The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase.

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering.

Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering.

The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid ß, interact with lipids, and this interaction can strongly favour the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to model membranes formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the model membranes take an active role in the reaction. The binding of α synuclein to the model membranes immediately induces a major structural change in the lipid assembly, which leads to a break-up into small and mostly disc- or rod-like lipid-protein particles. This transition can be reversed by temperature changes or proteolytic protein removal. Incubation of the small lipid-α-synuclein particles for several hours, however, leads to amyloid fibril formation, whereby the lipids are incorporated into the amyloid fibrils.

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering.

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular-dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

Semi-empirical Analysis of Complex ITC Data from Protein-Surfactant Interactions.

Isothermal titration calorimetry (ITC) is a widely used method to determine binding affinities and thermodynamics in ligand-receptor interactions, but it also has the capability of providing detailed information on much more complex events. However, the lack of available methods to analyze ITC data is limiting the use of the technique in such multifaceted cases. Here, we present the software ANISPROU. Through a semi-empirical approach that allows for extraction of quantitative information from complex ITC data, ANISPROU solves an inverse problem where three parameters describing a set of predefined functions must be found. In analogy to strategies adopted in other scientific fields, such as geophysics, imaging, and many others, it employs an optimization algorithm which minimizes the difference between calculated and experimental data. In contrast to the existing methods, ANISPROU provides automated and objective analysis of ITC data on sodium dodecyl sulfate (SDS)-induced protein unfolding, and in addition, more information can be extracted from the data. Here, data series on SDS-mediated protein unfolding is analyzed, and binding isotherms and thermodynamic information on the unfolding events are extracted. The obtained binding isotherms as well as the enthalpy of different events are similar to those obtained using the existing manual methods, but our methodology ensures a more robust result, as the entire data set is used instead of single data points. We foresee that ANISPROU will be useful in other cases with complex enthalpograms, for example, in cases with coupled interactions in biomolecular, polymeric, and amphiphilic systems including cases where both structural changes and interactions occur simultaneously.

Oligomerization of Pharmaceutically Relevant Insulin Analogues for Varying Concentration and Salinity Revealed by Small-Angle X-ray Scattering.

Two different insulin analogues, insulin degludec and lithocholyl insulin, were studied by small-angle X-ray scattering with respect to their self-assembly and interactions in solution at different concentrations of insulin and salt, NaCl. Very different behavior was observed for the two. Insulin degludec, linked to a hexadecanedioic acid, consistently formed di-hexamers, without any further oligomeric growth upon screening of electrostatic repulsions, indicating a stable di-hexamer unit without further oligomerization, as expected in the presence of phenol. The other insulin analogue, linked to the sterol lithocholic acid, formed n-hexamers with n ranging from 1 to 15, increasing with NaCl concentration and insulin concentration, indicating attractive forces in competition with the electrostatic repulsion and solution entropy. At the highest concentration of insulin and NaCl, a liquid crystal phase was observed, which has not previously been identified, featuring a quadratic structure organized into layers, which might hold interesting properties for pharmaceutical applications.

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

Combining molecular dynamics simulations with small-angle X-ray and neutron scattering data to study multi-domain proteins in solution.

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 small-angle 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 1H/2H isotope substitutions.

Aescin - a natural soap for the formation of lipid nanodiscs with tunable size.

The saponin ß-aescin from the seed extract of the horse chestnut tree Aesculus hippocastanum has demonstrated a beneficial role in clinical therapy which is in part related to its strong interaction with biological membranes. In this context the present work investigates the self-assembly of nm-sized discoidal lipid nanoparticles composed of ß-aescin and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The discoidal lipid nanoparticles reassemble from small discs into larger discs, ribbons and finally stacks of sheets upon heating from gel-phase to fluid phase DMPC. The morphological transition of the lipid nano-particles is mainly triggered by the phospholipid phase state change. The final morphology depends on the phospholipid-to-saponin ratio and the actual temperature. The study is conducted by small-angle X-ray scattering (SAXS) and transmission (TEM) and freeze fracture electron microscopy (FFEM) are used to cover larger length scales. Two different models, representing a disc and ribbon-like shape are applied to the SAXS data, evaluating possible geometries and molecular mixing of the nano-particles. The stacked sheets are analysed by the Caillé theory.

