Accepted Manuscript
Recent advances in neutron reflectivity studies of biological membranes
Jeremy H. Lakey
PII: S1359-0294(19)30007-X
DOI: https://doi.org/10.1016/j.cocis.2019.02.012
Reference: COCIS 1274
To appear in: Current Opinion in Colloid & Interface Science
Received Date: 7 February 2019
Revised Date: 26 February 2019
Accepted Date: 26 February 2019
Please cite this article as: Lakey JH, Recent advances in neutron reflectivity studies of biologicalmembranes, Current Opinion in Colloid & Interface Science, https://doi.org/10.1016/j.cocis.2019.02.012.
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Recent advances in neutron reflectivity studies of biological membranes
Jeremy H. Lakey
Institute for Cell and Molecular Bioscience, Newcastle University, Framlington Place,
Newcastle upon Tyne, NE2 4HH, UK
Abstract
Biological membranes are critical living interfaces which not only compartmentalise cells but
also provide specific surfaces upon which biochemical reactions take place. Their study is
essential for our understanding of life and disease, and also for the development of new
pharmaceuticals. Since they are dynamic structures reliant upon the surrounding aqueous
phase for their activity, techniques which can probe them at a molecular scale under
physiological conditions are crucial to understand their function. Here I describe some of the
recent developments in neutron reflectivity (NR) in this research area and suggest where
future developments in the technology might be usefully directed.
Keywords
Neutron reflectivity, model membranes, outer membrane, lipopolysaccharide, deuteration,
GISANS.
1. Introduction
It has been recounted many times that the geneticist J.B.S Haldane said that one of the
things you could learn from natural history about a Creator was that “he has an inordinate
fondness for beetles”. This conclusion was based upon the fact that there are over 300,000
beetle species compared to about 10,000 mammals. If a membrane biologist were asked the
same question then the answer might be “a great fondness for interfaces” since cells are
replete with boundaries and surfaces which govern how life runs at the molecular level.
However, if we look from the physics point of view, Wolfgang Pauli concluded that God
made the bulk and surfaces were the work of the devil. Due to the inherent difficulty in
studying cell membranes this conclusion would probably unite biologist and physicist alike.
Cells vary in the complexity of the membranes that they contain. The first cells were
presumably defined by the first cell or cytoplasmic membrane and therefore were likely to
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have been surrounded by a single lipid bilayer. This pattern persists in the prokaryotes, the
simplest cells alive on the planet which include the bacteria and cyanobacteria. Their very
name is based upon their membrane structure since prokaryon indicates that they represent
a state before the existence of the nucleus, the membrane bound organelle in which nucleic
acids are packaged in eukaryotes such as fungi, animals and plants. As cells get more
complicated, it is their membrane architecture which is the most visible example of their
development and there are many techniques available to examine their structure and
dynamics ranging from atomic force microscopy to x-ray methods. To study them in relevant
situations they must be hydrated, at physiological temperatures and able to interact with
soluble components such as proteins or drugs. Neutron reflectivity (NR) is a method that can
probe the structure and dynamics of a single model membrane within an enclosed sample
cell, under close to physiological conditions, where it can be exposed to controlled changes
in environment or bathing solution. This review tries to highlight recent papers in the field
which show what is possible and where this science may go.
2. Membranes as biological interfaces.
Eukaryotes not only have nuclear membranes but also an array of membrane bound
compartments. In addition to this complexity there are two special membrane bound
compartments, the mitochondria and the chloroplasts, which are derived not from modified
cytoplasmic membrane but from free living cells that were taken up by the eukaryotes early
in their evolution from the archaea. The mitochondrion and chloroplast have two membranes
with a biochemically important intermembrane space in which crucial reactions take place.
These double membranes reflect the original structure of the free living forms and, as we
shall see later, these remain important in bacteria to this day. These membranes not only
create separate compartments within cells but also provide interfaces where reactions and
other activities occur.
