Small angle scattering reveals the orientation of cytochrome P450 19A1 in lipoprotein nanodiscs.

Human aromatase (CYP19A1), the cytochrome P450 enzyme responsible for conversion of androgens to estrogens, was incorporated into lipoprotein nanodiscs (NDs) and interrogated by small angle X-ray and neutron scattering (SAXS/SANS). CYP19A1 was associated with the surface and centered at the edge of the long axis of the ND membrane. In the absence of the N-terminal anchor, the amphipathic A'- and G'-helices were predominately buried in the lipid head groups, with the possibly that their hydrophobic side chains protrude into the hydrophobic, aliphatic tails. The prediction is like that for CYP3A4 based on SAXS employing a similar modeling approach. The orientation of CYP19A1 in a ND is consistent with our previous predictions based on molecular dynamics simulations and lends additional credibility to the notion that CYP19A1 captures substrates from the membrane.

The Scottish Medical Imaging Archive: 57.3 Million Radiology Studies Linked to Their Medical Records.

Keywords: MRI, Imaging Sequences, Ultrasound, Mammography, CT, Angiography, Conventional Radiography Published under a CC BY 4.0 license. See also the commentary by Whitman and Vining in this issue.

Morphological Characterization of Self-Amplifying mRNA Lipid Nanoparticles.

The mRNA technology has emerged as a rapid modality to develop vaccines during pandemic situations with the potential to protect against endemic diseases. The success of mRNA in producing an antigen is dependent on the ability to deliver mRNA to the cells using a vehicle, which typically consists of a lipid nanoparticle (LNP). Self-amplifying mRNA (SAM) is a synthetic mRNA platform that, besides encoding for the antigen of interest, includes the replication machinery for mRNA amplification in the cells. Thus, SAM can generate many antigen encoding mRNA copies and prolong expression of the antigen with lower doses than those required for conventional mRNA. This work describes the morphology of LNPs containing encapsulated SAM (SAM LNPs), with SAM being three to four times larger than conventional mRNA. We show evidence that SAM changes its conformational structure when encapsulated in LNPs, becoming more compact than the free SAM form. A characteristic "bleb" structure is observed in SAM LNPs, which consists of a lipid-rich core and an aqueous RNA-rich core, both surrounded by a DSPC-rich lipid shell. We used SANS and SAXS data to confirm that the prevalent morphology of the LNP consists of two-core compartments where components are heterogeneously distributed between the two cores and the shell. A capped cylinder core-shell model with two interior compartments was built to capture the overall morphology of the LNP. These findings provide evidence that bleb two-compartment structures can be a representative morphology in SAM LNPs and highlight the need for additional studies that elucidate the role of spherical and bleb morphologies, their mechanisms of formation, and the parameters that lead to a particular morphology for a rational design of LNPs for mRNA delivery.

Temperature-induced DNA density transition in phage λ capsid revealed with contrast-matching SANS.

Structural details of a genome packaged in a viral capsid are essential for understanding how the structural arrangement of a viral genome in a capsid controls its release dynamics during infection, which critically affects viral replication. We previously found a temperature-induced, solid-like to fluid-like mechanical transition of packaged λ-genome that leads to rapid DNA ejection. However, an understanding of the structural origin of this transition was lacking. Here, we use small-angle neutron scattering (SANS) to reveal the scattering form factor of dsDNA packaged in phage λ capsid by contrast matching the scattering signal from the viral capsid with deuterated buffer. We used small-angle X-ray scattering and cryoelectron microscopy reconstructions to determine the initial structural input parameters for intracapsid DNA, which allows accurate modeling of our SANS data. As result, we show a temperature-dependent density transition of intracapsid DNA occurring between two coexisting phases-a hexagonally ordered high-density DNA phase in the capsid periphery and a low-density, less-ordered DNA phase in the core. As the temperature is increased from 20 °C to 40 °C, we found that the core-DNA phase undergoes a density and volume transition close to the physiological temperature of infection (~37 °C). The transition yields a lower energy state of DNA in the capsid core due to lower density and reduced packing defects. This increases DNA mobility, which is required to initiate rapid genome ejection from the virus capsid into a host cell, causing infection. These data reconcile our earlier findings of mechanical DNA transition in phage.

