Isothermal titration calorimetry and surface plasmon resonance methods to probe protein-protein interactions.

Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are two commonly used methods to probe biomolecular interactions. ITC can provide information about the binding affinity, stoichiometry, changes in Gibbs free energy, enthalpy, entropy, and heat capacity upon binding. SPR can provide information about the association and dissociation kinetics, binding affinity, and stoichiometry. Both methods can determine the nature of protein-protein interactions and help understand the physicochemical principles underlying complex biochemical pathways and communication networks. This methods article discusses the practical knowledge of how to set up and troubleshoot these two experiments with some examples.

Biophysical evolution of the receptor-binding domains of SARS-CoVs.

With hundreds of coronaviruses (CoVs) identified in bats that can infect humans, it is essential to understand how CoVs that affected the human population have evolved. Seven known CoVs have infected humans, of which three CoVs caused severe disease with high mortalities: severe acute respiratory syndrome (SARS)-CoV emerged in 2002, Middle East respiratory syndrome-CoV in 2012, and SARS-CoV-2 in 2019. SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor-binding domain (RBD) of spike protein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor-binding motif that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that, despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry toward ACE2 compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse receptor-binding motifs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrant further optimization of protein subunit vaccines that use RBD as an antigen by inserting stabilizing mutations or formulation screening.

Convergent Evolution of Multiple Mutations Improves the Viral Fitness of SARS-CoV-2 Variants by Balancing Positive and Negative Selection.

Multiple mutations have been seen to undergo convergent evolution in SARS-CoV-2 variants of concern. One such evolution occurs in Beta, Gamma, and Omicron variants at three amino acid positions K417, E484, and N501 in the receptor binding domain of the spike protein. We examined the physical mechanisms underlying the convergent evolution of three mutations K417T/E484K/N501Y by delineating the individual and collective effects of mutations on binding to angiotensin converting enzyme 2 receptor, immune escape from neutralizing antibodies, protein stability, and expression. Our results show that each mutation serves a distinct function that improves virus fitness supporting its positive selection, even though individual mutations have deleterious effects that make them prone to negative selection. Compared to the wild-type, K417T escapes Class 1 antibodies and has increased stability and expression; however, it has decreased receptor binding. E484K escapes Class 2 antibodies; however, it has decreased receptor binding, stability, and expression. N501Y increases receptor binding; however, it has decreased stability and expression. When these mutations come together, the deleterious effects are mitigated due to the presence of compensatory effects. Triple mutant K417T/E484K/N501Y has increased receptor binding, escapes both Class 1 and Class 2 antibodies, and has similar stability and expression as that of the wild-type. These results show that the convergent evolution of multiple mutations enhances viral fitness on different fronts by balancing both positive and negative selection and improves the chances of selection of mutations together.

Receptor binding, immune escape, and protein stability direct the natural selection of SARS-CoV-2 variants.

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.

2D NMR Analysis of the Effect of Asparagine Deamidation Versus Methionine Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Protein.

Two of the most common forms of chemical modifications that compromise the efficacy of therapeutic proteins are the deamidation of asparagine residues and oxidation of methionine residues. We probed how deamidation affects the structure, stability, aggregation, and function of interferon alpha-2a (IFNA2a), and compared with our earlier results on methionine oxidation. Upon deamidation, no significant changes were observed in the global secondary structure of IFNA2a with minor changes in its tertiary structure. However, deamidation destabilized the protein, and increased its propensity to aggregate under accelerated stress conditions. Cytopathic inhibition and antiproliferation assays showed drastic decrease in the functionality of deamidated IFNA2a compared to the wild-type. 2D NMR measurements showed structural changes in local protein regions, with no effect on the overall global structure of IFNA2a. These local protein regions corresponded well with the aggregation hot-spots predicted by computational programs, and the functional hot-spots identified by site-directed mutagenesis. When compared to the effects of methionine oxidation, deamidation caused lesser aggregation, because of lesser structural unfolding observed in aggregation hot-spots by 2D NMR. In comparison to oxidation, deamidation showed larger decrease in function, because deamidation affected key amino acid residues in functional hot-spots as observed by 2D NMR and structural modeling. Such quantitative comparison between the effects of deamidation and oxidation on a pharmaceutical protein has not been done before, and the high-resolution structural information on local protein regions obtained by 2D NMR provided a better insight compared to low-resolution methods that probe global protein structure.

Effect of Chemical Oxidation on the Higher Order Structure, Stability, Aggregation, and Biological Function of Interferon Alpha-2a: Role of Local Structural Changes Detected by 2D NMR.