The lipid bilayer forms the basis for all of these structures and has the important properties
of self-assembly, flexibility and fluidity which allow it to perform the many functions required
of membranes. In turn this means that the molecules which form these disparate structures
conform to a basic pattern of amphipathic structure, where a hydrophilic head group is
attached to one or more acyl chains composed mainly of –CH2– groups. This simple
architecture belies a wide array of molecular designs which confer changes in the physical
properties of the membranes, their surface interactions and even their metabolic function.
For example, membrane lipids normally quietly responsible for the structure of a cell
membrane can be converted after trauma, by enzymatic action, into powerful signalling
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molecules involved in wound healing [1]. This process involves removing one acyl chain
which makes the lipid sufficiently water soluble to allow it to leave the bilayer and act upon
targets elsewhere in the cell.
Thus the individual parts of the membrane lipid play critical roles in the function of the
bilayer. The headgroups provide charge, molecular interactions, cell signalling and many
other roles. The lipid chains control the fluidity, stability and also contribute to the
biochemistry of the cell. Membranes are also characterised by their protein content which
defines the type and level of biochemical or physiological activity which that each membrane
undertakes. For example the Schwann cell membrane of the myelin sheath, which
electrically insulates nerve fibres, has a very low protein content (approx. 18% by weight),
whilst the mitochondrial inner membrane, which is highly metabolically active, has almost
80% protein. To understand these varied biological interfaces it is therefore essential to be
able to simultaneously study these various components within the assembled membrane
and observe how they work together. It is in this observation of dynamic mixtures of
biological molecules that NR provides unique information.
3. Neutron reflection studies of membranes
If one spreads a colourless oil on the surface of water and illuminates it with white light, one
can see rainbow patterns due to interfering reflection and refraction from the layers of
differing thickness and refractive index. In a similar way, specular neutron reflection
measures the differences in chemical composition across the membrane by the way
differences in neutron scattering length density (SLD, the neutron analogue of refractive
index) alter the angular and wavelength dependent intensity of reflected neutrons. The data
is fitted to models of the transbilayer structure composed of series of layers each defined by
a thickness, roughness and SLD. Neutron reflection can be applied to lipid monolayers at the
air-water interface or lipid bilayers assembled on solid substrates such as silicon. There
have been several recent reviews on applications of neutron reflection to membranes or thin
films where comparisons with diffraction and details of the experimental methods are clearly
described [2-4].
4. What can neutrons tell us about dynamic membrane biology?
Figure 1 provides a guide to the questions that neutron scattering methods can address. In
Fig1 A the simple lipid bilayer is shown with gaps which can occur due to incomplete layer
assembly or due to intrinsic dynamics, for example when using unsaturated phospholipids
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[5]. As with all biological molecular assemblies, water is the common environment in which
all membrane studies take place. Here, the ability of neutrons to differentiate between the
distinct SLDs of hydrogen and deuterium is of great importance since the hydration state can
be measured more accurately than by other methods. In studying model bilayers, which
feature greatly in neutron reflection studies [3, 5-7], the use of different background solvent
contrasts, obtained by mixing H2O and D2O, allows the water content along the normal to the
membrane plane to be easily measured. In general the more well packed the membrane the
lower the water content of the hydrophobic core and so the quality of the membrane can be
readily measured. Subsequently the effects of membrane perturbing molecules such as
antibacterial peptides can be determined by the increasing water penetration into the
hydrophobic core of the bilayer even if the peptide itself cannot be visualised by neutron
reflection due to its small size, or insufficiently distinctive SLD, [8-11]. Finally, the right hand
side of Fig1A shows that lipid phase changes from solid or gel to liquid crystalline phase can
be revealed by thickness changes or water content. In Fig1B on the left, an asymmetric
membrane is shown where the lipid types are different on either side. This could be
determined by the different head group sizes which can be measured by NR. Such
asymmetry is found throughout nature but is most pronounced in bacterial outer membranes
OM discussed later in the review. However the measurement is made more accurate by
deuterating the lower leaflet which is then clearly resolved by neutrons [12]. Any subsequent
mixing of these two layers is quantifiable. In other cases different lipid types can be
selectively deuterated to examine their distribution [13] and the exchange between the
leaflets can be followed. Finally, on the right protein lipid interactions which lead to
alterations in lipid distribution can be followed or supported by small angle neutron scattering
measurements [14, 15]. In Fig 1C the possibilities of membrane protein studies are outlined.