Peptide Disc Mediated Control of Membrane Protein Orientation in Supported Lipid Bilayers for Surface-Sensitive Investigations.

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with ∼0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.

Orchestration of signaling by structural disorder in class 1 cytokine receptors.

BACKGROUND: Class 1 cytokine receptors (C1CRs) are single-pass transmembrane proteins responsible for transmitting signals between the outside and the inside of cells. Remarkably, they orchestrate key biological processes such as proliferation, differentiation, immunity and growth through long disordered intracellular domains (ICDs), but without having intrinsic kinase activity. Despite these key roles, their characteristics remain rudimentarily understood. METHODS: The current paper asks the question of why disorder has evolved to govern signaling of C1CRs by reviewing the literature in combination with new sequence and biophysical analyses of chain properties across the family. RESULTS: We uncover that the C1CR-ICDs are fully disordered and brimming with SLiMs. Many of these short linear motifs (SLiMs) are overlapping, jointly signifying a complex regulation of interactions, including network rewiring by isoforms. The C1CR-ICDs have unique properties that distinguish them from most IDPs and we forward the perception that the C1CR-ICDs are far from simple strings with constitutively bound kinases. Rather, they carry both organizational and operational features left uncovered within their disorder, including mechanisms and complexities of regulatory functions. CONCLUSIONS: Critically, the understanding of the fascinating ability of these long, completely disordered chains to orchestrate complex cellular signaling pathways is still in its infancy, and we urge a perceptional shift away from the current simplistic view towards uncovering their full functionalities and potential. Video abstract.

Structural Insight into the Self-Assembly of a Pharmaceutically Optimized Insulin Analogue Obtained by Small-Angle X-ray Scattering.

B29Nε-lithocholyl-γ-l-ßGlu-desB30 human insulin [NN344] belongs to a group of insulins with fatty acid or sterol modifications. These insulin analogues have been found to form subcutaneous depots upon injection and hereby have a protracted release profile in vivo. In the present study, B29Nε-lithocholyl-γ-l-Glu-desB30 human insulin was investigated using in-solution small-angle X-ray scattering (SAXS) at chemical conditions designed to mimic three stages during treatment in vivo: in-vial/pen, postinjection, and longer times after injection. We found that the specific insulin analogue formed a mixture of mono- and dihexamers under in-vial/pen conditions of low salt and stabilizing phenol. At postinjection, conditions mimicking a subcutaneous depot, B29Nε-lithocholyl-γ-l-Glu-desB30 human insulin, formed very long straight soluble hexamer-based rods stacked along the Zn(II)-axis. The self-assembly was triggered by an increase in salt concentration when going from vial to physiological conditions. Mimicking longer times after injection and the additional removal of phenol caused the length of the rods to decrease significantly. Finally, we found that the self-assembly could be controlled by varying the amount of modification at the interaction interface by making mixed hexamers of B29Nε-lithocholyl-γ-l-Glu-desB30 and desB30 human insulin. This opens extra possibilities for controlling the release profile of very-long-acting insulins.

Structure and Dynamics of the Central Lipid Pool and Proteins of the Bacterial Holo-Translocon.

The bacterial Sec translocon, SecYEG, associates with accessory proteins YidC and the SecDF-YajC subcomplex to form the bacterial holo-translocon (HTL). The HTL is a dynamic and flexible protein transport machine capable of coordinating protein secretion across the membrane and efficient lateral insertion of nascent membrane proteins. It has been hypothesized that a central lipid core facilitates the controlled passage of membrane proteins into the bilayer, ensuring the efficient formation of their native state. By performing small-angle neutron scattering on protein solubilized in "match-out" deuterated detergent, we have been able to interrogate a "naked" HTL complex, with the scattering contribution of the surrounding detergent micelle rendered invisible. Such an approach has allowed the confirmation of a lipid core within the HTL, which accommodates between 8 and 29 lipids. Coarse-grained molecular dynamics simulations of the HTL also demonstrate a dynamic, central pool of lipids. An opening at this lipid-rich region between YidC and the SecY lateral gate may provide an exit gateway for newly synthesized, correctly oriented, membrane protein helices, or even small bundles of helices, to emerge from the HTL.

