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The protein tau is involved in several neurogenerative diseases such as Alzheimer's Disease, where tau content and fibrillation have been linked to disease progression. Tau colocalizes with phospholipids and glycosaminoglycans in vivo. We investigated if and how tau fibrillation can be induced by two lysophospholipids, namely the zwitterionic 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) and the anionic 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG) as well as the glycosaminoglycan heparin. We used a range of biophysical techniques including small-angle X-ray scattering, Thioflavin T fluorescence, and SDS-PAGE, collecting data at various time points to obtain structural information on each phase of the fibrillation. We find that LPC does not induce fibrillation or interact with tau. Low concentrations of LPG induce fibrillation by formation of small hydrophobic clusters with monomeric tau. At higher LPG concentrations, a core-shell complex is formed where tau wraps around LPG micelles with regions extending away from the micelles. For heparin, loosely associated oligomers are formed rapidly with around ten tau molecules. Fibrils formed with either LPG or heparin show similar final cross-section dimensions. Furthermore, SDS-resistant oligomers are observed for both LPG and heparin. Our study demonstrates that tau fibrillation can be induced by two different biologically relevant cofactors leading to structurally different initial states but similar cross-sectional dimensions for the fibrils. Structural information about initial states prior to fibril formation is important both to gain a better understanding of the onset of fibrillation in vivo, and for the development of targeted drugs that can reduce or abolish tau fibrillation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bacterial functional amyloids contribute to biofilm development by bacteria and provide protection from the immune system and prevent antibiotic treatment. Strategies to target amyloid formation and interrupt biofilm formation have attracted recent interest due to their antimicrobial potential. Functional amyloid in Pseudomonas (Fap) includes FapC as the major component of the fibril while FapB is a minor component suggested to function as a nucleator of FapC. The system also includes the small periplasmic protein FapA, which has been shown to regulate fibril composition and morphology. The interplay between these three components is central in Fap fibril biogenesis. Here we present a comprehensive biophysical and spectroscopy analysis of FapA, FapB and FapC and provide insight into their molecular interactions. We show that all three proteins are primarily disordered with some regions with structural propensities for α-helix and β-sheet. FapA inhibits FapC fibrillation by targeting the nucleation step, whereas for FapB the elongation step is modulated. Furthermore, FapA alters the morphology of FapC (more than FapB) fibrils. Complex formation is observed between FapA and FapC, but not between FapA and FapB, and likely involves the N-terminus of FapA. We conclude that FapA is an intrinsically disordered chaperone for FapC that guards against fibrillation within the periplasm. This new understanding of a natural protective mechanism of Pseudomonas against amyloid formations can serve as inspiration for strategies blocking biofilm formation in infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Although the anionic surfactant sodium dodecyl sulfate, SDS, has been used for more than half a century as a versatile and efficient protein denaturant for protein separation and size estimation, there is still controversy about its mode of interaction with proteins. The term "rod-like" structures for the complexes that form between SDS and protein, originally introduced by Tanford, is not sufficiently descriptive and does not distinguish between the two current vying models, namely protein-decorated micelles a.k.a. the core-shell model (in which denatured protein covers the surface of micelles) versus beads-on-a-string model (where unfolded proteins are surrounded by surfactant micelles). Thanks to a combination of structural, kinetic and computational work particularly within the last 5-10 years, it is now possible to rule decisively in favor of the core-shell model. This is supported unambiguously by a combination of calorimetric and small-angle X-ray scattering (SAXS) techniques and confirmed by increasingly sophisticated molecular dynamics simulations. Depending on the SDS:protein ratio and the protein molecular mass, the formed structures can range from multiple partly unfolded protein molecules surrounding a single shared micelle to a single polypeptide chain decorating multiple micelles. We also have much new insight into how this species forms. It is preceded by the binding of small numbers of SDS molecules which subsequently grow by accretion. Time-resolved SAXS analysis reveals an asymmetric attack by SDS micelles followed by distribution of the increasingly unfolded protein around the micelle. The compactness of the protein chain continues to evolve at higher SDS concentrations according to single-molecule studies, though the protein remains completely denatured on the tertiary structural level. SDS denaturation can be reversed by addition of nonionic surfactants that absorb SDS forming mixed micelles, leaving the protein free to refold. Refolding can occur in parallel tracks if only a fraction of the protein is initially stripped of SDS. SDS unfolding is nearly always reversible unless carried out at low pH, where charge neutralization can lead to superclusters of protein-surfactant complexes. With the general mechanism of SDS denaturation now firmly established, it largely remains to explore how other ionic surfactants (including biosurfactants) may diverge from this path., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Bleeding is associated with a significantly increased morbidity and mortality. Bleeding events are often described in the unstructured text of electronic health records, which makes them difficult to identify by manual inspection., Objectives: To develop a deep learning model that detects and visualizes bleeding events in electronic health records., Patients/methods: Three hundred electronic health records with International Classification of Diseases, Tenth Revision diagnosis codes for bleeding or leukemia were extracted. Each sentence in the electronic health record was annotated as positive or negative for bleeding. The annotated sentences were used to develop a deep learning model that detects bleeding at sentence and note level., Results: On a balanced test set of 1178   sentences, the best-performing deep learning model achieved a sensitivity