Proteins including toxins [16, 17] can interact simply with membrane surfaces or with specific
protein (or lipid) receptors [18-20]. Furthermore the interaction of drugs or nanoparticles can
be readily measured [21]. Integral membrane proteins usually span the membrane and
naturally have a different SLD to the lipid component [22, 23], a difference that can be
enhanced by protein deuteration to reveal their individual structures within the membrane
[24]. In some cases these proteins are channel forming and they thus introduce water into
the hydrophobic core of the membrane, as shown by the blue channel. This can be
measured by solvent contrast methods but is difficult to differentiate from membrane defects
shown in Fig. 1A. Finally the dynamics of membrane proteins including multimerisation or
conformational change can be addressed. The limitation of specular reflection data to only
one dimension limits our ability to see such events and in the final section of this review I
discuss the potential developments which may make this essential next step achievable
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5. Deuteration of biological molecules.
Through the use of deuterated lipids and proteins, their positions within the membrane can
be selectively determined against a background of different molecules [25-27]. The different
chemical composition and hydration of phospholipid head groups compared to their acyl tails
is often enough to allow us to model their thickness and position but by selectively
deuterating these regions one can significantly enhance the contrast between one region
and another giving one confidence in the derived models. Selective deuteration of all or parts
of lipids is most easily achievable by purchasing commercially available materials but such
studies can be extended by bespoke deuteration of lipids and proteins relevant to the
membrane study in question [28].
Useful levels of deuteration can be achieved in any laboratory which purifies the native
material from bacteria and higher levels up to complete (per-) deuteration can be obtained
by working with dedicated deuteration laboratories often associated with major neutron
research centres [26]. Specific lipids, such as lipopolysaccharide, can be isolated from the
natural producer organism simply grown in media where water has been replaced by D2O or
also with deuterated carbon sources [26]. Where the lipids are found in mammals which
cannot be fed high concentrations of D2O modified yeast cells have been found to be good
sources of deuterated lipids such as cholesterol [29]. Deuterated proteins can be produced
recombinantly by having their genes expressed in bacteria or yeast which can be grown in
deuterated media. This can be made more specific by deuterating part of the protein as
elegantly shown for the interaction of the Parkinson’s disease related protein α‑Synuclein
with membranes [18]. Thus the different components of the membrane are purified as
separate samples and then recombined to reproduce the model of the natural membrane.
An important study has been carried out into the properties of deuterated lipids produced in
a natural host, in this case the yeast Pichia pastoris. It was shown that growth in deuterated
media altered the molecular composition of the natural membrane so that the model
membranes formed from them had different dimensions [30]. In another study the ratio of
phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in Escherichia coli lipid
extracts was found to be similar in normal and deuterated samples [7]. In the future it will be
interesting to see if deuterated lipids show specific isotope effects.
In an interesting alternative to combining purified proteins with lipids to construct the models,
it has been shown that proteins can be inserted into supported lipid bilayers using cell free
methods, where the proteins are inserted directly into the membrane from isolated
ribosomes using a cell free synthesis method [24] [31]. The proteins are made from amino
acids added to the solution, and it is possible to use deuterated versions so that the proteins
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have bespoke SLD values. In one example, the insertion of the hepatitis C p7 viroporin into
model bilayers was not clear when hydrogenated protein was used, but when deuterated
protein was synthesised the insertion became clearly measurable [24]. This insertion could
also be followed by electrochemical techniques since the protein increases the conductance
of lipid bilayers in which it is inserted.