Synthesis of Polymer Nanoweb via a Lipid Template.

We report a generalized platform for synthesizing a polymer nanoweb with a high specific surface area via a bicellar template, composed of 1,2-dipalmitoyl phosphocholine (DPPC), 1,2-dihexanoyl phosphocholine (DHPC), and 1,2-dipalmitoyl phosphoglycerol (DPPG). The pristine bicelle (in the absence of monomer or polymer) yields a variety of well-defined structures, including disc, vesicle, and perforated lamella. The addition of styrene monomers in the mixture causes bicelles to transform into lamellae. Monomers are miscible with DPPC and DPPG initially, while polymerization drives polymers to the DHPC-rich domain, resulting in a polymer nanoweb supported by the outcomes of small angle neutron scattering, differential scanning calorimetry, and transmission electron microscopy.

Enabling Efficient Design of Biological Formulations Through Advanced Characterization.

The present review summarizes the use of differential scanning calorimetry (DSC) and scattering techniques in the context of protein formulation design and characterization. The scattering techniques include wide angle X-ray diffractometry (XRD), small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). While DSC is valuable for understanding thermal behavior of the excipients, XRD provides critical information about physical state of solutes during freezing, annealing and in the final lyophile. However, as these techniques lack the sensitivity to detect biomolecule-related transitions, complementary characterization techniques such as small-angle scattering can provide valuable insights.

Protein-Surfactant and Protein-Protein Interactions During Freeze and Thaw: A Small-Angle Neutron Scattering Study of Lysozyme Solutions with Polysorbate and Poloxamer.

Protein structural changes during freezing and subsequent thawing are of great importance to a variety of biopharmaceutical applications. In this work, we studied the influence of non-ionic surfactants (polysorbate 20 and poloxamer 188) on protein structural changes during freeze and thaw using lysozyme as a model protein. Small-angle neutron scattering was employed to characterize protein structures in both liquid and frozen solution states. The results show minimal impact of polysorbate 20 on lysozyme structures during freeze and thaw using practically relevant concentrations. Polysorbate 20 used at 0.04% (w/w) completely prevents freeze-induced aggregation of lysozyme. Poloxamer 188 seems to interact with lysozyme; when applied at high concentrations (10% w/w), such interaction prevents protein crowding or close packing typically associated with freeze concentration. Despite such interactions, lysozyme aggregation is observed with 10% (w/w) of poloxamer 188 during freezing, although the aggregation is reversed upon thawing.

Machine learning models in trusted research environments - understanding operational risks.

Introduction: Trusted research environments (TREs) provide secure access to very sensitive data for research. All TREs operate manual checks on outputs to ensure there is no residual disclosure risk. Machine learning (ML) models require very large amount of data; if this data is personal, the TRE is a well-established data management solution. However, ML models present novel disclosure risks, in both type and scale. Objectives: As part of a series on ML disclosure risk in TREs, this article is intended to introduce TRE managers to the conceptual problems and work being done to address them. Methods: We demonstrate how ML models present a qualitatively different type of disclosure risk, compared to traditional statistical outputs. These arise from both the nature and the scale of ML modelling. Results: We show that there are a large number of unresolved issues, although there is progress in many areas. We show where areas of uncertainty remain, as well as remedial responses available to TREs. Conclusions: At this stage, disclosure checking of ML models is very much a specialist activity. However, TRE managers need a basic awareness of the potential risk in ML models to enable them to make sensible decisions on using TREs for ML model development.

A round-robin approach provides a detailed assessment of biomolecular small-angle scattering data reproducibility and yields consensus curves for benchmarking.

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å-1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å-1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å-1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.

Planning, executing and assessing the validity of SANS contrast variation experiments.