of 0.90, specificity of 0.90, and negative predictive value of 0.90. On a test set consisting of 700 notes, of which 49 were positive for bleeding, the model achieved a note-level sensitivity of 1.00, specificity of 0.52, and negative predictive value of 1.00. By using a sentence-level model on a note level, the model can explain its predictions by visualizing the exact sentence in a note that contains information regarding bleeding. Moreover, we found that the model performed consistently well across different types of bleedings., Conclusions: A deep learning model can be used to detect and visualize bleeding events in the free text of electronic health records. The deep learning model can thus facilitate systematic assessment of bleeding risk, and thereby optimize patient care and safety., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Lipoteichoic acid (LTA), a surface associated polymer amphiphile tethered directly to the Gram-positive bacterial cytoplasmic membrane, is a key structural and functional membrane component. Its composition in the membrane is regulated by bacteria under different physiological conditions. How such LTA compositional variations modulate the membrane structural stability and integrity is poorly understood. Here, we have investigated structural changes in mixed liposomes mimicking the lipid composition of Gram-positive bacteria membranes, in which the concentration of Bacillus Subtilis LTA was varied between 0-15 mol%. Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) measurements indicated formation of mixed unilamellar vesicles, presumably stabilized by the negatively charged LTA polyphosphates. The vesicle size increased with the LTA molar concentration up to ∼6.5 mol%, accompanied by a broadened size distribution, and further increasing the LTA concentration led to a decrease in the vesicle size. At 80 °C, SANS analyses showed the formation of larger vesicles with thinner shells. Complementary Cryo-TEM imaging confirmed the vesicle formation and the size increase with LTA addition, as well as the presence of interconnected spherical aggregates of smaller size at higher LTA concentrations. The results are discussed in light of the steric and electrostatic interactions of the bulky LTA molecules with increased chain fluidity at the higher temperature, which affect the molecular packing and interactions, and thus depend on the LTA composition, in the membrane., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The occurrence of an amorphous calcium phosphate layer covering the crystalline apatite core has been suggested to be an intrinsic feature of both bone mineral and synthetic biomimetic analogs. However, an exahustive quantitative picture of the amorphous-crystalline relationship in these materials is still missing. Here, we present a multiple scale modelling that combines small-angle X-ray scattering (SAXS) and synchrotron wide-angle X-ray total scattering (WAXTS) analyses to investigate the amorphous-crystalline spatial interplay in bone sample and biomimetic carbonated nano-apatites. SAXS analysis indicates the presence of a single morphology consisting of tiny nanoplates (NPLs) and provides a measure of their thickness (falling in the 3-5 nm range). WAXTS analysis was performed by developing atomistic models of apatite NPLs incorporating lattice strain, mostly attributed to the carbonate content, and calculating the X-ray patterns using the Debye Scattering Equation. Upon model optimization, the size and strain parameters of the crystalline platelets were derived and the amorphous component, co-existing with the crystalline one, separated and quantified (in the 23-33 wt% range). Notably, the thickness of the apatite core was found to exhibit nearly null (bone) or minor (< 0.5 nm, biomimetic samples) deviations from that of the entire NPLs, suggesting that the amorphous material remains predominantly distributed along the lateral sides of the NPLs, in a core-crown-like arrangement. The lattice strain analysis indicates a significant stiffness along the c axis, which is comparable in bone and synthetic samples, and larger deformations in the other directions. STATEMENT OF SIGNIFICANCE: Current models of bone mineral and biomimetic nanoapatites suggest the occurrence of an amorphous layer covering the apatitic crystalline nanoplates in a core-shell arrangement. By combining X-ray scattering techniques in the small and wide angle regions, we propose a joint atomic-to-nanometre scale modelling to investigate the amorphous-crystalline interplay within the nanoplates. Estimates are extracted for the thickness of the entire nanoplates and the crystalline core, together with the quantification of the amorphous fraction and apatite lattice strain. Based on the thickness matching, the location of the amorphous material mostly along the edges of the nanoplates is inferred, with a vanishing or very thin layer in the thickness direction, suggesting a core-crown-like arrangement, with possible implications on the mineral surface reactivity., Competing Interests: Declaration of Competing Interest There are no conflicts to declare. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript., (Copyright © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Atherosclerosis is the main killer in the west and therefore a major health challenge today. Total serum cholesterol and lipoprotein concentrations, used as clinical markers, fail to predict the majority of cases, especially between the risk scale extremes, due to the high complexity in lipoprotein structure and composition. In particular, low-density lipoprotein (LDL) plays a key role in atherosclerosis development, with LDL size being a parameter considered for determining the risk for cardiovascular diseases. Determining LDL size and structural parameters is challenging to address experimentally under physiological-like conditions. This article describes the biochemistry and ultrastructure of normolipidemic and hypertriglyceridemic LDL fractions and subfractions using small-angle X-ray scattering. Our results conclude that LDL particles of hypertriglyceridemic compared to healthy individuals 1) have lower LDL core melting temperature, 2) have lower cholesteryl ester ordering in their core, 3) are smaller, rounder and more spherical below melting temperature, and 4) their protein-containing shell is thinner above melting temperature., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Many proteins form complexes called liprotides with oleic acid and other cis-fatty acids under conditions where the protein is partially unfolded. The complexes vary in structure depending on the ratio of protein and lipid, but the most common structural organization is the core-shell structure, in which a layer of dynamic, partially unfolded