Distinct α-Synuclein:Lipid Co-Structure Complexes Affect Amyloid Nucleation through Fibril Mimetic Behavior.

A hallmark of Parkinson's disease is the presence of Lewy bodies consisting of lipids and proteins, mainly fibrillated α-synuclein (aSN). aSN is an intrinsically disordered protein exerting its physiological role in an ensemble of states, one of which coexists in large assemblies with lipids, recently termed co-structures. Here, we decipher the kinetics of aSN:lipid co-structure formation to decode its mechanism of formation, and we show that the co-structures form with a distinct stoichiometry. Through seeded fibrillation assays, we demonstrate that aSN:lipid co-structures accelerate aSN fibril nucleation compared to lipid vesicles alone. A small-angle X-ray scattering-based model is proposed in which aSN decorates the lipid vesicle surface, yielding properties similar to those of the fibril surface, enhancing fibril nucleation. The delicate balance of aSN structural states close to and on the membrane may under given conditions, e.g., increased local concentrations, be a crucial switching factor between functional and pathological behavior.

Aescin-Induced Conversion of Gel-Phase Lipid Membranes into Bicelle-like Lipid Nanoparticles.

Mixtures of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the saponin ß-aescin spontaneously form monodisperse, bilayered discoidal micelles (also known as "bicelles" or "nanodisks") in aqueous solution. Such bicelles form below the melting temperature of DMPC when the phospholipids are in the rigid Lß' state and are precursors of spontaneously formed vesicles. The aescin concentration must be far above the cmcaescin (≈0.3-0.4 mM). It was found that the shape and size of the bicelles are tunable by composition. High amounts of aescin decrease the size of the bicelles from diameters of ∼300 Å at 7 mol % to ∼120 Å at 30 mol % ß-aescin. The structures are scrutinized by complementary small-angle X-ray and neutron scattering experiments. The scattering curves are subsequently analyzed by a model-independent (indirect Fourier transform analysis) and a model-based approach where bicelles are described as polydisperse bilayer disks encircled by a ß-aescin rim. Moreover, the monomodal distribution and low polydispersity of the samples were confirmed by photon correlation spectroscopy. The discoidal structures were visualized by transmission electron microscopy.

The influence of pH, protein concentration and calcium ratio on the formation and structure of nanotubes from partially hydrolyzed bovine α-lactalbumin.

Formation of nanotubes from partially hydrolysed α-lactalbumin (α-La) was investigated at five pH values, two concentrations of α-La and two calcium levels. Nanotubes were formed under almost all combinations of the investigated factors, and for the first time the formation of nanotubes at low pH (4.0) and low protein concentration (10 g l-1) was observed. Only one sample (10 g l-1, calcium ratio 2.4, and pH 7.5) formed mainly fibrils instead of nanotubes. By altering the three investigated factors, fibrils and/or aggregates were sometimes formed together with nanotubes resulting in transparent, semi-transparent, or non-transparent gels, or sediments. However, structural modelling based on small-angle X-ray scattering data indicated that the formed nanotubes were only to a minor degree affected by the investigated factors. The majority of the nanotubes were found to have an outer diameter of around 19 nm, an inner diameter of 6.6 nm and a wall thickness of 6.0 nm, except for three samples at low α-La concentrations and high calcium levels which exhibited slightly smaller dimensions. These three factors affected the hydrolysis as well as the self-assembly rate, resulting in the observed differences. However, these factors did not influence the architecture of the self-assembled nanotubes, and the lateral spacing of the individual parallel ß-sheet motifs was found to be 1.05 ± 0. 03 nm for all nanotubes. This study provides novel fundamental knowledge of the formation and structure of α-La nanotubes under different conditions, which will facilitate future application of these nanotubes in food and pharmaceutical areas.

