli) membranes and hence relevant to studies of biomembrane function
and disruption. The chemical deuteration of heterocyclic and aromatic compounds has made possible a diverse
range of investigations including the study of molecular diffusion in thin film triple-layer organic light emitting
devices (OLEDS) by simultaneous neutron reflectometry and fluorescence measurements1, and neutron diffraction
of metal-organic frameworks (MOFs) used for gas-separation and sequestration - facilitating the localization of
hydrogen gas atoms inside the framework.
The approach to deuterating these molecules and case studies of their application in neutron experiments will be
presented.
[1]
Smith, ARG; Ruggles, JL; Cavaye, H; Shaw, PE; Darwish, TA; James, M; Gentle, IR and Burn, PL, Adv. Funct.
Mater. 21(12), 2225-2231 (2011)
ICNS 2013 International Conference on Neutron Scattering
P.032 The "protein corona" of silica- human serum albumin
A Jackson2, J White1, P Lindner3 and M Henderson1
1
Australian National University, Australia, 2European Spallation Source, Sweden, 3Institut Laue Langevin, France
In the recent review and front cover for Nature Nanotechnology 207, 779-786, 2012, K A Dawson and colleagues
describe biological consequences of the interaction of nanoparticles with proteins found in blood and other bodily
fluids. This model has proved to be a fruitful way of looking at this question.
How the particles are attached, how they might be directed by more knowledge of the structure of the “corona” are
questions put by Dawson et al. The present model for this attachment is a spherically shell of protein around the
nanoparticle with some peptide strands exposed to the surrounding fluid.
Having previously recognised the strong attachment between silica and b lactoglobulin [1], this paper is an interim
report on the first test of the model in solution -using contrast variation in neutron small angle scattering. We have
shown that a “corona”, of the type described above, is not found when the globular protein human serum albumin
(HSA) interacts with 8nm radius silica nanoparticles in standard phosphate buffer, pH7, at 25C.
Instead, our data are well fitted for solutions by a "pearls on a string" model of threads of protein and silica – which
subsequently coalesce. Such protein-nanoparticle aggregates may be toxic if they are biochemically nondegradable or trapped by bodily organs. This is relevant to the biological fate of silica in serum and plasma and may
indicate other types of "corona' of biological importance.
[1]
Ang J.C.,Lin J-M., Yaron P.N. and White J.W. Protein trapping of Silica Nanoparticles. Soft Matter, 6, 383 –
390, (2010)
P.033 The atomic structure of indole in solution
A Johnston1, Y Zhang2, S Busch1, M Sansom1, C Redfield1 and S McLain1
1
University of Oxford, UK, 2Princeton University, USA
Indole is an aromatic, heterocyclic, organic compound which is the functional group of the amino acid tryptophan.
Tryptophan is an amino acid essential to membrane proteins structures, where it is thought to anchor these proteins
to the membrane bi-layer. Indole is also a precursor to many pharmaceuticals, where its ring structure is often seen
in many synthetic drug molecules. Here the atomic structure of indole in methanol/water solutions has been
determined using a combination of NMR and neutron diffraction with isotopic substitution techniques,
complemented by computational techniques. The neutron diffraction data was modelled using Empirical Potential
Structure Refinement (EPSR) – a computational technique specifically designed to model disordered materials- and
further compared with independent molecular dynamics simulations.
This combination of techniques allows for a detailed understanding of the microscopic atomic structure of indole in
an ampliphilic solution. Measurements on indole in solutions of 1 indole :29 methanol:30 water molar ratio have
been performed in order to understand the hydration of indole in the presence of organic groups and water. This
allows for the hydrophilic versus hydrophobic interactions to be determined between indole and water in methanol
solutions, which provides insight into the role of indole as a functional group in tryptophan where this group is
thought to anchor proteins to the membrane bilayer at the water/lipid interface.
ICNS 2013 International Conference on Neutron Scattering
P.034 Application of neutron scattering to the problems of drug delivery through the skin
M Kiselev
Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Russia
Development the vesicular drug carriers through the skin are prospective medical project. Lipid matrix of the
outermost layer of the skin, stratum corneum (SC), is major skin barrier. Important scientific problems should be
solved for the project realization:
1. Nanostructure of the stratum corneum lipid matrix and organization of the drug diffusion road.
2. Development of stable unilamellar vesicles (ULVs) with high deformation properties and methods for
the ULVs characterization.