and extended proteins surrounds a micelle-like fatty acid core. This structure, first reported for α-lactalbumin together with OA, resembles complexes formed between proteins and anionic surfactants like SDS. Liprotides first rose to fame through their anti-carcinogenic properties which still remains promising for topical applications though not yet implemented in the clinic. In addition, liprotides show potential in drug delivery thanks to the ability of the micelle core to solubilize and stabilize hydrophobic compounds, though applications are challenged by their sensitivity to acidic pH and dynamic exchange of lipids which makes them easy prey for serum "hoovers" such as albumin. However, liprotides are also of fundamental interest as a generic "protein complex structure", demonstrating the many and varied structural consequences of protein-lipid interactions. Here we provide an overview of the different types of liprotide complexes, ranging from quasi-native complexes via core-shell structures to multi-layer structures, and discuss the many conditions under which they form. Given the many variable types of complexes that can form, rigorous biophysical analysis (stoichiometry, shape and structure of the complexes) remains crucial for a complete understanding of the mechanisms of action of this fascinating group of protein-lipid complexes both in vitro and in vivo., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Protein fibrillation is traditionally associated with misfolding, loss of functional phenotype, and gain of toxicity in neurodegenerative diseases. However, many organisms exploit fibrils in the form of functional amyloids (FA), as seen in bacteria, such as E. coli, Salmonella, Bacillus, and Pseudomonas. Here, we provide structural information and mechanistic data for fibrillation of the smallest amyloidogenic truncation unit along with the full-length version (FL) of the major amyloid protein FapC from Pseudomonas, predicted to consist of three β-hairpin-forming imperfect repeats separated by disordered regions. Using a series of truncation mutants, we establish that the putative loops (linkers) increase the rate of aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates in a pathway dominated by secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. SAXS on FapC FL, R3C, and remaining truncation constructs resolves two major coexisting species in the fibrillation process, namely pre-fibrillar loosely aggregated monomers, and cylindrical, elliptical cross-section fibrils. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα-Cβ chemical shifts, indicative of a predominant β-sheet topology with some α-helix or loop chemical shifts. Our work emphasizes the complex nature of FapC fibrillation. In addition, we are able to deduce the importance of non-repeat regions (i.e., predicted loops), which enhance the amyloid protein aggregation and their influence on the polymorphism of the fibril architecture., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C 12 E 8 ). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C 12 E 8 leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C 12 E 8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C 12 E 8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C 12 E 8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pseudomonas species export the amyloid-forming protein FapC to strengthen bacterial biofilm. P. species also produce the biosurfactant rhamnolipid (Rhl) and its outer membrane contains lipopolysaccharide (LPS). Given the possible contacts between FapC, Rhl and LPS, we here investigate how Rhl and LPS affect FapC fibrillation compared with SDS, known to promote fibrillation of proteins at sub-micellar concentrations. Micelles of all three surfactants help FapC bypass the nucleation lag phase, leading to rapid fibrillation, which persists even at high concentrations of micelles and incorporates almost all available FapC monomers. Fibrils formed at high micellar concentrations of Rhl and SDS seed fibrillation at low surfactant concentrations while retaining the original fibril structure. FapC interacts strongly with SDS to form a dense network of narrow fibrils. Small angle X-ray scattering (SAXS) analyses reveal that surfactants reduce the population of intermediates in the fibrillation process and detect a fast aggregation step over the first 2-4 h which precedes the main fibrillation monitored by Thioflavin T. An additional SAXS-detected rearrangement of early aggregates occurs after 4-10 h. At high Rhl concentrations, the micelles are decorated with protein fibrils. SDS induces FapC fibrillation so efficiently that epigallocatechin-3-gallate (EGCG) is unable to inhibit this process. However, EGCG stimulates FapC oligomer formation and inhibits fibrillation both on its own and in the presence of Rhl and LPS. This oligomer could be modelled as a compact core with a flexible shell. This suggests that EGCG can override the natural amyloid-stimulatory properties of these biosurfactants and thus target biofilm., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lipids often play an important role in the initial steps of fibrillation. The melanosomal protein Pmel17 forms amyloid in vivo and contains a highly amyloidogenic Repeat domain (RPT), important for melanin biosynthesis. RPT fibrillation is influenced by two lysolipids, the anionic lysophosphatidylglycerol (LPG) and zwitterionic lysophosphatidylcholine (LPC), both present in vivo at elevated concentrations in melanosomes, organelles in which Pmel17 aggregate. Here we investigate the interaction of RPT with both LPG and LPC using small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), electron microscopy, fluorescence and circular dichroism (CD) spectroscopy. Under non-shaking conditions, both lipids promote fibrillation but this is driven by different interactions with RPT. Each RPT binds >40 LPG molecules but only weak interactions are seen with LPC. Above LPG's criticial micelle concentration (cmc), LPG and RPT form connected micelles where RPT binds to the surface as beads on a string with core-shell structures. Binding to LPG only induces α-helical structure well above the cmc, while LPC has no measurable effect on the protein structure. While low (but still super-cmc) concentrations of LPG strongly promote aggregation, at higher LPG concentrations (10 mM), only ~ one RPT binds per micelle, inhibiting amyloid formation. ITC and SAXS reveal some interactions between the zwitterionic lipid LPC and RPT below the cmc but little above the cmc. Nevertheless, LPC only promotes aggregation above the cmc and this process is not inhibited by high LPC concentrations, suggesting that monomers and micelles cooperate to influence amyloid formation., (Copyright © 2018. Published by Elsevier B.V.)