Small angle neutron scattering (SANS) along with contrast variation (CV) can provide key information that is used to determine the shape and structure of biological complexes in solution. The successful SANS CV experiment is usually a result of judicious planning, careful execution and meticulous scrutiny of the resultant SANS data. A workflow for planning, executing and, importantly, assessing the validity of SANS CV data is presented here, along with tips to follow in order to perform a successful SANS CV experiment. Some knowledge of the basics of small angle scattering is assumed, including data reduction and standard analysis to obtain model independent parameters such as the radius of gyration and forward scattering intensity, and SANS CV theory is not covered in detail. Rather, the focus is on the SANS CV workflow from in silico experiment planning and execution to obtaining the SANS CV data, assessing its validity, and determining a model structure or ensemble of structures using the SANS CV data as constraints.

Small-angle neutron scattering contrast variation studies of biological complexes: Challenges and triumphs.

Small-angle neutron scattering (SANS) has been a beneficial tool for studying the structure of biological macromolecules in solution for several decades. Continued improvements in sample preparation techniques, including deuterium labeling, neutron instrumentation and complementary techniques such as small-angle x-ray scattering (SAXS), cryo-EM, NMR and x-ray crystallography, along with the availability of more powerful structure prediction algorithms and computational resources has made SANS more important than ever as a means to obtain unique information on the structure of biological complexes in solution. In particular, the contrast variation (CV) technique, which requires a large commitment in both sample preparation and measurement time, has become more practical with the advent of these improved resources. Here, challenges and recent triumphs as well as future prospects are discussed.

Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations.

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (CNaCl) and monoclonal antibody (mAb) concentration (CmAb) were assessed using the diffusion interaction parameter kD and second virial coefficient B22 measured from solutions with low to moderate CmAb. The effective structure factor S(q)eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with CNaCl, but also with increasing CmAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including kD and S(q)eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low CmAb.

Structures and Dynamics of Anionic Lipoprotein Nanodiscs.

Nanolipoprotein particles known as nanodiscs (NDs) have emerged as versatile and powerful tools for the stabilization of membrane proteins permitting a plethora of structural and biophysical studies. Part of their allure is their flexibility to accommodate many types of lipids and precise control of the composition. However, little is known about how variations in lipid composition impact their structures and dynamics. Herein, we investigate how the introduction of the anionic lipid POPG into POPC NDs impacts these features. Small-angle X-ray and neutron scattering (SAXS and SANS) of variable-composition NDs are complemented with molecular dynamics simulations to interrogate how increasing the concern of POPG impacts the ND shape, structure of the lipid core, and the dynamics of the popular membrane scaffold protein, MSP1D1(-). A convenient benefit of including POPG is that it eliminates D2O-induced aggregation observed in pure POPC NDs, permitting studies by SANS at multiple contrasts. SAXS and SANS data could be globally fit to a stacked elliptical cylinder model as well as an extension of the model that accounts for membrane curvature. Fitting to both models supports that the introduction of POPG results in strongly elliptical NDs; however, MD simulations predict the curvature of the membrane, thereby supporting the use of the latter model. Trends in the model-independent parameters suggest that increases in POPG reduce the conformational heterogeneity of the MSP1D1(-), which is in agreement with MD simulations that show that the incorporation of sufficient POPG suppresses disengagement of the N-terminal helix from the lipid core. These studies highlight novel structural changes in NDs in response to an anionic lipid and will inform the interpretation of future structural studies of membrane proteins embedded in NDs of mixed lipid composition.

Head on Comparison of Self- and Nano-assemblies of Gamma Peptide Nucleic Acid Amphiphiles.

