Clare L. Armstrong

The Observation of Highly Ordered Domains in Membranes with Cholesterol.

Rafts, or functional domains, are transient nano- or mesoscopic structures in the exoplasmic leaflet of the plasma membrane, and are thought to be essential for many cellular processes. Using neutron diffraction and computer modelling, we present evidence for the existence of highly ordered lipid domains in the cholesterol-rich (32.5 mol%) liquid-ordered () phase of dipalmitoylphosphatidylcholine membranes. The liquid ordered phase in one-component lipid membranes has previously been thought to be a homogeneous phase. The presence of highly ordered lipid domains embedded in a disordered lipid matrix implies non-uniform distribution of cholesterol between the two phases. The experimental results are in excellent agreement with recent computer simulations of DPPC/cholesterol complexes [Meinhardt, Vink and Schmid (2013). Proc Natl Acad Sci USA 110(12): 4476–4481], which reported the existence of nanometer size domains in a liquid disordered lipid environment.

Structure of Cholesterol in Lipid Rafts.

Rafts, or functional domains, are transient nano-or mesoscopic structures in the plasma membrane and are thought to be essential for many cellular processes such as signal transduction, adhesion, trafficking, and lipid or protein sorting. Observations of these membrane heterogeneities have proven challenging, as they are thought to be both small and short lived. With a combination of coarse-grained molecular dynamics simulations and neutron diffraction using deuterium labeled cholesterol molecules, we observe raftlike structures and determine the ordering of the cholesterol molecules in binary cholesterol-containing lipid membranes. From coarse-grained computer simulations, heterogenous membranes structures were observed and characterized as small, ordered domains. Neutron diffraction was used to study the lateral structure of the cholesterol molecules. We find pairs of strongly bound cholesterol molecules in the liquid-disordered phase, in accordance with the umbrella model. Bragg peaks corresponding to ordering of the cholesterol molecules in the raftlike structures were observed and indexed by two different structures: a monoclinic structure of ordered cholesterol pairs of alternating direction in equilibrium with cholesterol plaques, i.e., triclinic cholesterol bilayers.

Partitioning of ethanol into lipid membranes and its effect on fluidity and permeability as seen by X-ray and neutron scattering

We present a combined neutron and X-ray scattering investigation to study the effect of ethanol on the molecular structure and dynamics of lipid membranes. 1,2-Dimyristoyl-sn-glycero-3-phoshatidylcholine (DMPC) powder hydrated with a 5 wt% ethanol solution (corresponding to 2 mol% of ethanol) was used in this study. From high-resolution X-ray experiments the position and partitioning of the ethanol molecules in the phospholipid bilayers was determined in their gel and fluid phases. We find that the ethanol molecules reside in the head group region of the bilayers, with 1.6 ethanol molecules per lipid molecule in the gel phase and 1.2 ethanol molecules per lipid molecule in the fluid phase. We find evidence for enhanced permeability in both fluid and gel phases of the phospholipid bilayers in the presence of ethanol molecules. Elastic and quasi-elastic neutron scattering data, obtained using a neutron backscattering spectrometer, was used to study slow, nanosecond molecular dynamics on length scales corresponding to lipid diffusion, acyl chain dynamics and solvent dynamics. While the presence of ethanol molecules had no observable effect on these types of dynamics in the fluid (Lα) phase, the membranes appeared to have a higher degree of order in gel (Lβ) and ripple (Pβ′) phases. In particular, lipid diffusion was found to be slower by a factor of two in the more rigid gel phase when ethanol was present.

Co-existence of gel and fluid lipid domains in single-component phospholipid membranes

Lateral nanostructures in membranes, so-called rafts, are believed to strongly influence membrane properties and functions. The experimental observation of rafts has proven difficult as they are thought to be dynamic structures that likely fluctuate on nano- to microsecond time scales. Using neutron diffraction we present direct experimental evidence for the co-existence of gel and fluid lipid domains in a single-component phospholipid membrane made of DPPC as it undergoes its main phase transition. The coherence length of the neutron beam sets a lower limit for the size of structures that can be observed. Neutron coherence lengths between 30 and 242 A used in this study were obtained by varying the incident neutron energy and the resolution of the neutron spectrometer. We observe Bragg peaks corresponding to co-existing nanometer sized structures, both in out-of-plane and in-plane scans, by tuning the neutron coherence length. During the main phase transition, instead of a continuous transition that shows a pseudo-critical behavior, we observe the co-existence of gel and fluid domains.