Interaction between the activating NKG2D receptor on lymphocytes and its ligands MICA, MICB, and ULBP1-6 modulate T and NK cell activity and may contribute to the pathogenesis of Crohn's disease (CD). NKG2D ligands are generally not expressed on the cell surface of normal, non-stressed cells, but expression of MICA and MICB in CD intestine has been reported. In this exploratory study, we further characterize the expression of NKG2D and its ligands, including the less well-described ULBP4-6, in CD, and test if NKG2D ligand interactions are involved in the migration of activated T cells into the affected mucosal compartments. Intestinal tissue from CD patients and healthy controls were analyzed by flow cytometry, mass cytometry, and immunohistochemistry for expression of NKG2D and ligands, and for cytokine release. Furthermore, NKG2D-dependent chemotaxis of activated CD8 + T cells across a monolayer of ligand-expressing human intestinal endothelial cells was examined. Activated lymphocytes down-regulated NKG2D expression upon accumulation in inflamed CD intestine. NKG2D expression on CD56 + T and γδ T cells from inflamed tissue seemed inversely correlated with CRP levels and cytokine release. B cells, monocytes, mucosal epithelium, and vascular endothelium expressed NKG2D ligands in inflamed CD intestine. The expression of NKG2D ligands was correlated with cytokine release, but was highly variable between patients. Stimulation of vascular intestinal endothelial cells in vitro induced expression of NKG2D ligands, including MICA/B and ULBP2/6. Blockade of NKG2D on CD8 + T cells inhibited the migration over ligand-expressing endothelial cells. Intestinal induction of NKG2D ligands and ligand-induced down-regulation of NKG2D in CD suggest that the NKG2D-ligand interaction may be involved in both the activation and recruitment of NKG2D + lymphocytes into the inflamed CD intestine., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Liprotides are protein-lipid complexes in which the fatty acids form a micelle-like core surrounded by a shell of partially unfolded protein molecules. These complexes can be formed in different ways. The simplest approach is a thermal treatment where protein and fatty acid are mixed and then incubated at elevated temperatures. Using this approach we here demonstrate that we can monitor liprotide formation in real time using Small-Angle X-ray Scattering (SAXS). Optimal conditions for liprotide formation, i.e. temperature and incubation times, as well as liprotide stability and structure, vary for different proteins. The apo form of α-lactalbumin (aLA) forms liprotides at room temperature, however, Ovalbumin (Ova) and Bovine Serum Albumin (BSA) require elevated temperatures (≥60°C) to form liprotides, and in addition, they need to be returned to lower temperatures to remain stable; repeated cycles of heating and cooling gradually dissociate the liprotides in parallel with the formation of disulfide-bonded aggregates. Real-time tracking of the formation of liprotides of BSA or Ova with OA at 60-65°C showed that liprotide formation takes place within a period of 12-18min and is preceded by a loss of secondary structure of the protein and binding of OA to the protein. Our SAXS-based approach provides a straightforward strategy to optimize liprotide formation for a wide range of different proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The formulation glatiramer acetate (GA) is widely used in therapy of multiple sclerosis. GA consists of random copolymers of four amino acids, in ratios that produce a predominantly positive charge and an amphipathic character. With the extraordinary complexity of the drug, several pharmacological modes-of-action were suggested, but so far none, which rationalizes the cationicity and amphipathicity as part of the mode-of-action. Here, we report that GA rapidly kills primary human T lymphocytes and, less actively, monocytes. LL-37 is a cleavage product of human cathelicidin with important roles in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45+ debris, derived from cell membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic contacts, which is critical for the lytic properties. In our study, SAXS showed that GA also forms this type of contacts. Taken together, our study offers new insight on the immunomodulatory mode-of-action of positively charged co-polymers. The comparison of LL-37 and GA highlights a consistent requirement of certain oligomeric and chemical properties to support cytotoxic effects of cationic polymers targeting human leukocytes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bioinspired in vitro collagen mineralization experiments have been performed in the presence of citrate and the combined role of the two bone organic matrix components in controlling mineral formation was investigated for the first time. Mineralized and non-mineralized collagen fibrils have been in depth characterized by combining small- and wide-angle X-ray scattering (SAXS/WAXS) techniques with Atomic Force Microscopy (AFM) imaging. A synergic effect of collagen and citrate in driving the formation of long-term stable amorphous calcium phosphate (ACP) nanoparticles with platy morphology was found. AFM images on mineralized collagen fibrils revealed that some of the ACP nanoparticles were deposited on the intramolecular nanoscopic holes of collagen fibrils., Statement of Significance: Citrate is an important component of the bone organic matrix but its specific role in bone mineralization is presently unclear. In this work, bioinspired in vitro collagen mineralization experiments in the presence of citrate have been carried out and the combined role of collagen and citrate in controlling mineral formation has been addressed for the first time. Through X-ray scattering and Atomic Force Microscopy characterizations on mineralized and non-mineralized collagen fibrils, we have found that citrate in synergy with collagen stabilizes an amorphous calcium phosphate (ACP) phase with platy morphology over one week and controls its deposition on collagen fibrils., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Micro-flow imaging (MFI(™) ) is an increasingly important technique for the characterization of subvisible particles during the development of biopharmaceutical products. Protein particles are highly variable in size, appearance, and translucency posing challenges to optical detection techniques. We have developed a set of standard statistical tests applicable for routine evaluation of MFI™ particle dataset quality. The tests evaluate the spatial randomness of particles using nearest neighbor and quadrat analysis. Using case studies of stressed antibody samples, we demonstrate how the tests uncover fragmentation artifacts and uneven detector sensitivity for translucent particles. To improve the detection of translucent particles, a new local pixel intensity variance particle detection algorithm has been developed. The improved algorithm significantly decreases fragmentation artifacts, and also increases sensitivity toward translucent particles in general. Our results highlight current limitations