Presented report relates to these problems. Main results are:
i.
ii.
[1]
[2]
[3]
[4]
Significance of the ceramide 6 molecules in the creation of super-strong interaction in the SC lipid
matrix have been demonstrated via neutron diffraction on the oriented model SC membranes [1,2].
Method of separated form factors (SFF) was developed for the characterization of the unilamellar
vesicles via small-angle neutron scattering. Application of the SFF method for DMPC vesicles
demonstrates the dependence of the bilayer structure and hydration on the membrane curvature [3,4].
M.A. Kiselev, et al. European Biophys. J. 34 [2005] 1030–1040
M.A. Kiselev. Crystallography Reports 52 (2007) 525–528
М.А. Kiselev, et al., European Biophys. J. 35 [2006] 477-493
M.A. Kiselev, et al. Chemical Physics 345 (2008) 185-190
P.035 Stealth carriers for structural studies of membrane proteins in solution
S Maric1, N Skar-Gislinge1, M Boas Thygesen2, J Schiller3, M Moulin4, M Haertlein4, T Forsyth4, T Günther Pomorski2
and L Arleth1
1
The Niels Bohr Institute, Denmark, 2University of Copenhagen, Denmark, 3University of Leipzig, Germany, 4Institut
Laue Langevin, France
Structural studies of membrane proteins remain a great experimental challenge. The reconstitution of such proteins
into carriers that mimic the native bilayer environment allows the use of small-angle scattering techniques to carry
out fast and reliable structural analyses. One problem with this approach is that the carriers contribute to the
measured scattering intensity in a highly non-trivial fashion, making subsequent data analysis very challenging.
Here we present an elegant solution to circumvent the intrinsic complexity brought about by the presence of the
carrier. This approach relies on the use of a well-tested nanodisc platform in conjunction with small-angle neutron
scattering (SANS) studies that exploit deuterium labelling and contrast variation methods. We demonstrate that it is
possible to prepare a specifically deuterated, stealth version of these nanodiscs that renders it invisible to neutrons
in 100% D2O at the length scales relevant to SANS . This stealth carrier can then be used as a platform for lowresolution structural studies of membrane proteins using well-established data analysis tools originally developed
for soluble proteins.
ICNS 2013 International Conference on Neutron Scattering
P.036 SANS study of the interaction of αB-crystallin with prefibrillar α-synuclein
A Rekas1, A Sokolova1 and J Carver2
1
ANSTO, Australia, 2University of Adelaide, Australia
The molecular chaperone, αB-crystallin, has the ability to prevent amorphous and fibrillar aggregation of proteins
implicated in human diseases, e.g. cataract and amyloidoses. Co-localization of αB-crystallin with α-synuclein in
Lewy bodies and evidence for their interaction in vitro suggest an involvement of αB-crystallin in the response to
Parkinson's disease.
Australian National Deuteration Facility offers biological and chemical deuteration of molecules for the neutron
beam and NMR user communities. 73% deuterated αB-crystallin was produced with structural and functional
characteristics consistent with those of hydrogenated αB-crystallin.
We studied interactions of αB-crystallin with α-synuclein by SANS on NG7 beamline at NCNR. Each component
was measured at three contrast points (40%, 70% and 99% D2O). No structural changes were seen in either
protein over 4 hr at 37oC. Consistent with previous models, αB-crystallin molecule formed a globular oligomer. Ab
initio shape models of α-synuclein were consistent with previous data showing elongated shapes.
Data of the complex in 40% and 70% D2O indicated a progressive increase in dimensions with time. This might
indicate formation of a complex in which α-synuclein molecules intercalate and displace αB-crystallin subunits.