Peptide nucleic acids (PNAs) are nucleic acid analogs with superior hybridization properties and enzymatic stability than deoxyribonucleic acid (DNA). In addition to gene targeting applications, PNAs have garnered significant attention as bio-polymer due to the Watson-Crick -based molecular recognition and flexibility of synthesis. Here, we engineered PNA amphiphiles using chemically modified gamma PNA (8 mer in length) containing hydrophilic diethylene glycol units at the gamma position and covalently conjugated lauric acid (C12) as a hydrophobic moiety. Gamma PNA (γPNA) amphiphiles self-assemble into spherical vesicles. Further, we formulate nano-assemblies using the amphiphilic γPNA as a polymer via ethanol injection-based protocols. We perform comprehensive head-on comparison of the physicochemical and cellular uptake properties of PNA derived self- and nano-assemblies. Small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) analysis reveal ellipsoidal morphology of γPNA nano-assemblies that results in superior cellular delivery compate to the spherical self-assembly. Next, we compare the functional activities of γPNA self-and nano-assemblies in lymphoma cells via multiple endpoints, including gene expression, cell viability, and apoptosis-based assays. Overall, we establish that γPNA amphiphile is a functionally active bio-polymer to formulate nano-assemblies for a wide range of biomedical applications.

Styrene-Maleic Acid Copolymer Nanodiscs to Determine the Shape of Membrane Proteins.

Lipid nanodiscs can be used to solubilize functional membrane proteins (MPs) in nativelike environments. Thus, they are promising reagents that have been proven useful to characterize MPs. Both protein and non-protein molecular belts have shown promise to maintain the structural integrity of MPs in lipid nanodiscs. Small-angle neutron scattering (SANS) can be used to determine low-resolution structures of proteins in solution, which can be enhanced through the use of contrast variation methods. We present theoretical contrast variation SANS results for protein and styrene-maleic acid copolymer (SMA) belt 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) nanodiscs with and without additional bound or transmembrane proteins. The predicted scattering properties are derived from atomistic molecular dynamics simulations to account for conformational fluctuations, and we determine deuterium-labeling conditions such that SANS intensity profiles only include contributions from the scattering of the MP of interest. We propose strategies to tune the neutron scattering length densities (SLDs) of the SMA and DMPC using selective deuterium labeling such that the SLD of the nanodisc becomes homogeneous and its scattering can essentially be eliminated in solvents containing an appropriate amount of D2O. These finely tuned labeled polymer-based nanodiscs are expected to be useful to extract the size and molecular shape information of MPs using SANS-based contrast variation experiments, and they can be used with MPs of any molecular weight.

Reversible Self-Association in Lactate Dehydrogenase during Freeze-Thaw in Buffered Solutions Using Neutron Scattering.

The aims of this work were to evaluate the effect of freezing and thawing stresses on lactate dehydrogenase (LDH) stability under three conditions. (i) In a solution buffered with sodium phosphate (NaP; 10 and 100 mM). The selective crystallization of disodium hydrogen phosphate during freezing caused a pronounced pH shift. (ii) In a solution buffered with histidine, where there was no pH shift due to buffer salt crystallization. (iii) At different concentrations of LDH so as to determine the self-stabilizing ability of LDH. The change in LDH tetrameric conformation was measured by small-angle neutron scattering (SANS). The pH of the phosphate buffer solutions was monitored as a function of temperature to quantify the pH shift. The conditions of buffer component crystallization from solution were identified using low-temperature X-ray diffractometry. Dynamic light scattering (DLS) enabled us to determine the effect of freeze-thawing on the protein aggregation behavior. LDH, at a high concentration (1000 µg/mL; buffer concentration 10 mM), has a pronounced self-stabilizing effect and did not aggregate after five freeze-thaw cycles. At lower LDH concentrations (10 and 100 µg/mL), only with the selection of an appropriate buffer, irreversible aggregation could be avoided. While SANS provided qualitative information with respect to protein conformation, the insights from DLS were quantitative with respect to the particle size of the aggregates. SANS is the only technique which can characterize the protein both in the frozen and thawed states.

Direct localization of detergents and bacteriorhodopsin in the lipidic cubic phase by small-angle neutron scattering.