PURPOSE: Oxidized interferons have been shown to aggregate and cause immunogenicity. In this study, the structural mechanisms underlying oxidation-induced interferon alpha-2a (IFNA2a) aggregation and loss of function were examined. METHODS: IFNA2a was oxidized using 0.037% vol/vol hydrogen peroxide. Oxidized protein was probed using biophysical methods that include denaturant melts, particle counting, proteolysis-coupled mass spectrometry, and 2D NMR. RESULTS: Oxidized IFNA2a did not show major changes in its secondary structure, but showed minor changes in tertiary structure when compared to the unoxidized protein. In addition, a significant loss of conformational stability was observed upon oxidation. Correspondingly, increased protein aggregation was observed resulting in the formation of sub-visible particles. Oxidized protein showed decreased biological function in terms of its anti-viral potency and cytopathic inhibition efficacy. Proteolysis-coupled mass spectrometry identified five methionine residues that were oxidized with no correlation between the extent of oxidation and their accessible surface area. 2D 15N-1H HSQC NMR identified residue-level local structural changes in the protein upon oxidation, which were not detectable by global probes such as far-UV circular dichroism and fluorescence. CONCLUSIONS: Increased protein aggregation and decreased function of IFNA2a upon oxidation correlated with the site of modification identified by proteolysis-coupled mass spectrometry and local structural changes in the protein detected by 2D NMR.

The N-Terminal Flanking Region Modulates the Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain.

Despite sharing a high degree of sequence similarity, the tandem calponin-homology (CH) domain of utrophin binds to actin 30 times stronger than that of dystrophin. We have previously shown that this difference in actin binding affinity could not be ascribed to the differences in inter-CH-domain linkers [Bandi, S., et al. (2015) Biochemistry 54, 5480-5488]. Here, we examined the role of the N-terminal flanking region. The utrophin tandem CH domain contains a 27-residue flanking region before its CH1 domain. We examined its effect by comparing the structure and function of full-length utrophin tandem CH domain Utr(1-261) and its truncated Utr(28-261) construct. Both full-length and truncated constructs are monomers in solution, with no significant differences in their secondary or tertiary structures. Truncated construct Utr(28-261) binds to actin 30 times weaker than that of the full-length Utr(1-261), similar to that of the dystrophin tandem CH domain with a much shorter flanking region. Deletion of the N-terminal flanking region stabilizes the CH1 domain. The magnitude of the change in binding free energy upon truncation is similar to that of the change in thermodynamic stability. The isolated N-terminal peptide by itself is significantly random coil and does not bind to F-actin in the affinity range of Utr(1-261) and Utr(28-261). These results indicate that the N-terminal flanking region significantly affects the actin binding affinity of tandem CH domains. This observation further stresses that protein regions other than the three actin-binding surfaces identified earlier, irrespective of whether they directly bind to actin, also contribute to the actin binding affinity of tandem CH domains.

Interdomain Linker Determines Primarily the Structural Stability of Dystrophin and Utrophin Tandem Calponin-Homology Domains Rather than Their Actin-Binding Affinity.

Tandem calponin-homology (CH) domains are the most common actin-binding domains in proteins. However, structural principles underlying their function are poorly understood. These tandem domains exist in multiple conformations with varying degrees of inter-CH-domain interactions. Dystrophin and utrophin tandem CH domains share high sequence similarity (∼82%), yet differ in their structural stability and actin-binding affinity. We examined whether the conformational differences between the two tandem CH domains can explain differences in their stability and actin binding. Dystrophin tandem CH domain is more stable by ∼4 kcal/mol than that of utrophin. Individual CH domains of dystrophin and utrophin have identical structures but differ in their relative orientation around the interdomain linker. We swapped the linkers between dystrophin and utrophin tandem CH domains. Dystrophin tandem CH domain with utrophin linker (DUL) has similar stability as that of utrophin tandem CH domain. Utrophin tandem CH domain with dystrophin linker (UDL) has similar stability as that of dystrophin tandem CH domain. Dystrophin tandem CH domain binds to F-actin ∼30 times weaker than that of utrophin. After linker swapping, DUL has twice the binding affinity as that of dystrophin tandem CH domain. Similarly, UDL has half the binding affinity as that of utrophin tandem CH domain. However, changes in binding free energies due to linker swapping are much lower by an order of magnitude compared to the corresponding changes in unfolding free energies. These results indicate that the linker region determines primarily the structural stability of tandem CH domains rather than their actin-binding affinity.

The N- and C-Terminal Domains Differentially Contribute to the Structure and Function of Dystrophin and Utrophin Tandem Calponin-Homology Domains.

