Modulation of biophysical properties of nucleocapsid protein in the mutant spectrum of SARS-CoV-2.

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

Like other types of RNA viruses, the genetic material of SARS-CoV-2 (the agent responsible for COVID-19) is formed of an RNA molecule which is prone to accumulating mutations. This gives SARS-CoV-2 the ability to evolve quickly, and often to remain one step ahead of treatments. Understanding how these mutations shape the behavior of RNA viruses is therefore crucial to keep diseases such as COVID-19 under control. The gene that codes for the protein that 'packages' the genetic information inside SARS-CoV-2 is particularly prone to mutations. This nucleocapsid (N) protein participates in many key processes during the life cycle of the virus, including potentially interfering with the immune response. Exactly how the physical properties of the N-Protein are impacted by the mutations in its genetic sequence remains unclear. To investigate this question, Nguyen et al. predicted the various biophysical properties of different regions of the N-protein based on a computer-based analysis of SARS-CoV-2 genetic databases. This allowed them to determine if specific protein regions were positively or negatively charged in different mutants. The analyses showed that some domains exhibited great variability in their charge between protein variants ­ reflecting the fact that the corresponding genetic sequences showed high levels of plasticity. Other regions remained conserved, however, including across related coronaviruses. Nguyen et al. also conducted biochemical experiments on a range of N-proteins obtained from clinically relevant SARS-CoV-2 variants. Their results highlighted the importance of protein segments with no fixed three-dimensional structure. Mutations in the related sequences created high levels of variation in the physical properties of these 'intrinsically disordered' regions, which had wide-ranging consequences. Some of these genetic changes even gave individual N-proteins the ability to interact with each other in a completely new way. These results shed new light on the relationship between genetic mutations and the variable physical properties of RNA virus proteins. Nguyen et al. hope that this knowledge will eventually help to develop more effective treatments for viral infections.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2.

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also exhibiting functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38±10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Size Exclusion Chromatography-Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization.

Over the past decade, adeno-associated viruses (AAVs) have attained significant prominence in gene therapy and genome editing applications, necessitating the development of robust and precise methodologies to ensure the quality and purity of AAV products. Existing AAV characterization techniques have proven effective for the analysis of pure and homogeneous AAV samples. However, there is still a demand for a rapid and low-sample-consumption method suitable for the characterization of lower purity or heterogeneous AAV samples commonly encountered in AAV products. Addressing this challenge, we propose the SEC-MP method, which combines size exclusion chromatography (SEC) with mass photometry (MP). In this novel approach, SEC effectively separates monomeric AAV particles from impurities, while the UV detector determines the virus particle concentration. MP complements this process by estimating the fraction of fully packaged AAVs in the total population of AAV particles. This combined methodology enables accurate determination of the titer of effective, fully packaged AAVs in samples containing aggregates, incorrectly packaged AAVs with incomplete genomes, protein or DNA fragments, and other impurities. Our experimental results demonstrate that SEC-MP provides valuable guidance for sample quality control and subsequent applications in the field of AAV research.

Mass photometry: A powerful tool for carbohydrates-proteins conjugation monitoring and glycoconjugates molecular mass determination.

Glycoconjugate vaccines are important additions to the existing means for prevention of diseases caused by bacterial and viral pathogens. Conjugating carbohydrates to proteins is a crucial step in the development of these vaccines. Traditional mass spectrometry techniques, such as MALDI-TOF and SELDI-TOF, have difficulties in detecting glycoconjugates with high molecular masses. Mass photometry (MP) is a single-molecule technique that has been recently developed, which allows mass measurements of individual molecules and generates mass distributions based on hundreds to thousands of these measurements. In this study, we evaluated the performance of MP in monitoring carbohydrate-protein conjugation reactions and characterization of conjugates. Three different glycoconjugates were prepared from carrier protein BSA, and one from a large protein complex, a virus capsid with 3.74 MDa molecular mass. The masses measured by MP were consistent with those obtained by SELDI-TOF-MS and SEC-MALS. The conjugation of BSA dimer to carbohydrate antigen was also successfully characterized. This study shows that the MP technique is a promising alternative to methods developed earlier for monitoring glycoconjugation reactions and characterization of glycoconjugates. It measures intact molecules in solution and it is highly accurate over a wide mass range. MP requires only a very small amount of sample and has no specific buffer constraints. Other MP advantages include minimal cost of consumables and rapid data collection and analysis. Its advantages over other methods make it a valuable tool for researchers in the glycoconjugation field.

A conserved oligomerization domain in the disordered linker of coronavirus nucleocapsid proteins.