Lipidic cubic phase (LCP) crystallization methods have been essential in obtaining crystals of certain membrane proteins, particularly G-protein-coupled receptors. LCP crystallization is generally optimized across a large number of potential variables, one of which may be the choice of the solubilizing detergent. A better fundamental understanding of the behavior of detergents in the LCP may guide and simplify the detergent selection process. This work investigates the distribution of protein and detergent in LCP using the membrane protein bacteriorhodopsin (bR), with the LCP prepared from highly deuterated monoolein to allow contrast-matched small-angle neutron scattering. Contrast-matching allows the scattering from the LCP bilayer itself to be suppressed, so that the distribution and behavior of the protein and detergent can be directly studied. The results showed that, for several common detergents, the detergent micelle dissociates and incorporates into the LCP bilayer essentially as free detergent monomers. In addition, the detergent octyl glucoside dissociates from bR, and neither the protein nor detergent forms clusters in the LCP. The lack of detergent assemblies in the LCP implies that, upon incorporation, micelle sizes and protein/detergent interactions become less important than they would be in solution crystallization. Crystallization screening confirmed this idea, with crystals obtained from bR in the presence of most detergents tested. Thus, in LCP crystallization, detergents can be selected primarily on the basis of protein stabilization in solution, with crystallization suitability a lesser consideration.

Phase Behavior of Poloxamer 188 Aqueous Solutions at Subzero Temperatures: A Neutron and X-ray Scattering Study.

Phase transitions of poloxamer 188 (P188) aqueous solutions at freezing temperatures are investigated using small-angle neutron scattering (SANS) and small- and wide-angle X-ray scatterings (SAXS and WAXS). It is shown that P188 solution (10%) undergoes the following sequence of phase transitions during cooling from 25 to -150 °C: micelle solution, solution of monomers, two-phase mixture of liquid crystalline mesophase + ice, and finally crystalline P188 + ice. Formation of the liquid crystalline phase during freezing is likely to be triggered by water freezing to ice and corresponding freeze concentration of the remaining solution. During heating of the frozen solutions, the sequence of the phase transitions is reversed: crystalline P188 + ice, liquid crystalline mesophase + ice, monomer solution + ice, monomer solution, and finally micelle solution. Similar phase transitions are detected for dilute solutions of P188 (1%) except that micelle formation is not observed at 25 °C, consistent with the literature reported critical micelle concentration (CMC) at this temperature. The present study provides new insight into P188 aqueous solutions at freezing temperatures and has practical implications on the design and development of pharmaceutical formulations.

Structural Characterization and Modeling of a Respiratory Syncytial Virus Fusion Glycoprotein Nanoparticle Vaccine in Solution.

The respiratory syncytial virus (RSV) fusion (F) protein/polysorbate 80 (PS80) nanoparticle vaccine is the most clinically advanced vaccine for maternal immunization and protection of newborns against RSV infection. It is composed of a near-full-length RSV F glycoprotein, with an intact membrane domain, formulated into a stable nanoparticle with PS80 detergent. To understand the structural basis for the efficacy of the vaccine, a comprehensive study of its structure and hydrodynamic properties in solution was performed. Small-angle neutron scattering experiments indicate that the nanoparticle contains an average of 350 PS80 molecules, which form a cylindrical micellar core structure and five RSV F trimers that are arranged around the long axis of the PS80 core. All-atom models of full-length RSV F trimers were built from crystal structures of the soluble ectodomain and arranged around the long axis of the PS80 core, allowing for the generation of an ensemble of conformations that agree with small-angle neutron and X-ray scattering data as well as transmission electron microscopy (TEM) images. Furthermore, the hydrodynamic size of the RSV F nanoparticle was found to be modulated by the molar ratio of PS80 to protein, suggesting a mechanism for nanoparticle assembly involving addition of RSV F trimers to and growth along the long axis of the PS80 core. This study provides structural details of antigen presentation and conformation in the RSV F nanoparticle vaccine, helping to explain the induction of broad immunity and observed clinical efficacy. Small-angle scattering methods provide a general strategy to visualize surface glycoproteins from other pathogens and to structurally characterize nanoparticle vaccines.

SurA is a cryptically grooved chaperone that expands unfolded outer membrane proteins.

The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the ß-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network.