Comprehensive Study of the Self-Assembly of Phospholipid Nanodiscs: What Determines Their Shape and Stoichiometry?

Phospholipid nanodiscs have quickly become a widely used platform for studies of membrane proteins. However, the molecular self-assembly process that ultimately should place a membrane protein inside a nanodisc is not well understood. This poses a challenge for a successful high-yield reconstitution of general membrane proteins into nanodiscs. In the present work, the self-assembly process of POPC-MSP1D1 nanodiscs was carefully investigated by systematically modulating the reconstitution parameters and probing the effect with a small-angle X-ray scattering analysis of the resulting nanodiscs. First, it was established that nanodiscs prepared using the standard protocol followed a narrow but significant size distribution and that the formed nanodiscs were stable at room temperature over a time range of about a week. Systematic variation of the POPC/MSP1D1 stoichiometry of the reconstitution mixture showed that a ratio of less than 75:1 resulted in lipid-poor nanodiscs, whereas ratios of 75:1 and larger resulted in nanodiscs with constant POPC/MSP1D1 ratios of 60:1. A central step in the self-assembly process consists in adding detergent-absorbing resin beads to the reconstitution mixture to remove the reconstitution detergent. Surprisingly, it was found that this step did not play a significant role for the shape and stoichiometry of the formed nanodiscs. Finally, the effect of the choice of detergent used in the reconstitution process was investigated. It was found that detergent type is a central determining factor for the shape and stoichiometry of the formed nanodiscs. A significantly increasing POPC/MSP1D1 stoichiometry of the formed nanodiscs was observed as the reconstitution detergent type is changed in the order: Tween80, DDM, Triton X-100, OG, CHAPS, Tween20, and Cholate, but with no simple correlation to the characteristics of the detergent. This emphasizes that the detergents optimal for solution storage and crystallization of membrane proteins, in particular DDM, should not be used alone for nanodisc reconstitution. However, our data also show that when applying mixtures of the reconstitution detergent cholate and the storage detergents DDM or OG, cholate dominates the reconstitution process and nanodiscs are obtained, which resemble those formed without storage detergents.

Dimeric peptides with three different linkers self-assemble with phospholipids to form peptide nanodiscs that stabilize membrane proteins.

Three dimers of the amphipathic α-helical peptide 18A have been synthesized with different interhelical linkers inserted between the two copies of 18A. The dimeric peptides were denoted 'beltides' where Beltide-1 refers to the 18A-dimer without a linker, Beltide-2 is the 18A-dimer with proline (Pro) as a linker and Beltide-3 is the 18A-dimer linked by two glycines (Gly-Gly). The self-assembly of the beltides with the phospholipid DMPC was studied with and without the incorporated membrane protein bacteriorhodopsin (bR) through a combination of coarse-grained MD simulations, size-exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, small-angle scattering (SAS), static light scattering (SLS) and UV-Vis spectroscopy. For all three beltides, MD and combined small-angle X-ray and -neutron scattering were consistent with a disc structure composed by a phospholipid bilayer surrounded by a belt of peptides and with a total disc diameter of approximately 10 nm. CD confirmed that all three beltides were α-helical in the free form and with DMPC. However, as shown by SEC the different interhelical linkers clearly led to different properties of the beltides. Beltide-3, with the Gly-Gly linker, was very adaptable such that peptide nanodiscs could be formed for a broad range of different peptide to lipid stoichiometries and therefore also possible disc-sizes. On the other hand, both Beltide-2 with the Pro linker and Beltide-1 without a linker were less adaptable and would only form discs of certain peptide to lipid stoichiometries. SLS revealed that the structural stability of the formed peptide nanodiscs was also highly affected by the linkers and it was found that Beltide-1 gave more stable discs than the other two beltides. With respect to membrane protein stabilization, each of the three beltides in combination with DMPC stabilizes the seven-helix transmembrane protein bacteriorhodopsin significantly better than the detergent octyl glucoside, but no significant difference was observed between the three beltides. We conclude that adaptability, size, and structural stability can be tuned by changing the interhelical linker while maintaining the properties of the discs with respect to membrane protein stabilization.