P.037 Analysis of Size and Distribution of Korean Human Compact Bone by Small Angle Neutron Scattering
E J Shin2, Y Choi1, B S Seong2 and Y S Han2
1
Dankook University, Korea, 2KAERI, Korea
Small angle neutron scattering (SANS) technique obtained by thin film evaluation was applied to evaluate bony
canliculi of compact bone of human to gain important information about growth and degradation of the human
bone. In this study, various types of bone of the Korean body with different physiological histories were selected to
fit to the size of a normal SANS sample-holder of HANARO. Directional distribution of bony lacuna of the human
compact bone was observed. The lacuna of the normal and osteoporosis bones were periodically distributed in
compact bone with about 7.3μm intervals. The amount of canliculi of lacuna was larger in normal bone than in
osteoporosis bone. Microstructure observation by transmission electron microscopy and measurement of bone
density also support the fact that SANS is one of the useful techniques to study in-situ evaluation of the thin bony
canliculi of compact bone of humans and bio-bodies.
P.038 Polyelectrolyte thin films as a platform for pH-responsive lipid membranes
S Singh, A Junghans and J Majewski
Los Alamos National Laboratory, USA
We present a robust and simple method for the preparation of pH-responsive lipid membranes. Polymeric cushions
to support a lipid bilayer were obtained by performing layer-by-layer (LbL) deposition of a polycation and a
polyanion on quartz, with polycation as a capping layer. A model lipidic membrane was obtained by depositing
1,2-Dipalmitoyl-sn-glycero-3-phophoethanolamine (DPPE) bilayer using Langmuir-Blodgett and Langmuir-Schäfer
technique. Neutron Reflectivity experiments revealed that the separation distance between the polymeric cushion
and the DPPE bilayer can be reversibly adjusted by varying the pH of the aqueous environment. We believe that
electrostatic interactions between the polymeric cushion and the DPPE bilayer play an important role in affecting
the separation distance between the same. Similar pH-dependent lift-off was also observed with DPPC bilayer
deposited on similar polymeric cushions. We believe that this novel system offers great potential for fundamental
biophysical studies of membrane properties decoupled from the underlying solid support.
ICNS 2013 International Conference on Neutron Scattering
P.039 Subunit kinetics in protein complexes revealed by deuteration-assisted small-angle neutron scattering
M Sugiyama1, E Kurimoto2, H Yagi3, M Hirai4, G Zaccai5 and K Kato6
1
Kyoto University, Japan, 2Meijo University, Japan, 3Nagoya City University, Japan, 4Gumma University,
Japan, 5Institut Laue Langevin, France, 6Okazaki Institute for Integrative Bioscience, Japan
Protein complexes, which are homo- or hetero-oligomers, have kinetics related to their formation/deformation and
also functions. However, the kinetics, such as “subunit exchange” between homo-oligomers and/or heterooligomers consisting of similar subunits, is invisible because it makes no/little structural change. Under this
situation, deuteration and small-angle neutron scattering (SANS) are good combination to reveal the kinetics is
hidden: deuteration labels the subunits with less deformation and SANS can distinguish them from non-deuterated
ones.
With this technique, we revealed kinetics of subunit exchange between proteasome α7 rings (homo-oligomer)
[1]: proteasome α7 ring is a heptamer with seven fold symmetry and α7 subunit is one of components of the 20S
Proteasome. We prepared for deuterated proteasome α7 rings and natural ones, and mixed them in 81% D2O
solvent, where deuterated and natural oligomer has same absolute scattering contrast. Then, we performed its timeresolved SANS experiment. The zero-angle scattering intensity gradually decreased indicating the subunit exchange
proceeded and generated the oligomers with deuterated and natural subunits which has lower scattering contrast.
By analyzing the time evolution of the zero-angle scattering intensity, the subunit exchange of this oligomer has two
steps processes.
In the presentation, we will show subunit exchange between proteasome α7 rings and proteasome α6 subunits,
which could relate to formation mechanism of the 20S proteasome. In addition, we will show another type of
subunit exchange in α-crystallin (eye lens protein).