Dystrophin and utrophin are two muscle proteins involved in Duchenne/Becker muscular dystrophy. Both proteins use tandem calponin-homology (CH) domains to bind to F-actin. We probed the role of N-terminal CH1 and C-terminal CH2 domains in the structure and function of dystrophin tandem CH domain and compared with our earlier results on utrophin to understand the unifying principles of how tandem CH domains work. Actin cosedimentation assays indicate that the isolated CH2 domain of dystrophin weakly binds to F-actin compared to the full-length tandem CH domain. In contrast, the isolated CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain. Thus, the obvious question is why the dystrophin tandem CH domain requires CH2, when its actin binding is determined primarily by CH1. To answer, we probed the structural stabilities of CH domains. The isolated CH1 domain is very unstable and is prone to serious aggregation. The isolated CH2 domain is very stable, similar to the full-length tandem CH domain. These results indicate that the main role of CH2 is to stabilize the tandem CH domain structure. These conclusions from dystrophin agree with our earlier results on utrophin, indicating that this phenomenon of differential contribution of CH domains to the structure and function of tandem CH domains may be quite general. The N-terminal CH1 domains primarily determine the actin binding function whereas the C-terminal CH2 domains primarily determine the structural stability of tandem CH domains, and the extent of stabilization depends on the strength of inter-CH domain interactions.

The C-terminal domain of the utrophin tandem calponin-homology domain appears to be thermodynamically and kinetically more stable than the full-length protein.

Domains are in general less stable than the corresponding full-length proteins. Human utrophin tandem calponin-homology (CH) domain seems to be an exception. Reversible, equilibrium denaturant melts indicate that the isolated C-terminal domain (CH2) is thermodynamically more stable than the tandem CH domain. Thermal melts show that CH2 unfolds at a temperature higher than that at which the full-length protein unfolds. Stopped-flow kinetics indicates that CH2 unfolds slower than the full-length protein, indicating its higher kinetic stability. Thus, the utrophin tandem CH domain may be one of the few proteins in which an isolated domain is more stable than the corresponding full-length protein.

The actin binding affinity of the utrophin tandem calponin-homology domain is primarily determined by its N-terminal domain.

The structural determinants of the actin binding function of tandem calponin-homology (CH) domains are poorly understood, particularly the role of individual domains. We determined the actin binding affinity of isolated CH domains from human utrophin and compared them with the affinity of the full-length tandem CH domain. Traditional cosedimentation assays indicate that the C-terminal CH2 domain binds to F-actin much weaker than the full-length tandem CH domain. The N-terminal CH1 domain is less stable and undergoes severe protein aggregation; therefore, traditional actin cosedimentation assays could not be used. To address this, we have developed a folding-upon-binding method. We refolded the CH1 domain from its unfolded state in the presence of F-actin. This results in a competition between actin binding and aggregation. A differential centrifugation technique was used to distinguish actin binding from aggregation. Low-speed centrifugation pelleted CH1 aggregates, but not F-actin or its bound protein. Subsequent high-speed centrifugation resulted in the cosedimentation of bound CH1 along with F-actin. The CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain, unlike the CH2 domain. The actin binding cooperativity between the two domains was quantitatively calculated from the association constants of the full-length tandem CH domain and its CH domains, and found to be much smaller than the association constant of the CH1 domain alone. These results indicate that the actin binding affinity of the utrophin tandem CH domain is primarily determined by its CH1 domain, when compared to that of its CH2 domain or the cooperativity between the two CH domains.

High yield soluble bacterial expression and streamlined purification of recombinant human interferon α-2a.

Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is clinically used to modulate hairy cell leukemia as well as hepatitis c. Historically, IFNA2 has been purified from human leukocytes as well as bacterial expression systems. In most cases, bacterial expression of IFNA2 resulted in inclusion body formation, or required numerous purification steps that decreased the protein yield. Here, we describe an expression and purification scheme for IFNA2 using a pET-SUMO bacterial expression system and a single purification step. Using the SUMO protein as the fusion tag achieved high soluble protein expression. The SUMO tag was cleaved with the Ulp1 protease leaving no additional amino acids on the fusion terminus following cleavage. Mass spectrometry, circular dichroism, 2D heteronuclear NMR, and analytical ultracentrifugation confirmed the amino acid sequence identity, secondary and tertiary protein structures, and the solution behavior of the purified IFNA2. The purified protein also had antiviral and anti-proliferative activities comparable to the WHO International Standard, NIBSC 95/650, and the IFNA2 standard available from PBL Assay Science. Combining the expression and purification protocols developed here to produce IFNA2 on a laboratory scale with the commercial fermenter technology commonly used in pharmaceutical industry may further enhance IFNA2 yields, which will promote the development of interferon-based protein drugs to treat various disorders.

Missense mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-beta aggregates.

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations--L54R, A168D, A171P, and Y231N--on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded alpha-helical protein in solution, as is evident from its alpha-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR (15)N-(1)H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal (T(1)) and transverse (T(2)) relaxation experiments. Compared to WT, three mutants--L54R, A168D, and A171P--show a decreased alpha-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a "molten globule" state. In contrast, Y231N has an alpha-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-beta structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.