6. A biological case study - the Gram-negative bacterial outer membrane (OM).
Here, partly due to my own research but also highlighting that of several other groups, I
discuss a series of recent papers on bacterial outer membranes (OM) which also highlight
general trends in reflectometry studies of membranes. As stated in the introduction, Gram-
negative bacteria possess a double membrane structure which provides extra protection
from the outside environment since the outer membrane acts as a molecular sieve. Not only
is this of biological importance but these bacteria are increasingly important causes of
antibiotic resistant infections from species such as Escherichia coli, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp. This has meant
that there has been great interest in studying this structure in vitro since, due to the very
small size of bacterial cells, many of the methods successfully used to study membranes of
eukaryotes in vivo are not applicable. The particular challenge of the OM is that it is highly
asymmetric with “normal” phospholipids present on the inner face and complex glycolipids
called lipopolysaccharides (LPS) on the outer face. This asymmetry is an essential feature of
the membrane enabling the robust outer layer to resist environmental challenges in the gut,
such as bile salts and proteases. LPS consists of a lipid A section with 5-7 acyl chains to
which is attached the core oligosaccharide of the head group with about ten sugar units. This
can be extended to include many hundreds of units in so called smooth strains of bacteria. It
has been shown that we can gain an insight into OM function using symmetric bilayers
containing mixtures of LPS and phospholipids [32, 33] but the full capabilities of NR are
exploited on fully asymmetric models.
One way to create asymmetric surfaces is to use hybrid bilayer membranes where the solid
surface is covered with a hydrophobic layer upon which a lipid monolayer is created. This is
shown in Figure 2 reproduced from [8]. An example of long chain “smooth” LPS being
studied in such a system has been published by Schneck’s group [34]. Building upon
previous work [35, 36] they were able to create layers containing both short and long LPS
chains and thus use neutrons to measure how these extend into the aqueous environment.
Furthermore they were also able to measure the interactions of two layers by creating
another opposing one, across a small water layer, at the air-water interface. This, together
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with floating bilayer studies [5, 6], further demonstrates the possibilities of investigating
several interacting membranes using NR.
We have created complete asymmetric models by combining two monolayers, one of
phospholipid and one of LPS, formed sequentially at the air-water interface and co -
assembled using Langmuir-Blodgett and Langmuir-Schaefer techniques onto silicon or gold
surfaces. In Figure 3 a complete asymmetric outer membrane model assembled on a silicon
surface is presented with a layer of bound antibacterial protein (colicin N). An alternative
model in which the outer membrane floats on a ≈ 2nm water layer has also been developed
[6].
As outlined in Figure 1 the great strength of neutron reflectivity is the ability to resolve, in Fig
3, the hydrogenous LPS (either purified from bacteria or commercially available) and the
deuterated phospholipid (commercially available), allowing the asymmetry to be not only
observed but accurately quantified, along with a measure of the % surface coverage from
solvent contrast experiments (Figure 3). The hydrophobic tails in either deuterated or
hydrogenated form show SLDs which do not change much when the background solvent is
exchanged from H2O to D2O. However, the solvated headgroups of the core
oligosaccharide, and to a lesser extent the phospholipid, show SLD values which partially
track the bulk solvent and reveal the level of hydration. In this study the layer of bound
protein occupied about 40% of the surface by volume and this is shown by the high degree
of SLD change upon solvent exchange. In the same paper we were also able to measure the
thickness of core oligosaccharide from two versions of LPS and show that this acts as an
insulator to reduce the binding of toxic proteins to the bacterial surface [17]. In a subsequent
study, it was shown that the lipopeptide antibiotic polymyxin only penetrates into membranes
when the LPS is in the liquid crystalline form [8]. The phase change could be measured both
by NR and FTIR, and, provided biophysical support to biological observations that bacteria
maintain their LPS in a liquid crystalline rather than gel state. The combined use of NR and
FTIR is a promising development in the field of membrane research [37].
These models have also been used to support the development of new polymyxin type
antibiotics which are needed to overcome resistance to this important class of antibiotics.