and the potential for improvements in the optical detection techniques for subvisible protein aggregates., (© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0-6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s(20,w)(0) were determined to be 3.64 and 5.51±0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. [6]) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p(r) function indicated an elongated conformation supported by a radius of gyration of 37.5±0.2 Å and a maximum dimension of ~130 Å. The effects of glycerol on the formation of dimers are discussed. This new model of aIF5B in solution shows that there are universal structural differences between aIF5B and the homologous protein IF2 from Escherichia coli., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catalytic domain, most serine proteases have N-terminal A-chains with independently folded domains. Little is known about how zymogen activation affects the interplay between domains. This question is investigated with urokinase-type plasminogen activator (uPA), which has an epidermal growth factor domain and a kringle domain, connected to the catalytic domain by a 15-residue linker. uPA has been implicated under several pathological conditions, and one possibility for pharmacological control is targeting the conversion of the zymogen pro-uPA to active uPA. Therefore, a small-angle X-ray scattering study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived using available atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect to the serine protease domain. In contrast, pro-uPA was observed to be rigid, with the amino-terminal fragment domain in a fixed position with respect to the serine protease domain. Analytical ultracentrifugation analysis supported the observed difference between pro-uPA and uPA in overall shape and size seen with small-angle X-ray scattering. Upon association of either of two monoclonal Fab (fragment antigen-binding) fragments that are directed against the catalytic domain of, respectively, pro-uPA and uPA, rigid structures were formed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Human transforming growth factor β induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compromise transparency. The complete three-dimensional structure of TGFBIp is not yet available, with the exception of a partial X-ray structure of the archetype FAS1 domain derived from Drosophila fasciclin-1. In this study, small-angle X-ray scattering (SAXS) models of intact wild-type (WT) human TGFBIp and a mutant (R124H) are presented. The mutation R124H leads to a variant of granular CD. The deduced structure of the TGFBIp monomer consists of four FAS1 domains in a simple "beads-on-a-string" arrangement, constructed by the superimposition of four consecutive Drosophila fasciclin domains. The SAXS-based model of the TGFBIp R124H mutant displayed no structural differences from WT. Both WT TGFBIp and the R124H mutant formed trimers at higher protein concentrations. The similar association properties and three-dimensional shape of the two proteins suggest that the mutation does not induce any major structural rearrangements, but points towards the role of other corneal-specific factors in the formation of corneal R124H deposits., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in Parkinson's disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably different from the fibrillation pathway in the absence of SDS. Fibrillation occurs most extensively and most rapidly (starting within 45 min) under conditions where 12 SDS molecules are bound per alphaSN molecule, which is also the range where SDS binding is associated with the highest enthalpy. Fibrillation is only reduced in proportion to the fraction of SDS below 25 mol% SDS in mixed surfactant mixtures with nonionic surfactants and is inhibited by formation of bulk micelles and induction of alpha-helical structure. In this fibrillogenic complex, 4 alphaSN molecules initially associate with 40-50 SDS molecules to form a shared micelle that gradually grows in size. The complex initially exhibits a mixture of random coil and alpha-helix, but incubation results in a structural conversion into beta-sheet structure and concomitant formation of thioflavin-T-binding fibrils over a period of several hours. Based on small-angle X-ray scattering, the aggregates elongate as a beads-on-a-string structure in which individual units of ellipsoidal SDS-alphaSN are bridged by strings of the protein, so that aggregates nucleate around the surface of protein-stabilized micelles. Thus, fibrillation in this case occurs by a process of continuous accretion rather than by the rate-limiting accumulation of a distinct nucleus. The morphology of the SDS-induced fibrils does not exhibit the classical rod-like structures formed by alphaSN when aggregated by agitation in the absence of SDS. The SDS-induced fibrils have a flexible worm-like appearance, which can be converted into classical straight fibrils by continuous agitation. SDS-induced fibrillation represents an alternative and highly reproducible mechanism for fibrillation where protein association is driven by the formation of shared micelles, which subsequently allows the formation of beta-sheet structures that presumably link individual micelles. This illustrates that protein fibrillation may occur by remarkably different mechanisms, testifying to the versatility of this process., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Solution and adsorption properties of both charged and uncharged bottle-brush polymers have been investigated. The solution conformation and interactions in solution have been investigated by small-angle scattering techniques. The association of the bottle-brush polymers with anionic surfactants has also been studied. Surfactant binding isotherm measurements, NMR, surface tension measurements, as well as SAXS, SANS and light scattering techniques were utilized for understanding the association behaviour in bulk solutions. The adsorption of the bottle-brush polymers onto oppositely charged surfaces has been explored using a battery of techniques, including reflectometry, ellipsometry, quartz crystal microbalance, and neutron reflectivity. The combination of these techniques allowed determination of adsorbed mass, layer thickness, water content, and structural changes occurring during layer formation. The adsorption onto mica was found to be very different to that on silica, and an explanation for this was sought by employing a lattice mean-field theory. The model was able to reproduce a number of salient experimental features characterizing the adsorption of the bottle-brush polymers over a wide range of compositions, spanning from uncharged bottle-brushes to linear polyelectrolytes. This allowed us to shed light on the importance of electrostatic surface properties and non-electrostatic surface-polymer affinity for the adsorption. The interactions between bottle-brush polymers and anionic surfactants in adsorbed layers have also been elucidated using ellipsometry, neutron reflectivity and surface force measurements., (Copyright 2010 Elsevier B.V. All rights reserved.)

