Widespread amyloidogenicity potential of multiple myeloma patient-derived immunoglobulin light chains.

BACKGROUND: In a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity. RESULTS: We introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins. CONCLUSIONS: We show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.

Differentiation of subnucleus-sized oligomers and nucleation-competent assemblies of the Aß peptide.

A significant feature of Alzheimer's disease is the formation of amyloid deposits in the brain consisting mainly of misfolded derivatives of proteolytic cleavage products of the amyloid precursor protein amyloid-ß (Aß) peptide. While high-resolution structures already exist for both the monomer and the amyloid fibril of the Aß peptide, the mechanism of amyloid formation itself still defies precise characterization. In this study, low and high molecular weight oligomers (LMWOs and HMWOs) were identified by sedimentation velocity analysis, and for the first time, the temporal evolution of oligomer size distributions was correlated with the kinetics of amyloid formation as determined by thioflavin T-binding studies. LMWOs of subnucleus size contain fewer than seven monomer units and exist alongside a heterogeneous group of HMWOs with 20-160 monomer units that represent potential centers of nucleus formation due to high local monomer concentrations. These HMWOs already have slightly increased ß-strand content and appear structurally similar regardless of size, as shown by examination with a range of fluorescent dyes. Once fibril nuclei are formed, the monomer concentration begins to decrease, followed by a decrease in oligomer concentration, starting with LMWOs, which are the least stable species. The observed behavior classifies the two LMWOs as off pathway. In contrast, we consider HMWOs to be on-pathway, prefibrillar intermediates, representing structures in which nucleated conformational conversion is facilitated by high local concentrations. Aß40 and Aß42 M35ox take much longer to form nuclei and enter the growth phase than Aß42 under identical reaction conditions, presumably because both the size and the concentration of HMWOs formed are much smaller.

Met/Val129 polymorphism of the full-length human prion protein dictates distinct pathways of amyloid formation.

Methionine/valine polymorphism at position 129 of the human prion protein, huPrP, is tightly associated with the pathogenic phenotype, disease progress, and age of onset of neurodegenerative diseases such as Creutzfeldt-Jakob disease or Fatal Familial Insomnia. This raises the question of whether and how the amino acid type at position 129 influences the structural properties of huPrP, affecting its folding, stability, and amyloid formation behavior. Here, our detailed biophysical characterization of the 129M and 129V variants of recombinant full-length huPrP(23-230) by amyloid formation kinetics, CD spectroscopy, molecular dynamics simulations, and sedimentation velocity analysis reveals differences in their aggregation propensity and oligomer content, leading to deviating pathways for the conversion into amyloid at acidic pH. We determined that the 129M variant exhibits less secondary structure content before amyloid formation and higher resistance to thermal denaturation compared to the 129V variant, whereas the amyloid conformation of both variants shows similar thermal stability. Additionally, our molecular dynamics simulations and rigidity analyses at the atomistic level identify intramolecular interactions responsible for the enhanced monomer stability of the 129M variant, involving more frequent minimum distances between E196 and R156, forming a salt bridge. Removal of the N-terminal half of the 129M full-length variant diminishes its differences compared to the 129V full-length variant and highlights the relevance of the flexible N terminus in huPrP. Taken together, our findings provide insight into structural properties of huPrP and the effects of the amino acid identity at position 129 on amyloid formation behavior.

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects.

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

Versatile Route toward Hydrophobically Polymer-Grafted Gold Nanoparticles from Aqueous Dispersions.

Stabilization of gold nanoparticles in organic solvents is a key challenge in making them available for a wider range of material applications. Polymers are often used as stabilizing ligands because they also allow for the introduction of new properties and functionalities. Many of the established synthesis protocols for gold nanoparticles are water-based. However, the insolubility of many synthetic polymers in water renders the direct functionalization of aqueous particle dispersions with these ligands difficult. Here, we report on an approach for the functionalization of gold nanoparticles, which were prepared by aqueous synthesis, with hydrophobic polymer ligands and their characterization in nonpolar, organic dispersions. Our method employs an auxiliary ligand to first transfer gold nanoparticles from an aqueous to an organic medium. In the organic phase, the auxiliary ligand is then displaced by thiolated polystyrene ligands to form a dense polymer brush on the particle surface. We characterize the structure of the ligand shell using electron microscopy, scattering techniques, and ultracentrifugation and analyze the influence of the molecular weight of the polystyrene ligands on the structure of the polymer brush. We further investigate the colloidal stability of polystyrene-functionalized gold nanoparticles in various organic solvents. Finally, we extend the use of our protocol from small, spherical gold nanoparticles to larger gold nanorods and nanocubes.

An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis.

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.

Farnesylation of human guanylate-binding protein 1 as safety mechanism preventing structural rearrangements and uninduced dimerization.

Human guanylate-binding protein 1 (hGBP1) belongs to the family of dynamin-like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post-translationally modified by attachment of a farnesyl group yielding farn-hGBP1. In this study, hydrodynamic differences in farn-hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size-exclusion chromatography (SEC). In addition, we performed small-angle X-ray scattering (SAXS) experiments coupled with a SEC setup (SEC-SAXS) to investigate structural properties of nonmodified hGBP1 and farn-hGBP1 in solution. SEC-SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal-like conformation in nucleotide-free solution, whereas unmodified hGBP1 forms a monomer-dimer equilibrium both in the inactive ground state in nucleotide-free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn-hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn-hGBP1 that occur in the presence of nucleotides. DATABASE: All SEC-SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8.

Toward the Mode of Action of the Clinical Stage All-d-Enantiomeric Peptide RD2 on Aß42 Aggregation.

The aggregation of amyloid-ß (Aß) into oligomers and fibrillary structures is critical for the pathogenesis of Alzheimer's disease (AD). Recently, research effort has been focused on developing novel agents that can preferentially suppress Aß oligomer mediated toxicities, for example, by directly targeting these toxic assemblies. The compound RD2 has been developed and optimized for Aß42 monomer binding and stabilization of the monomer in its native intrinsically disordered conformation. It has been demonstrated to improve and even reverse the cognitive and behavioral deficits in AD mouse models, while the detailed mechanism of action is not fully clarified. Here we focused on exploring the interaction between RD2 and Aß42 monomers and its consequences for the fibrillation of Aß42. RD2 binds to Aß42 monomers with nanomolar affinities, according to microscale thermophoresis and surface plasmon resonance measurements. Complexes between RD2 and Aß42 monomers are formed at 1:1 and other stoichiometries, as revealed by analytical ultracentrifugation. At substoichiometric levels, RD2 slows down the secondary structure conversion of Aß42 and significantly delays the fibril formation. Our research provides experimental evidence in supporting that RD2 eliminates toxic Aß assemblies by stabilizing Aß monomers in their native intrinsically disordered conformation. The study further supports the promising application of RD2 in counteracting Aß aggregation related pathologies.

Solution-Based Determination of Dissociation Constants for the Binding of Aß42 to Antibodies.

Amyloid ß-peptides (Aß) play a major role in the pathogenesis of Alzheimer's disease. Therefore, numerous monoclonal antibodies against Aß have been developed for basic and clinical research. The present study applied fluorescence based analytical ultracentrifugation and microscale thermophoresis to characterize the interaction between Aß42 monomers and three popular, commercially available antibodies, namely 6E10, 4G8 and 12F4. Both methods allowed us to analyze the interactions at low nanomolar concentrations of analytes close to their dissociation constants (K D) as required for the study of high affinity interactions. Furthermore, the low concentrations minimized the unwanted self-aggregation of Aß. Our study demonstrates that all three antibodies bind to Aß42 monomers with comparable affinities in the low nanomolar range. K D values for Aß42 binding to 6E10 and 4G8 are in good agreement with formerly reported values from SPR studies, while the K D for 12F4 binding to Aß42 monomer is reported for the first time.

Posttranslational Modification of the NADP-Malic Enzyme Involved in C4 Photosynthesis Modulates the Enzymatic Activity during the Day.

Evolution of the C4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C4 pathway. Here, we show that regulation of maize (Zea mays) C4-NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C4-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. Phosphorylation of ZmC4-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C4-NADP-ME indicated that Ser419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC4-NADP-ME at Ser419 during the first hours in the light is a cellular mechanism that fine tunes the enzymatic activity to coordinate the carbon concentration mechanism with the CO2 fixation rate, probably to avoid CO2 leakiness from bundle sheath cells.

Interference with Amyloid-ß Nucleation by Transient Ligand Interaction.

Amyloid-ß peptide (Aß) is an intrinsically disordered protein (IDP) associated with Alzheimer's disease. The structural flexibility and aggregation propensity of Aß pose major challenges for elucidating the interaction between Aß monomers and ligands. All-D-peptides consisting solely of D-enantiomeric amino acid residues are interesting drug candidates that combine high binding specificity with high metabolic stability. Here we characterized the interaction between the 12-residue all-D-peptide D3 and Aß42 monomers, and how the interaction influences Aß42 aggregation. We demonstrate for the first time that D3 binds to Aß42 monomers with submicromolar affinities. These two highly unstructured molecules are able to form complexes with 1:1 and other stoichiometries. Further, D3 at substoichiometric concentrations effectively slows down the ß-sheet formation and Aß42 fibrillation by modulating the nucleation process. The study provides new insights into the molecular mechanism of how D3 affects Aß assemblies and contributes to our knowledge on the interaction between two IDPs.

Molecular adaptations of NADP-malic enzyme for its function in C4 photosynthesis in grasses.

In C4 grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C4-NADP-ME). The activity of C4-NADP-ME was optimized by natural selection to efficiently deliver CO2 to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C4-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C4 adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME. Site-directed mutagenesis and structural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C4 metabolism.

Structure of the SLy1 SAM homodimer reveals a new interface for SAM domain self-association.

Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5', of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop-end-helix interface.

Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

Inhibition of amyloid Aß aggregation by high pressures or specific d-enantiomeric peptides.

Pressure can shift the polymer-monomer equilibrium of Aß, increasing pressure first leads to a release of Aß-monomers, surprisingly at pressures higher than 180 MPa repolymerization is induced. By high pressure NMR spectroscopy, differences of partial molar volumes ΔV0 and compressibility factors Δß' of polymerization were determined at different temperatures. The d-enantiomeric peptides RD2 and RD2D3 bind to monomeric Aß with affinities substantially higher than those determined for fibril formation. By reducing the Aß concentration below the critical concentration for polymerization they inhibit the formation of toxic oligomers. Chemical shift perturbation allows the identification of the binding sites. The d-peptides are candidates for drugs preventing Alzheimer's disease. We show that RD2D3 has a positive effect on the cognitive behaviour of transgenic (APPSwDI) mice prone to Alzheimer's disease. The heterodimer complexes have a smaller Stokes radius than Aß alone indicating the recognition of a more compact conformation of Aß identified by high pressure NMR before.

Stoichiometric Zn2+ interferes with the self-association of Aß42: Insights from size distribution analysis.

The abnormal aggregation of amyloid ß (Aß) peptides in the brain has been recognized as a central event in Alzheimer's disease (AD). Divalent metal ions such as Zn2+ have been shown to be closely involved in modulating Aß self-association. Although the link between Zn2+ dyshomeostasis and brain Aß deposition has been established, the effect of Zn2+ on the aggregation of Aß is still incompletely clarified. By combining analytical ultracentrifugation (AUC), circular dichroism (CD) spectroscopy, thioflavin T (ThT) assay and atomic force microscopy (AFM) imaging, we analyzed the impact of stoichiometric Zn2+ on the aggregation process of Aß42, the main toxic isoform of Aß species in the brain. Aß42 aggregates found in the presence of Zn2+ were smaller in size, non-fibrillary and showed less ß-sheet structures than aggregates formed in absence of Zn2+. AUC showed that Zn2+ was capable of retaining monomeric Aß42 in solution. Zn2+ chelation by EDTA totally reversed the inhibitory effect of Zn2+ on Aß42 fibrillation. Our results provide further evidence that Zn2+ shifts the self-association of Aß42 toward a non-fibrillary pathway by interfering with the aggregation process at multiple levels.