The resistance mechanism can involve modifications of the LPS and the effect that these
changes have on the structure and dynamics of the membrane can be observed by X-ray
[11] and neutron reflectivity [9]. It is also possible to observe the behaviour of novel
antibiotics [10] or anti-microbial peptides in the membrane model [38]. These models lack
integral outer membrane proteins [14] and their role in the penetration of this class of
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antibiotics has not been measured. The significant challenge is to introduce the proteins in
an oriented form so that they conform to the overall asymmetry of the OM. One way is to fix
the proteins to the solid substrate, which then acts as a symmetry breaker but it may
preclude the subsequent use of successive monolayer assembly to achieve asymmetric lipid
membranes [39, 40]. It has been shown that molecularly complete outer membranes can be
assembled on surfaces using outer membrane vesicles and that they show asymmetry [41].
It would be interesting to apply NR to these models, since they should mimic the OM more
closely. However, since NR averages the contributions it may be necessary to start with
bacterial preparations with simplified OM such as reduced outer membrane protein variety
and one short form of LPS. Use of deuterated antibiotics etc. may allow these to be
observed against the natural background or the OM may be made using deuterated bacteria
[41]. Furthermore native eukaryotic biological membranes are also being used to create
planar membrane models which can be investigated by many methods including NR [42]
7. Conclusions.
For the membrane biochemist, knowledge of the lateral motions of lipids and membrane
proteins in the plane of the bilayer is of great interest. The fluid mosaic model proposed in
the 1960’s has been modified over the years to include possible phase separations, rafts
and protein–lipid complexes but understanding the mobility and dynamics of the membrane
is all four dimensions, including time [43], remains a serious challenge. The time dimension
is being addressed by faster beamlines so that changes can be followed with resolution of
minutes or shorter [43, 44]. Resolution in the plane of the bilayer is currently less than
afforded by X-ray grazing incidence diffraction [45] or small angle scattering at the air-water
interface but we need to use neutrons since they are capable of penetrating the
experimental cells where we can construct complete bilayer models under controlled
conditions (Figure 4). Off specular NR signals have been recorded for LPS membranes [46]
whilst experiments with grazing incidence small angle neutron scattering (GISANS), which
can potentially resolve nm sized structures have shown promise with self-organising colloidal
materials [47] and block copolymers [48, 49]. Furthermore grazing incidence neutron spin
echo spectroscopy (GINSES) has been applied to measure the depth dependent dynamics
of extended polymer brushes on silicon substrates [50]. Neutron reflection has contributed
significantly to our understanding of biological membranes but we have only scratched the
surface of what could be measured. Sample preparation is developing fast e.g. [42] and
since three or four dimensional [43] experiments are clearly possible we shall be able to
probe areas of membrane behaviour inaccessible to all other methods. If we can develop
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reliable applications in these areas we will certainly solve some crucial basic science
questions and probably save lives too.
Acknowledgements.
I wish to thank my patient collaborators in neutron scattering for their help and support over
the years in developing realistic biological models that have had influence beyond the world
of neutron science. Particular thanks go to Nico Paracini and Dan Peters for helpful
comments on the manuscript. All opinions and mistakes in this review are my own.
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[34] *Rodriguez-Loureiro I, Latza VM, Fragneto G, Schneck E. Conformation of Single and
Interacting Lipopolysaccharide Surfaces Bearing O-Side Chains. Biophysical Journal.
2018;114:1624-35.
Demonstrates the ability to observe extended, hydrated polysacharide structures
which occur on the surfaces of cells. Such molecules are unlikley to be accurately
observed by other methods and are critical for many intercellular interactions in both
eukaryotes and prokaryotes.
[35] Herrmann M, Schneck E, Gutsmann T, Brandenburg K, Tanaka M. Bacterial
lipopolysaccharides form physically cross-linked, two-dimensional gels in the presence of
divalent cations. Soft Matter. 2015;11:6037-44.
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[36] Schneck E, Schubert T, Konovalov OV, Quinn BE, Gutsmann T, Brandenburg K, et al.
Quantitative determination of ion distributions in bacterial lipopolysaccharide membranes by
grazing-incidence X-ray fluorescence. Proceedings of the National Academy of Sciences of
the United States of America. 2010;107:9147-51.