Mixtures of the two non-ionic surfactants hexaoxyethylene dodecyl ether (C(12)E(6)) and n-dodecyl-beta-D-maltoside (beta-C(12)G(2)) were studied with regard to surface properties, bulk properties, foam films, and foams. The reason for studying a mixture of an ethylene oxide (C(i)E(j)) and a sugar (C(n)G(m)) based surfactant is that despite being non-ionic, these two surfactants behave quite differently. Firstly, the physico-chemical properties of aqueous solutions of C(n)G(m) surfactants are less temperature-sensitive than those of C(i)E(j) solutions. Secondly, the surface charge density q(0) of foam films stabilized by C(n)G(m) surfactants is pH insensitive down to the so-called isoelectric point, while that of foam films stabilized by C(i)E(j) surfactants changes linearly with the pH. The third difference is related to interaction forces between solid surfaces. Under equilibrium conditions very high forces are needed to expel beta-C(12)G(2) from between thiolated gold surfaces, while for C(12)E(6) low loads are sufficient. Fourthly, the adsorption of C(12)E(6) and beta-C(12)G(2) on hydrophilic silica and titania, respectively, is inverted. While the surface excess of C(12)E(6) is large on silica and negligible on titania, beta-C(12)G(2) adsorbs very little on silica but has a large surface excess on titania. What is the reason for this different behaviour? Under similar conditions and for comparable head group sizes, it was found that the hydration of C(i)E(j) surfactants is one order of magnitude higher but on average much weaker than that of C(n)G(m) surfactants. Moreover, C(n)G(m) surfactants possess a rigid maltoside unit, while C(i)E(j) surfactants have a very flexible hydrophilic part. Indeed, most of the different properties mentioned above can be explained by the different hydration and the head group flexibilities. The intriguing question of how mixtures of C(i)E(j) and C(n)G(m) surfactants would behave arises organically. Thus various properties of C(12)E(6)+beta-C(12)G(2) mixtures in aqueous solution have been studied with a focus on the 1:1 mixture. The results are compared with those of the single surfactants and are discussed accordingly., (Copyright 2009 Elsevier B.V. All rights reserved.)

We have combined spectroscopy, chromatography, calorimetry, and small-angle X-ray scattering (SAXS) to provide a comprehensive structural and stoichiometric description of the sodium dodecyl sulfate (SDS)-induced denaturation of the 86-residue alpha-helical bovine acyl-coenzyme-A-binding protein (ACBP). Denaturation is a multistep process. Initial weak binding of 1-3 SDS molecules per protein molecule below 1.3 mM does not perturb the tertiary structure. Subsequent binding of approximately 13 SDS molecules per ACBP molecule leads to the formation of SDS aggregates on the protein and changes in both tertiary and secondary structures. SAXS data show that, at this stage, a decorated micelle links two ACBP molecules together, leaving about half of the polypeptide chain as a disordered region protruding into the solvent. Further titration with SDS leads to the additional uptake of 26 SDS molecules, which, according to SAXS, forms a larger decorated micelle bound to a single ACBP molecule. At the critical micelle concentration, we conclude from reduced mobility and increased fluorescence anisotropy that each ACBP molecule becomes associated with more than one micelle. At this point, 56-60 SDS molecules are bound per ACBP molecule. Our data provide key structural insights into decorated micelle complexes with proteins, revealing a remarkable diversity in the different conformations they can stabilize. The data highlight that a minimum decorated micelle size, which may be a key driving force for intermolecular protein association, exists. This may also provide a structural basis for the known ability of submicellar surfactant concentrations to induce protein aggregation and fibrillation.