Construction of Insulin 18-mer Nanoassemblies Driven by Coordination to Iron(II) and Zinc(II) Ions at Distinct Sites.

Controlled self-assembly (SA) of proteins offers the possibility to tune their properties or to create new materials. Herein, we present the synthesis of a modified human insulin (HI) with two distinct metal-ion binding sites, one native, the other abiotic, enabling hierarchical SA through coordination with two different metal ions. Selective attachment of an abiotic 2,2'-bipyridine (bipy) ligand to HI, yielding HI-bipy, enabled Zn(II)-binding hexamers to SA into trimers of hexamers, [[HI-bipy]6]3, driven by octahedral coordination to a Fe(II)  ion. The structures were studied in solution by small-angle X-ray scattering and on surfaces with AFM. The abiotic metal ligand had a higher affinity for Fe(II) than Zn(II)  ions, enabling control of the hexamer formation with Zn(II) and the formation of trimers of hexamers with Fe(II)  ions. This precise control of protein SA to give oligomers of oligomers provides nanoscale structures with potential applications in nanomedicine.

Small-angle X-ray scattering of the cholesterol incorporation into human ApoA1-POPC discoidal particles.

Structural and functional aspects of high-density lipoproteins have been studied for over half a century. Due to the plasticity of this highly complex system, new aspects continue to be discovered. Here, we present a structural study of the human Apolipoprotein A1 (ApoA1) and investigate the role of its N-terminal domain, the so-called globular domain of ApoA1, in discoidal complexes with phospholipids and increasing amounts of cholesterol. Using a combination of solution-based small-angle x-ray scattering (SAXS) and molecular constrained data modeling, we show that the ApoA1-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-based particles are disk shaped with an elliptical cross section and composed by a central lipid bilayer surrounded by two stabilizing ApoA1 proteins. This structure is very similar to the particles formed in the so-called nanodisc system, which is based on N-terminal truncated ApoA1 protein. Although it is commonly agreed that the nanodisc is plain disk shaped, several more advanced structures have been proposed for the full-length ApoA1 in combination with POPC and cholesterol. This prompted us to make a detailed comparative study of the ApoA1 and nanodisc systems upon cholesterol uptake. Based on the presented SAXS analysis it is found that the N-terminal domains of ApoA1-POPC-cholesterol particles are not globular but instead an integrated part of the protein belt stabilizing the particles. Upon incorporation of increasing amounts of cholesterol, the presence of the N-terminal domain allows the bilayer thickness to increase while maintaining an overall flat bilayer structure. This is contrasted by the energetically more strained and less favorable lens shape required to fit the SAXS data from the N-terminal truncated nanodisc system upon cholesterol incorporation. This suggests that the N-terminal domain of ApoA1 actively participates in the stabilization of the ApoA1-POPC-cholesterol discoidal particle and allows for a more optimal lipid packing upon cholesterol uptake.

Small-angle scattering determination of the shape and localization of human cytochrome P450 embedded in a phospholipid nanodisc environment.

Membrane proteins reconstituted into phospholipid nanodiscs comprise a soluble entity accessible to solution small-angle X-ray scattering (SAXS) studies. It is demonstrated that using SAXS data it is possible to determine both the shape and localization of the membrane protein cytochrome P450 3A4 (CYP3A4) while it is embedded in the phospholipid bilayer of a nanodisc. In order to accomplish this, a hybrid approach to analysis of small-angle scattering data was developed which combines an analytical approach to describe the multi-contrast nanodisc with a free-form bead-model description of the embedded protein. The protein shape is then reconstructed ab initio to optimally fit the data. The result of using this approach is compared with the result obtained using a rigid-body description of the CYP3A4-in-nanodisc system. Here, the CYP3A4 structure relies on detailed information from crystallographic and molecular-dynamics studies of CYP3A4. Both modelling approaches arrive at very similar solutions in which the α-helical anchor of the CYP3A4 systematically stays close to the edge of the nanodisc and with the large catalytic domain leaning over the outer edge of the nanodisc. The obtained distance between the globular domains of CYP3A4 is consistent with previously published theoretical calculations.