[37] Skoda MWA, Thomas B, Hagreen M, Sebastiani F, Pfrang C. Simultaneous neutron
reflectometry and infrared reflection absorption spectroscopy (IRRAS) study of mixed
monolayer reactions at the air-water interface. Rsc Advances. 2017;7:34208-14.
[38] Michel JP, Wang YX, Kiesel I, Gerelli Y, Rosilio V. Disruption of Asymmetric Lipid
Bilayer Models Mimicking the Outer Membrane of Gram-Negative Bacteria by an Active
Plasticin. Langmuir. 2017;33:11028-39.
[39] Hoogerheide DP, Noskov SY, Kuszak AJ, Buchanan SK, Rostovtseva TK, Nanda H.
Structure of voltage-dependent anion channel-tethered bilayer lipid membranes determined
using neutron reflectivity. Acta Crystallographica Section D-Structural Biology.
2018;74:1219-32.
[40] Holt SA, Le Brun AP, Majkrzak CF, McGillivray DJ, Heinrich F, Lösche M, et al. An ion
channel containing model membrane: structural determination by magnetic contrast neutron
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Bacterial Outer Membrane Platform. Scientific Reports. 2016;6.
[42] **Pace HP, Hannestad JK, Armonious A, Adamo M, Agnarsson B, Gunnarsson A, et al.
Structure and Composition of Native Membrane Derived Polymer-Supported Lipid Bilayers.
Analytical Chemistry. 2018;90:13065-72.
An excellent demonstration of the possibilities to move closer to the native state in
membrane models and be able to analyse them with a range of biophysical methods
[43] **Koutsioubas A, Appavou M-S, Lairez D. Time-Resolved Neutron Reflectivity during
Supported Membrane Formation by Vesicle Fusion. Langmuir. 2017;33:10598-605.
Uses both specular and off specular methods to follow kinetics of vesicle fusion at a
solid surface
[44] Adlmann FA, Gutfreund P, Ankner JF, Browning JF, Parizzi A, Vacaliuc B, et al.
Towards neutron scattering experiments with sub-millisecond time resolution. Journal of
Applied Crystallography. 2015;48:220-6.
[45] Le Brun AP, Clifton LA, Halbert CE, Lin B, Meron M, Holden PJ, et al. Structural
Characterization of a Model Gram-Negative Bacterial Surface Using Lipopolysaccharides
from Rough Strains of Escherichia coli. Biomacromolecules. 2013;14:2014-22.
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[46] Schneck E, Oliveira RG, Rehfeldt F, Deme B, Brandenburg K, Seydel U, et al.
Mechanical properties of interacting lipopolysaccharide membranes from bacteria mutants
studied by specular and off-specular neutron scattering. Phys Rev E. 2009;80:9.
[47] Nouhi S, Hellsing MS, Kapaklis V, Rennie AR. Grazing-incidence small-angle neutron
scattering from structures below an interface. Journal of Applied Crystallography.
2017;50:1066-74.
[48] Muller-Buschbaum P. GISAXS and GISANS as metrology technique for understanding
the 3D morphology of block copolymer thin films. European Polymer Journal. 2016;81:470-
93.
[49] Wolff M, Herbel J, Adlmann F, Dennison AJC, Liesche G, Gutfreund P, et al. Depth-
resolved grazing-incidence time-of-flight neutron scattering from a solid-liquid interface.
Journal of Applied Crystallography. 2014;47:130-5.
[50] Wellert S, Hubner J, Boyaciyan D, Ivanova O, von Klitzing R, Soltwedel O, et al. A
grazing incidence neutron spin echo study of near surface dynamics in p(MEO(2)MA-co-
OEGMA) copolymer brushes. Colloid and Polymer Science. 2018;296:2005-14.
[51] Baboolal TG, Conroy MJ, Gill K, Ridley H, Visudtiphole V, Bullough PA, et al. Colicin N
binds to the periphery of its receptor and translocator, outer membrane protein F. Structure.
2008;16:371-9.