Protein amyloid formation proceeds through a number of different stages. Oligomeric species observed at early stages have aroused particular interest because of evidence for their involvement in cytotoxic processes such as membrane permeabilization. It is unclear whether these oligomers are obligate precursors to fibrils or represent "dead-end" species that impede fibrillation. Because of the many interconverting species present during amyloid formation, it is important to study the process as non-invasively as possible. Small angle X-ray scattering (SAXS) measurements allow us to monitor structural changes in solution for a population of different species over time. Here, SAXS was used to provide a detailed structural description of the fibrillation of the 29 residue peptide hormone glucagon at pH 2.5 from the monomer and early oligomers to mature fibers. Investigation of the pseudo-equilibrium behavior in the lag phase before fibrillation at several concentrations showed that glucagon is present in a monomeric form below about 5.1 mg/mL, while larger oligomers with average aggregation numbers of about three and seven, are formed at 6.4 and 10.7 mg/mL, respectively. Applying several modeling tools to the experimental data, it is shown that the early oligomerization states can be described as associations between glucagon molecules. After the lag phase, a short rod-like protofibril (radius of ~16 A and length >300 A) is formed and subsequently grows to N1000 A in length and assembles into long triple-bundled mature fibers. The protofibril shares many features with the elongated oligomer proposed to be the structural nucleus for insulin fibrils. We propose that on-pathway fibrillar intermediates share this elongated shape that easily allows them to be incorporated into mature fibrils. This contrasts with the annular shape, which is suggested to be involved in cytotoxic membrane permeabilization and may represent a dead-end species off the fibrillar pathway.

The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the alpha5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

This study describes the evaluation and characterization of a self-nanoemulsifying drug delivery system (SNEDDS) consisting of a nonionic surfactant (Cremophor RH40), a mixture of long chain mono-, di-, and triacylglycerides (Maisine 35-1 and Sesame oil) and ethanol. Compositions containing 10% (w/w) ethanol, 40%-60% (w/w) lipid content, and 30%-50% (w/w) Cremophor RH40 were identified as pharmaceutically relevant, robust, and self-nanoemulsifying when dispersed in aqueous media. The influence of adding three different lipophilic model drug compounds (danazol, halofantrine, and probucol) to the SNEDDS was evaluated. While danazol precipitated from the SNEDDS after dispersion in aqueous media, halofantrine and procubol remained solubilized. Halofantrine- and procubol-loaded SNEDDS were evaluated in both saline and in media simulating fasted and fed-state intestinal fluid (FaSSIF and FeSSIF) using dynamic light scattering and small-angle X-ray scattering (SAXS) techniques. Stable nanoemulsions with droplet sizes in the range of 20-50 nm were formed in all media and with and without drugs. The mean size of the droplets was neither affected significantly by being dispersed into the media simulating gastro intestinal fluid, nor by addition of the drug., ((c) 2007 Wiley-Liss, Inc.)

Cyclase-associated protein (CAP) is a highly conserved modular protein implicated in the regulation of actin filament dynamics and a variety of developmental and morphological processes. The protein exists as a high molecular weight complex in cell extracts and purified protein possesses a high tendency to aggregate, a major obstacle for crystallisation. Using a mutagenesis approach, we show that two structural features underlie the mechanism of oligomerisation in Dictyostelium discoideum CAP. Positively charged clusters on the surface of the N-terminal helix-barrel domain are involved in inter-molecular interactions with the N or C-terminal domains. Abolishing these interactions mainly renders dimers due to a domain swap feature in the extreme C-terminal region of the protein that was previously described. Based on earlier studies with yeast CAP, we also generated constructs with mutations in the extreme N-terminal region of Dictyostelium CAP that did not show significantly altered oligomerisation behaviour. Constructs with mutations in the earlier identified protein-protein interaction interface on the N-terminal domain of CAP could not be expressed as soluble protein. Assessment of the soluble proteins indicates that the mutations did not affect their overall fold. Further studies point to the correlation between stability of full-length CAP with its multimerisation behaviour, where oligomer formation leads to a more stable protein.

We have established a time-resolved fluorescence assay to study fibrillation of the 29 residue peptide hormone glucagon under a variety of different conditions in a high-throughput format. Fibrils formed at pH 2.5 differ in fibrillation kinetics, morphology, thioflavin T staining and FTIR/CD spectra depending on salts, glucagon concentration and fibrillation temperature. Apparent fibrillar stability correlates with spectral and kinetic properties; generally, fibrils formed under conditions favourable for rapid fibrillation (ambient temperatures, high glucagon concentration or high salt concentration) appear less thermostable than those formed under more challenging conditions (high temperatures, low glucagon or low salt concentrations). Properties of preformed fibrils used for seeding are inherited in a prion-like manner. Thus, we conclude that the structure of fibrils formed by glucagon is not the result of the global energy minimization, but rather kinetically controlled by solvent conditions and seed-imprinting. Fibrillar polymorphism, which is being reported for an increasing number of proteins, probably reflects that fibrils have not been under evolutionary constraints to retain a single active conformation. Our results highlight the complexity of the fibrillation mechanism of glucagon, since even subtle changes in fibrillation conditions can alter the type of fibrils formed, or result in formation of mixtures of several types of fibrils.