[1]
M.Sugiyama, et al., Biophysical J. 101, 2037-2042, (2011)
P.040 Abstract withdrawn
P.041 The ultrastructure and flexibility of thylakoid membranes in intact leaves and isolated chloroplasts as
revealed by small angle neutron scattering
R Ünnep1, O Zsiros2, K Solymosi3, L Kovács2, P Lambrev2, T Tóth2, L Rosta4, G Nagy5 and G Garab2
1
Wigner Research Centre for Physics, Hungary, 2Biological Research Center, Institute of Plant Biology, Hungarian
Academy of Sciences, Hungary, 3Eötvös Loránd University, Department of Plant Anatomy, Hungary, 4Wigner
Research Centre for Physics, Institute for Solid State Physics and Optics, Hungarian Academy of Sciences,
Hungary, 5Wigner Research Centre for Physics, Institute for Solid State Physics and Optics, Hungarian Academy of
Sciences, Hungary and Paul Scherrer Institute, Switzerland
Performing kinetic structural studies on the thylakoid membranes in intact photosynthetic organisms is a
challenging task due to the limited number of available non-invasive experimental techniques applicable for realtime in vivo measurements. Earlier we studied the multi-lamellar thylakoid membrane systems in cyanobacteria,
diatoms and isolated plant thylakoid membranes with small-angle neutron scattering (SANS) under different,
physiologically relevant environmental conditions and have revealed small ( 1-2 nm) but well discernible lightinduced reversible changes in the lamellar repeat distances occurring on the time scale of 10 s – 1-2 min [1-4].
Interestingly, the observed reorganizations differed significantly from each other in isolated plant thylakoid
membranes and in algal cells. This called our attention to the importance of measuring SANS on whole leaves,
where key regulatory processes occur. Thus, we have extended our investigations and found correlation between the
SANS profiles of intact leaves and the electron microscopic ultrastructure of their thylakoid membranes but also
revealed that the widely applied isolation methods perturb the membrane ultrastructure [5]. The application
ICNS 2013 International Conference on Neutron Scattering
of SANS on intact leaves has opened the possibility of identifying thylakoid membrane reorganizations associated
with different regulatory mechanisms of photosynthesis, such as the adaptation of plants to excess light, studied
here.
[1]
[2]
[3]
[4]
Nagy G. et al. 2011 Biochem J 436:225
Nagy G. et al. 2012 Photosynth Res 111:71
Posselt D. et al. 2012 Biochim Biophys Acta Bioenerg 1817:1220
Nagy G. et al. submitted [5] Ünnep R. et al. in preparation
P.042 Mechanism of cyclotide interaction and pore formation in lipid membranes
H Wacklin1, C Wang2 and D Craik2
1
ESS AB, Sweden, 2Institute for Molecular Biosciences,University of Queensland, Australia
Cyclotides are a family of plant-derived circular peptides with many potential therapeutic applications arising from
their remarkable stability, broad sequence diversity, and range of bioactivities. Their membrane-binding activity is
believed to be a critical component of their mechanism of action. We have studied the binding of the prototypical
cyclotides kalata B1 and kalata B2 to bilayer membranes using a combination of dissipative quartz-crystal
microbalance measurements and neutron reflectometry. Our results show that initial binding to the membrane
surface is followed by a gradual insertion and pore formation process consistent with toroidal pores, and provide a
structural insight into how cyclotides exert their biological activities.
P.043 Interfacial layer structure of human serum albumin nano-silica solutions
J White1, J-C Ang2, R Campbell3 and M Henderson1
1
Australian National University, Australia, 2Department of Materials Science , Berkeley, USA, 3Institut Laue
Langevin, France
The idea of a "corona" [1] formed when proteins and nanoparticles interact in body fluids when accidentally or
purposely brought together has been a very stimulating way of thinking about that composite particle's subsequent
reactivity and ultimate fate.
We have shown that these interactions can be very sensitively detected using x-ray and neutron reflectivity [2] as
they give rise, almost instantaneously, to composite nanometre films at the air-water interface.
This paper is a current report of the structure of films formed by 8nm silica particles and human serum albumin at
25 C and pH 7 using neutron reflectivity and contrast variation. This provides information on the layering of the two
components and the composition of those layers for comparison with parallel experiments using small angle
scattering on the solution species produced in mixtures of the same compositions as used for reflectivity. The data
report the onset of layer structures for very low concentrations of protein (<0.003mg/ml).
[1]
[2]
Monopoli M.P., Arberg C., Salvati A., and Dawson K. A., Biomolecular Coronas Provide the Biological
Identity of Nano-sized Materials. Nature Nanotechnology 207, 779-786, (2012)
Ang J.C.,Lin J-M., Yaron P.N. and White J.W. Protein trapping of Silica Nanoparticles. Soft Matter, 6, 383 –
390, (2010)
ICNS 2013 International Conference on Neutron Scattering