Figure legends
Figure 1. Biological membrane features addressable by neutron reflection methods
Studies of simple lipid bilayers can reveal features such as the hydration of the interior which
can be caused by incomplete assembly of the bilayer or physiologically important effects of
certain lipid mixtures. Neutron reflection is uniquely sensitive to replacement of H2O by D2O
and this can probe hydration of the interior and surface headgroups. As shown, this lipid
packing can be affected by the insertion of amphiphilic molecules such as detergents or
lipopeptide antibiotics. The physical state of the lipids, shown on the right as a phase
change, can also be observed by changes in thickness or hydration. B) More complex lipid
mixtures can be studied using differences in headgroups size to follow the components but
clearly if certain lipids can be deuterated (blue) then variations in symmetry or other
distributions are easier to follow. Protein-lipid interactions (green) are also an area yet to be
fully addressed by NR. C) Proteins can interact either with the surface of the model
membrane or with integral membrane proteins acting as receptors. Ion channel proteins
(centre) are a particular challenge since their water filled centres can look like gaps in the
membrane seen in (A). Finally protein-protein structure in the plane of the membrane will
require data from GISANS and related methods (see conclusions).
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Figure 2. Gram-negative outer membrane and models. (A) Schematic representation of
the Gram-negative cell envelope. The cytoplasm is enclosed by a phospholipid inner
membrane (proteins not shown) surrounded by a thin peptidoglycan cell wall. The outer layer
is the asymmetric outer membrane composed of phospholipids in the inner leaflet and
lipopolysaccharides in the outer leaflet. It contains water-filled channel proteins (porins)
through which most antibiotics enter the cell. The LPS is shown as two forms, a rough LPS
(red) containing the lipid A and core oligosaccharide, and variable lengths of O-antigen
present in smooth strains of Gram-negative bacteria in orange. Stabilizing divalent cations
are shown in green. (B) Simple OM model formed by a hybrid bilayer membrane containing
a monolayer of LPS self-assembled on a hydrophobic monolayer. (C) Asymmetric OM model
assembled in vitro on a silicon substrate by sequential deposition of synthetic phospholipids
and purified LPS using a Langmuir trough. (D) Chemical structure of polymyxin B.
Reproduced from [8].
Figure 3. Study of binding of the antibacterial protein colicin N to a model bacterial
outer membrane. The model was composed of short chain Rd-LPS and deuterated
dipalmitoyl-phosphatidylcholine (d-DPPC) on a silicon substrate. Neutron reflectivity profiles
and model data fits (A−C) and the scattering length density profiles these fits describe (D) for
equilibrium Colicin N adsorbed asymmetric DPPC (inner leaflet)/Rd LPS (outer leaflet)
bilayers in the presence of 20 mM HEPES pH 7.2 buffer with 20 µM CaCl2. The three
simultaneously fitted isotopic contrasts shown are (A) d-DPPC/Rd LPS in D2O (red line), (B)
d-DPPC/Rd LPS in a mixture of H2O/D2O that has an SLD equal to that of the silicon
substrate, effectively removing this interface from the data (black line), and (C) d-DPPC/Rd
LPS in H2O (green line). A representation of the interfacial structure determined from these
fits is shown (E) with the silicon substrate to the left (Black) covered with the SiO2 oxide layer
(grey) , lipid head group (green), deuterated tails (red), hydrogenous LPS tails (brown), LPS
core head group (green) and colicin N protein (pink). Reproduced from [17]
Figure 4. In plane data from neutron reflection experiments on model membranes.
Most current NR experiments collect data in the specular range (solid arrows) which
provides profiles of material distribution across the z axis of the membrane (see fig 2).
Methods which acquire data from the y-axis can provide information on the arrangement of
molecules, such as the outer membrane protein F shown here which forms lattice structures
observable by electron microscopy [51], scale bar 10 nm. Such structures are often in
dynamic equilibrium between different forms and regulate many processes in the cell.
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Neutron reflection could provide a unique view of how the individual molecular components
rearrange and contribute to these equilibria.
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