The N-terminal actin-binding tandem calponin-homology (CH) domain of dystrophin is in a closed conformation in solution and when bound to F-actin.

Deficiency of the vital muscle protein dystrophin triggers Duchenne/Becker muscular dystrophy, but the structure-function relationship of dystrophin is poorly understood. To date, molecular structures of three dystrophin domains have been determined, of which the N-terminal actin-binding domain (N-ABD or ABD1) is of particular interest. This domain is composed of two calponin-homology (CH) domains, which form an important class of ABDs in muscle proteins. A previously determined x-ray structure indicates that the dystrophin N-ABD is a domain-swapped dimer, with each monomer adopting an extended, open conformation in which the two CH domains do not interact. This structure is controversial because it contradicts functional studies and known structures of similar ABDs from other muscle proteins. Here, we investigated the solution conformation of the dystrophin N-ABD using a very simple and elegant technique of pyrene excimer fluorescence. Using the wild-type protein, which contains two cysteines, and the corresponding single-cysteine mutants, we show that the protein is a monomer in solution and is in a closed conformation in which the two CH domains seem to interact, as observed from the excimer fluorescence of pyrene-labeled wild-type protein. Excimer fluorescence was also observed in its actin-bound form, indicating that the dystrophin N-ABD binds to F-actin in a closed conformation. Comparison of the dystrophin N-ABD conformation with other ABDs indicates that the tandem CH domains in general may be monomeric in solution and predominantly occur in closed conformation, whereas their actin-bound conformations may differ.

Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin-binding domains of utrophin and dystrophin.

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne, and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-ß aggregates, indicating a possible role for utrophin mutations in disease mechanisms.

Biophysical Fitness Landscape of the SARS-CoV-2 Delta Variant Receptor Binding Domain.

Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased mortality among infected people. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single as well as the double mutations on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than increased receptor binding, is the main biophysical parameter that determined the fitness landscape of the Delta variant RBD.

FAD-deficient P187S mutation of NAD(P)H:quinone oxidoreductase 1 (NQO1*2) binds and accelerates ß-amyloid aggregation.

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-ß peptide (Aß1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aß1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aß1-42 peptide, the aggregation kinetics of Aß1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aß1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aß1-42. Aß1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aß1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aß1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aß1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.

Antibody-Dependent Complement Responses toward SARS-CoV-2 Receptor-Binding Domain Immobilized on "Pseudovirus-like" Nanoparticles.

Many aspects of innate immune responses to SARS viruses remain unclear. Of particular interest is the role of emerging neutralizing antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 in complement activation and opsonization. To overcome challenges with purified virions, here we introduce "pseudovirus-like" nanoparticles with ∼70 copies of functional recombinant RBD to map complement responses. Nanoparticles fix complement in an RBD-dependent manner in sera of all vaccinated, convalescent, and naïve donors, but vaccinated and convalescent donors with the highest levels of anti-RBD antibodies show significantly higher IgG binding and higher deposition of the third complement protein (C3). The opsonization via anti-RBD antibodies is not an efficient process: on average, each bound antibody promotes binding of less than one C3 molecule. C3 deposition is exclusively through the alternative pathway. C3 molecules bind to protein deposits, but not IgG, on the nanoparticle surface. Lastly, "pseudovirus-like" nanoparticles promote complement-dependent uptake by granulocytes and monocytes in the blood of vaccinated donors with high anti-RBD titers. Using nanoparticles displaying SARS-CoV-2 proteins, we demonstrate subject-dependent differences in complement opsonization and immune recognition.

Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors.

Adeno-associated virus (AAV) has emerged as a leading platform for gene delivery for treating various diseases due to its excellent safety profile and efficient transduction to various target tissues. However, the large-scale production and long-term storage of viral vectors is not efficient resulting in lower yields, moderate purity, and shorter shelf-life compared to recombinant protein therapeutics. This review provides a comprehensive analysis of upstream, downstream and formulation unit operation challenges encountered during AAV vector manufacturing, and discusses how desired product quality attributes can be maintained throughout product shelf-life by understanding the degradation mechanisms and formulation strategies. The mechanisms of various physical and chemical instabilities that the viral vector may encounter during its production and shelf-life because of various stressed conditions such as thermal, shear, freeze-thaw, and light exposure are highlighted. The role of buffer, pH, excipients, and impurities on the stability of viral vectors is also discussed. As such, the aim of this review is to outline the tools and a potential roadmap for improving the quality of AAV-based drug products by stressing the need for a mechanistic understanding of the involved processes.

Role of partial protein unfolding in alcohol-induced protein aggregation.

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as nondisease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins.