Phospholipid bilayers host and support the function of membrane proteins and may be stabilized in disk-like nano structures allowing for unprecedented solution studies of assembly, structure and function of membrane proteins. Based on small-angle neutron scattering in combination with variable temperature studies of synchrotron small-angle x-ray scattering on nanodiscs1 in solution we show that the fundamental nanodisc unit, consisting of a lipid bilayer surrounded by amphiphilic scaffold proteins, posses intrinsically an elliptical shape. The temperature dependence of the curvature of the nanodiscs prepared with two different phospholipid types (DLPC and POPC) shows that it is the scaffold protein that determines the overall elliptical shape and that the nanodiscs become more circular with increasing temperature. Our data also show that the hydrophobic bilayer thickness is to a large extent dictated by the scaffolding protein and adjusted to minimize the hydrophobic mismatch between protein and phospholipid. Our conclusions result from a new comprehensive and molecular based model of the nanodisc structure and the use of this to analyze the experimental scattering profile from nanodiscs. The model paves the way for future detailed structural studies of functional membrane proteins encapsulated in nanodiscs.

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It is well established that small sugars exert different types of stabilization of biomembranes both in vivo and in vitro. However, the essential question of whether sugars are bound to or expelled from membrane surfaces, i.e., the sign and size of the free energy of the interaction, remains unresolved, and this prevents a molecular understanding of the stabilizing mechanism. We have used smallangle neutron scattering and thermodynamic measurements to show that sugars may be either bound or expelled depending on the concentration of sugar. At low concentration, small sugars bind quite strongly to a lipid bilayer, and the accumulation of sugar at the interface makes the membrane thinner and laterally expanded. Above ∼0.2 M the sugars gradually become expelled from the membrane surface, and this repulsive mode of interaction counteracts membrane thinning. The dual nature of sugar-membrane interactions offers a reconciliation of conflicting views in earlier reports on sugar-induced modulations of membrane properties.membrane interface | membrane structure | preferential binding | preferential exclusion | interaction free energy S mall sugars such as the disaccharides sucrose and trehalose are among the so-called osmolytes (1) or compensatory solutes (2), which are accumulated in response to environmental stress in virtually all taxa. Their function is to act as inert regulators of the osmotic pressure, but they also optimize the physical properties of the cytosol (3) and stabilize biomolecular conformations against cold, drought, and heat (4-7). The same small carbohydrates have also proven useful in vitro as protectants or excipients for biopreservation (8). Many reports have shown that membranous structures are particularly stabilized by small sugars (4, 6, 9), but the definition of stabilization covers a wide range of biological and physical parameters. Thus, studies on intact cells have documented improved survival following exposure to heat, cold, drought, or chemical stressors (6,10,11). Other works have analyzed stabilization on the basis of phenomenological properties of model membranes, for example, the leakage or intermixing of probes in liposomes (12, 13). Finally, stability has been discussed with respect to rigorous physical parameters such as the structure or mechanical properties of lipid bilayers (14, 15). The current work addresses membrane dimensions and the thermodynamics of interaction with the purpose of elucidating fundamental aspects of membrane-sugar interrelationships. The different observations of sugar stabilization have sparked a large number of studies on sugars and model membranes (usually phospholipid bilayers) over the past 30 y. Investigations of fully hydrated membranes show an interesting tendency to fall into two groups with mutually conflicting conclusions. Thus, many investigations have suggested direct (favorable) interaction of sugars and the phospholipid interface (16-23), and it is obvious that such interactions could be the origin of sugar effects, for example, through int...

A fully open source software program for automated two‐dimensional and one‐dimensional data reduction and preliminary analysis of isotropic small‐angle X‐ray scattering (SAXS) data is presented. The program is freely distributed, following the open‐source philosophy, and does not rely on any commercial software packages. BioXTAS RAW is a fully automated program that, via an online feature, reads raw two‐dimensional SAXS detector output files and processes and plots data as the data files are created during measurement sessions. The software handles all steps in the data reduction. This includes mask creation, radial averaging, error bar calculation, artifact removal, normalization and q calibration. Further data reduction such as background subtraction and absolute intensity scaling is fast and easy via the graphical user interface. BioXTAS RAW also provides preliminary analysis of one‐dimensional data in terms of the indirect Fourier transform using the objective Bayesian approach to obtain the pair‐distance distribution function, PDDF, and is thereby a free and open‐source alternative to existing PDDF estimation software. Apart from the TIFF input format, the program also accepts ASCII‐format input files and is currently compatible with one‐dimensional data files from SAXS beamlines at a number of synchrotron facilities. BioXTAS RAW is written in Python with C++ extensions.

SDS wormlike micelles in water with NaBr are studied using small-angle neutron scattering. SDS concentrations ranging from 0.08 to 8.6 % vol in NaBr aqueous solutions at salinities from 0.6 to 1.0 M are covered. The scattering data are analyzed using a novel approach based on polymer theory and the results of Monte Carlo simulations. The method makes it possible to give a full interpretation of the scattering data, even for the entangled micellar solutions occurring at high concentrations and high salinities. Analysis of the scattering data at zero scattering angle demonstrates that the length of the micelles increases according to a power law as a function of concentration in the studied interval. The analysis furthermore shows that the length of the micelles increases exponentially with increasing salinity. The scattering data in the full range of scattering angles are analyzed using a model for polydisperse wormlike micelles where excluded volume effects are taken into account via an expression based on the polymer reference interaction site model (PRISM). This part of the analysis show that the micelles become more flexible as the salinity increases, which is due to an increased screening of the ionic micelles.

New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self-assemble in combination with phospholipids to form discoidal shaped particles that can stabilize membrane proteins. In the present study, we have investigated an ApoA1 mimetic peptide with respect to its solution structure when in complex with phospholipids. This was achieved using a powerful combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently into phospholipid nanodiscs.

Synaptic vesicles (SVs) are small, membrane-bound organelles that are found in the synaptic terminal of neurons, and which are crucial in neurotransmission. After a rise in internal [Ca(2+)] during neuronal stimulation, SVs fuse with the plasma membrane releasing their neurotransmitter content, which then signals neighboring neurons. SVs are subsequently recycled and refilled with neurotransmitter for further rounds of release. Recently, tremendous progress has been made in elucidating the molecular composition of SVs, as well as putative protein-protein interactions. However, what is lacking is an empirical description of SV structure at the supramolecular level-which is necessary to enable us to fully understand the processes of membrane fusion, retrieval, and recycling. Using small-angle x-ray scattering, we have directly investigated the size and structure of purified SVs. From this information, we deduced detailed size and density parameters for the protein layers responsible for SV function, as well as information about the lipid bilayer. To achieve a convincing model fit, a laterally anisotropic structure for the protein shell is needed, as a rotationally symmetric density profile does not explain the data. Not only does our model confirm many of the preexisting ideas concerning SV structure, but also for the first time, to our knowledge, it indicates structural refinements, such as the presence of protein microdomains.

Many proteins contain multiple folded domains separated by flexible linkers, and the ability to describe the structure and conformational heterogeneity of such flexible systems pushes the limits of structural biology. Using the three-domain protein TIA-1 as an example, we here combine coarse-grained molecular dynamics simulations with previously measured smallangle scattering data to study the conformation of TIA-1 in solution. We show that while the coarse-grained potential (Martini) in itself leads to too compact conformations, increasing the strength of protein-water interactions results in ensembles that are in very good agreement with experiments. We show how these ensembles can be refined further using a Bayesian/Maximum Entropy approach, and examine the robustness to errors in the energy function. In particular we find that as long as the initial simulation is relatively good, reweighting against experiments is very robust. We also study the relative information in X-ray and neutron scattering experiments and find that refining against the SAXS experiments leads to improvement in the SANS data. Our results suggest a general strategy for studying the conformation of multi-domain proteins in solution that combines coarse-grained simulations with small-angle X-ray scattering data that are generally most easy to obtain. These results may in turn be used to design further small-angle neutron scattering experiments that exploit contrast variation through 1 H/ 2 H isotope substitutions.