The Production of Fibers and Films from Solubilized Hagfish Slime Thread Proteins

Hagfish slime threads, which make up the fibrous component of the defensive slime of hagfishes, consist primarily of proteins from the intermediate filament family of proteins and possess impressive mechanical properties that make them attractive biomimetic models. To investigate whether solubilized intermediate filament proteins can be used to make high-performance, environmentally sustainable materials, we cast thin films on the surface of electrolyte buffers using solubilized hagfish slime thread proteins. The films were drawn into fibers, and the tensile properties were measured. Fiber mechanics depended on casting conditions and postspinning processing. Postsecondary drawing resulted in fibers with improved material properties similar to those of regenerated silk fibers. Structural analyses of the fibers revealed increased molecular alignment resulting from the second draw, but no increase in crystallinity. Our findings show promise for intermediate filament proteins as an alternative source for the design and production of high performance protein-based fibers.

Diffusion in single supported lipid bilayers studied by quasi-elastic neutron scattering

It seems to be increasingly accepted that the diversity and composition of lipids play an important role in the function of biological membranes. A prime example of this is the case of lipid rafts; regions enriched with certain types of lipids which are speculated to be relevant to the proper functioning of membrane embedded proteins. Although the dynamics of membrane systems have been studied for decades, the microscopic dynamics of lipid molecules, even in simple model systems, is still an active topic of debate. Neutron scattering has proven to be an important tool for accessing the relevant nanometre length scale and nano to picosecond time scales, thus providing complimentary information to macroscopic techniques. Despite their potential relevance for the development of functionalized surfaces and biosensors, the study of single supported membranes using neutron scattering poses the challenge of obtaining relevant dynamic information from a sample with minimal material. Using state of the art neutron instrumentation we were, for the first time, able to model lipid diffusion in single supported lipid bilayers. We find that the diffusion coefficient for the single bilayer system is comparable to the multi-lamellar lipid system. More importantly, the molecular mechanism for lipid motion in the single bilayer was found to be a continuous diffusion, rather than the flow-like ballistic motion reported in the stacked membrane system. We observed an enhanced diffusion at the nearest neighbour distance of the lipid molecules. The enhancement and change of character of the diffusion can most likely be attributed to the effect the supporting substrate has on the lipid organization.

Short range ballistic motion in fluid lipid bilayers studied by quasi-elastic neutron scattering

Diffusion is the primary mechanism for movement of lipids and proteins in the lateral direction of a biological membrane. In this paper we have used quasi-elastic neutron scattering to examine the diffusion process of lipid molecules in fluid DMPC membranes. We found that the motion over length scales greater than the lipid diameter could be characterized as a continuous diffusion process, with a diffusion coefficient of D = 64 × 10−12 m2/s. The continuous diffusion model has been successfully used in the past to describe the motion of lipid over long length scales. However, the focus of this measurement was to determine how the character of the molecular motion changes on length scales shorter than the nearest neighbour distance. At very short length scales (<2.37 A), we see first experimental evidence for a short-range flow-like ballistic motion.

Protein-protein interactions in membranes.

In this article we review the current status of our understanding of membrane mediated interactions from theory and experiment. Phenomenological mean field and molecular models will be discussed and compared to recent experimental results from dynamical neutron scattering and atomic force microscopy.

Incoherent Neutron Spin-Echo Spectroscopy as an Option to Study Long-Range Lipid Diffusion

Diffusion is the fundamental mechanism for lipids and other molecules to move in a membrane. It is an important process to consider in modelling the formation of membrane structures, such as rafts. Lipid diffusion is mainly studied by two different techniques: incoherent neutron scattering and fluorescence microscopy. Both techniques access distinctly different length scales. While neutron scattering measures diffusion over about 3 lipid diameters, microscopic techniques access motions of lipids over micrometer distances. The diffusion constants which are determined by these two methods often differ by about an order of magnitude, with the neutrons usually seeing a faster lipid diffusion. Different theories are used to describe lipid diffusion in the two experiments. In order to close the “gap” between these two techniques, we propose to study lipid diffusion at mesoscopic length scales using a neutron spin-echo (NSE) spectrometer. We have conducted an experiment in highly oriented, solid supported lipid bilayers to prove the feasibility of performing incoherent NSE on biological samples. Lateral lipid diffusion was measured in a fluid phase model membrane system at a length scale of 12 A. Using the high-energy resolution of the NSE technique, we find evidence for two dynamic processes.

Hydration Water Freezing in Single Supported Lipid Bilayers

We present a high-temperature and high-energy resolution neutron scattering investigation of hydration water freezing in single supported lipid bilayers. Single supported lipid bilayers provide a well-defined biological interface to study hydration water dynamics and coupling to membrane degrees of freedom. Nanosecond molecular motions of membrane and hydration water were studied in the temperature range 240 K < T < 290 K in slow heating and cooling cycles using coherent and incoherent elastic neutron scattering on a backscattering spectrometer. Several freezing and melting transitions were observed. From the length scale dependence of the elastic scattering, these transitions could be assigned to freezing and melting of hydration water dynamics, diffusive lipid, and lipid acyl-tail dynamics. Coupling was investigated by comparing the different freezing and melting temperatures. While it is often speculated that membrane and hydration water dynamics are strongly coupled, we find that membrane and hydration water dynamics are at least partially decoupled in single bilayers.