The nucleocapsid (N-)protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a key role in viral assembly and scaffolding of the viral RNA. It promotes liquid-liquid phase separation (LLPS), forming dense droplets that support the assembly of ribonucleoprotein particles with as-of-yet unknown macromolecular architecture. Combining biophysical experiments, molecular dynamics simulations, and analysis of the mutational landscape, we describe a heretofore unknown oligomerization site that contributes to LLPS, is required for the assembly of higher-order protein-nucleic acid complexes, and is coupled to large-scale conformational changes of N-protein upon nucleic acid binding. The self-association interface is located in a leucine-rich sequence of the intrinsically disordered linker between N-protein folded domains and formed by transient helices assembling into trimeric coiled-coils. Critical residues stabilizing hydrophobic and electrostatic interactions between adjacent helices are highly protected against mutations in viable SARS-CoV-2 genomes, and the oligomerization motif is conserved across related coronaviruses, thus presenting a target for antiviral therapeutics.

Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD.

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV-2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

The dimeric form of bacterial l-asparaginase YpAI is fully active.

l-asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity. Using multiple biophysical techniques that investigate enzymatic properties and quaternary structure at either high or low protein concentration, we found that dimeric YpAI is fully active, suggesting that the tetrameric form of this subfamily of enzymes does not bear significant enzymatic relevance. In this process, we extensively characterized YpAI, showing that it is a cooperative enzyme, although the mechanism of allostery is still not definitely established. We showed that, like most type I ASNases, the substrate affinity of YpAI is low and this enzyme is very similar in terms of both the structure and enzymatic properties to homologous type I ASNase from Escherichia coli (EcAI). We extended these studies to more medically relevant type II ASNases, used as anti-leukaemia drugs. We confirmed that type II ASNases are not allosteric, and that they might also be functional in a dimeric form. However, the determination of the accurate tetramer⇆dimer dissociation constants of these enzymes that most likely lie in the picomolar range is not possible with currently available biophysical techniques.

A New Structural Model of Apolipoprotein B100 Based on Computational Modeling and Cross Linking.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.

Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein.

Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by three to four different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced coassembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.

Soluble angiotensin-converting enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses using ACE2 as their receptor. Using structure-guided approaches, we developed a series of bivalent ACE2-Fcs harboring functionally and structurally validated mutations that enhance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain recognition by up to ~12-fold and remove angiotensin enzymatic activity. The lead variant M81 potently cross-neutralized SARS-CoV-2 variants of concern (VOCs), including Omicron, at subnanomolar half-maximal inhibitory concentration and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis, and complement deposition. When tested in a stringent K18-hACE2 mouse model, Fc-enhanced ACE2-Fc delayed death by 3 to 5 days or effectively resolved lethal SARS-CoV-2 infection in both prophylactic and therapeutic settings via the combined effects of neutralization and Fc-effector functions. These data add to the demonstrated utility of soluble ACE2 as a valuable SARS-CoV-2 antiviral and indicate that Fc-effector functions may constitute an important component of ACE2-Fc therapeutic activity.

Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein.

Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by 3-4 different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced co-assembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.

Virus-like Particle Display of Vibrio choleraeO-Specific Polysaccharide as a Potential Vaccine against Cholera.

Vibrio cholerae, a noninvasive mucosal pathogen, is endemic in more than 50 countries. Oral cholera vaccines, based on killed whole-cell strains of Vibrio cholerae, can provide significant protection in adults and children for 2-5 years. However, they have relatively limited direct protection in young children. To overcome current challenges, in this study, a potential conjugate vaccine was developed by linking O-specific polysaccharide (OSP) antigen purified from V. cholerae O1 El Tor Inaba strain PIC018 with Qß virus-like particles efficiently via squarate chemistry. The Qß-OSP conjugate was characterized with mass photometry (MP) on the whole particle level. Pertinent immunologic display of OSP was confirmed by immunoreactivity of the conjugate with convalescent phase samples from humans with cholera. Mouse immunization with the Qß-OSP conjugate showed that the construct generated prominent and long-lasting IgG antibody responses against OSP, and the resulting antibodies could recognize the native lipopolysaccharide from Vibrio cholerae O1 Inaba. This was the first time that Qß was conjugated with a bacterial polysaccharide for vaccine development, broadening the scope of this powerful carrier.

Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry.

Recombinant adeno-associated viruses (rAAV) are used extensively as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing; however, better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of rAAV preparations. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or require a large amount of time and material. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high-resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations but also identify partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Sub-picomole quantities of sample are sufficient for MP analysis, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor the physical attributes of rAAV vectors.

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.

Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses utilizing ACE2 as their receptor. Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbor structurally validated mutations that enhance spike (S) binding and remove angiotensin enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC 50 and was capable of robust Fc-effector functions, including antibody-dependent-cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, it delayed death or effectively resolved lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids.

Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here, we use biophysical tools to study N-protein interactions with oligonucleotides of different lengths, examining the size, composition, secondary structure, and energetics of the resulting states. We observe the formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant change in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within macromolecular condensates.

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.