The Aß oligomer eliminating D-enantiomeric peptide RD2 improves cognition without changing plaque pathology.

While amyloid-ß protein (Aß) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer's disease (AD), soluble oligomeric Aß has been hypothesized to be responsible for synapse damage, neurodegeneration, learning, and memory deficits in AD. Here, we investigate the in vitro and in vivo efficacy of the D-enantiomeric peptide RD2, a rationally designed derivative of the previously described lead compound D3, which has been developed to efficiently eliminate toxic Aß42 oligomers as a promising treatment strategy for AD. Besides the detailed in vitro characterization of RD2, we also report the results of a treatment study of APP/PS1 mice with RD2. After 28 days of treatment we observed enhancement of cognition and learning behaviour. Analysis on brain plaque load did not reveal significant changes, but a significant reduction of insoluble Aß42. Our findings demonstrate that RD2 was significantly more efficient in Aß oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aß oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aß oligomer elimination by RD2 treatment may be also beneficial for AD patients.

Aß42 pentamers/hexamers are the smallest detectable oligomers in solution.

Amyloid ß (Aß) oligomers may play a decisive role in Alzheimer's disease related neurodegeneration, but their structural properties are poorly understood. In this report, sedimentation velocity centrifugation, small angle neutron scattering (SANS) and molecular modelling were used to identify the small oligomeric species formed by the 42 amino acid residue long isoform of Aß (Aß42) in solution, characterized by a sedimentation coefficient of 2.56 S, and a radius of gyration between 2 and 4 nm. The measured sedimentation coefficient is in close agreement with the sedimentation coefficient calculated for Aß42 hexamers using MD simulations at µM concentration. To the best of our knowledge this is the first report detailing the Aß42 oligomeric species by SANS measurements. Our results demonstrate that the smallest detectable species in solution are penta- to hexamers. No evidences for the presence of dimers, trimers or tetramers were found, although the existence of those Aß42 oligomers at measurable quantities had been reported frequently.

EDTA aggregates induce SYPRO orange-based fluorescence in thermal shift assay.

Ethylenediaminetetraacetic acid (EDTA) is widely used in the life sciences as chelating ligand of metal ions. However, formation of supramolecular EDTA aggregates at pH > 8 has been reported, which may lead to artifactual assay results. When applied as a buffer component at pH ≈ 10 in differential scanning fluorimetry (TSA) using SYPRO Orange as fluorescent dye, we observed a sharp change in fluorescence intensity about 20°C lower than expected for the investigated protein. We hypothesized that this change results from SYPRO Orange/EDTA interactions. TSA experiments in the presence of SYPRO Orange using solutions that contain EDTA-Na+ but no protein were performed. The TSA experiments provide evidence that suggests that at pH > 9, EDTA4- interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal yielding a "denaturation temperature" of ~68°C. Titrating Ca2+ to SYPRO Orange and EDTA solutions quenched fluorescence. Ethylene glycol tetraacetic acid (EGTA) behaved similarly to EDTA. Analytical ultracentrifugation corroborated the formation of EDTA aggregates. Molecular dynamics simulations of free diffusion of EDTA-Na+ and SYPRO Orange of in total 27 µs suggested the first structural model of EDTA aggregates in which U-shaped EDTA4- arrange in an inverse bilayer-like manner, exposing ethylene moieties to the solvent, with which SYPRO Orange interacts. We conclude that EDTA aggregates induce a SYPRO Orange-based fluorescence in TSA. These results make it relevant to ascertain that future TSA results are not influenced by interference between EDTA, or EDTA-related molecules, and the fluorescent dye.