Background/aims: Chronic hepatitis B (CHB) and C (CHC) are commonly associated with hepatic steatosis. The aims of this study were to investigate predictors of hepatic steatosis, and their impact on inflammation and fibrosis in CHB and CHC., Methods: Consecutive patients with either CHB or CHC who underwent a liver biopsy at The Alfred Hospital between April and September 2002 were included. Histological analysis of liver biopsies was performed by two hepatopathologists blinded to the clinical data., Results: Ninety-one patients were analysed including 17 patients with CHB and 74 with CHC. CHC genotype 3, C-peptide, glucose and waist circumference were independent predictors of extent of Brunt steatosis grade, while CHC genotype 3, C-peptide and waist circumference were independent predictors of microvesicular steatosis grade. Alcohol intake and age were predictors of hepatic fibrosis. There was a trend toward a correlation between both Brunt steatosis and microvesicular steatosis grades and fibrosis progression rate in CHC genotype non-3., Conclusions: Hepatic steatosis is common in chronic hepatitis B and C, and is associated with waist circumference, glucose, C-peptide and chronic hepatitis C genotype 3. Steatosis grade appears to relate to hepatic fibrosis progression rate in chronic hepatitis C genotype non-3.

We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus. When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl. Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively. However, its tertiary structure appears to be more plastic than at neutral pH, and some regions of the protein may be partially unstructured. At 42 degrees C, there is a lag phase of several days after which fibrillation takes place over several hours. Data from the fibrillation behaviour of a comprehensive series of single and double mutants of S6 suggests that several factors control the onset of fibrillation. Firstly, there appears to be a contiguous region of "gatekeeper" residues that inhibit fibrillation, since their truncation significantly reduces the duration of the lag phase. This region overlaps extensively with the partially unstructured region of the protein, suggesting that residues with enhanced flexibility and solvent-accessibility are important for the initiation of fibrillation. Secondly, longer lag phases correlate with faster rates of unfolding. We interpret this to mean that kinetic stability also controls fibrillation but in the sense that the quasi-native state, rather than the denatured state, is the species that participates in nucleation. This implies that fibrillation can also occur from a quasi-native state as opposed to an ensemble of highly fluctuating structures, and highlights the delicate balance between flexibility and structure required to form organized assemblies of polypeptide chains.

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability. Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.

Selectively deuterated 70 S E. coli ribosomes and isolated 30 S and 50 S subunits were analyzed by X-ray and neutron solution scattering. The resulting contrast variation data set (42 curves in total) was proven to be consistent in describing the ribosome as a four-phase system composed of the protein and rRNA moieties of both subunits. This data set thus provides ten times more information than a single scattering curve. A solid body four-phase model of the 70 S ribosome at low resolution was built from the envelope functions of the 30 S and 50 S subunits and of those of the corresponding RNA moieties. The four envelopes were parameterized at a resolution of 3.5 nm using spherical harmonics and taking into account interface layers between the phases. The initial approximation for the envelopes of the subunits was taken from electron microscopic data presented recently by J. Frank and co-workers (Albany); the rRNA envelopes were initially approximated by spheres. The optimization and the refinement of the model proceeded by non-linear least squares minimization fitting the available experimental data. The refined envelopes of the subunits differ by about 10% from the starting approximation and the shape of the final 70 S model lies between the outer envelopes of the models by Frank and by M. von Heel & R. Brimacombe (Berlin). The rRNA moiety in the 30 S subunit is more anisometric than the subunit itself, whereas the rRNA of the 50 S subunit forms a compact core. The rRNAs protrude to the surfaces of the subunits and occupy approximately 30 to 40% of the corresponding surface areas. X-ray scattering curves of the two main functional elongation 70 S complexes (pre- and post-translocational) differ only marginally from those of the non-programmed ribosomes, suggesting that the low resolution four-phase model is also valid for the elongating 70 S ribosome.

Solutions of selectively deuterated 70 S Escherichia coli ribosomes and of free 30 S and 50 S subunits were studied by neutron scattering using contrast variation. The integrity of the partially deuterated particles was controlled by parallel X-ray measurements. Integral parameters of the entire ribosome, of its subunits and of the protein and rRNA moieties were evaluated. The data allow an experimental validation of the two most recent electron microscopy reconstructions of the 70 S ribosome presented by the groups of J. Frank (Albany) and of M. van Heel & R. Brimacombe (Berlin). For each reconstruction, integral parameters and theoretical scattering curves from the 70 S and its subunits were calculated and compared with the experimental data. Although neither of the two models yields a comprehensive agreement with the experimental data, Frank's model provides a better fit. For the 50 S subunit of van Heel & Brimacombe's model the fit with the experimental data improves significantly when the internal channels and tunnels are filled up. The poorer fit of the latter model is thus caused by its "sponge"-like structure which may partly be due to an enhancement of high frequency contributions in some of the steps of the three-dimensional image reconstruction. It seems therefore unlikely that the ribosome has a "sponge"-like structure with a pronounced network of channels.

The X-ray and neutron contrast variation data of the 50 S ribosomal subunit of Escherichia coli in solution are interpreted in the frame of a two-phase model described by the shapes of the 50 S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50 S subunit and of the RNA-rich core are evaluated with a resolution of about 4 nm. The shape of the envelope is in good agreement with the models of the 50 S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.