Thermodynamic and molecular dynamic insights into how fusion influences peptide-tag recognition of an antibody.

To understand the effect of protein fusion on the recognition of a peptide-tag by an antibody, we fused a CCR5-derived peptide-tag (pep1) to GFP and investigated its recognition by an anti-pep1 antibody, 4B08. First, to characterize the thermodynamic properties associated with the pep1-4B08 binding, isothermal titration calorimetry experiments were conducted. It was found that pep1 fused to the C-terminus of GFP (GFP-CT) enhanced the enthalpic gain by 2.1 kcal mol-1 and the entropic loss only by 0.9 kcal mol-1, resulting in an 8-fold increase in the binding affinity compared to the unfused pep1. On the other hand, pep1 fused to the N-terminus of GFP (GFP-NT) enhanced the enthalpic gain by 3.0 kcal mol-1 and the entropic loss by 3.2 kcal mol-1, leading to no significant enhancement of the binding affinity. To gain deeper insights, molecular dynamics simulations of GFP-NT, GFP-CT, and pep1 were performed. The results showed that the location of the fusion point sensitively affects the interaction energy, the solvent accessible surface area, and the fluctuation of pep1 in the unbound state, which explains the difference in the experimental thermodynamic properties.

Functional insights of Tyr37 in framework region 2 directly contributing to the binding affinities and dissociation kinetics in single-domain VHH antibodies.

Single-domain VHH antibody is regarded as one of the promising antibody classes for therapeutic and diagnostic applications. VHH antibodies have amino acids in framework region 2 that are distinct from those in conventional antibodies, such as the Val37Phe/Tyr (V37F/Y) substitution. Correlations between the residue type at position 37 and the conformation of the CDR3 in VHH antigen recognition have been previously reported. However, few studies focused on the meaning of harboring two residue types in position 37 of VHH antibodies, and the concrete roles of Y37 have been little to be elucidated. Here, we investigated the functional states of position 37 in co-crystal structures and performed analyses of three model antibodies with either F or Y at position 37. Our analysis indicates that Y at position 37 enhances the dissociation rate, which is highly correlated with drug efficacy. Our findings help to explain the molecular mechanisms that distinguish VHH antibodies from conventional antibodies.

Next-Generation Anti-TNFα Agents: The Example of Ozoralizumab.

Biologic therapy involving anti-tumor necrosis factor-α (anti-TNFα) agents has fundamentally changed the management of patients with immune-mediated inflammatory diseases, including rheumatoid arthritis, thus benefiting many patients. Nevertheless, the inability of some patients to achieve low disease activity or clinical remission remains a major concern. To address such concerns, next-generation anti-TNFα agents that differ from the immunoglobulin G-format anti-TNFα agents that have been used to date are being developed using antibody-engineering technology. Their unique design employing novel molecular characteristics affords several advantages, such as early improvement of clinical symptoms, optimization of drug bioavailability, enhancement of tissue penetration, and a reduction in side effects. This holds promise for a new paradigm shift in biologic therapy via the use of next-generation anti-TNFα agents. Ozoralizumab, a next-generation anti-TNFα agent that was recently approved in Japan, comprises a variable region heavy-chain format. It has a completely different structure from conventional therapeutic antibodies, such as a small molecular size, an albumin-binding module, and a unique format that produces an avidity effect. Ozoralizumab exhibited rapid biodistribution into joints, provided attenuation of Fcγ receptor-mediated inflammatory responses, and had a high binding affinity to TNFα in non-clinical studies. In clinical trials, ozoralizumab yielded an early improvement in clinical symptoms, a sustained efficacy for up to 52 weeks, and an acceptable tolerability in patients with rheumatoid arthritis. This review focuses on the results of pre-clinical and clinical trials for ozoralizumab and outlines the progress in next-generation antibody development.

THOUSAND-GRAIN WEIGHT 6, which is an IAA-glucose hydrolase, preferentially recognizes the structure of the indole ring.

An indole-3-acetic acid (IAA)-glucose hydrolase, THOUSAND-GRAIN WEIGHT 6 (TGW6), negatively regulates the grain weight in rice. TGW6 has been used as a target for breeding increased rice yield. Moreover, the activity of TGW6 has been thought to involve auxin homeostasis, yet the details of this putative TGW6 activity remain unclear. Here, we show the three-dimensional structure and substrate preference of TGW6 using X-ray crystallography, thermal shift assays and fluorine nuclear magnetic resonance (19F NMR). The crystal structure of TGW6 was determined at 2.6 Å resolution and exhibited a six-bladed ß-propeller structure. Thermal shift assays revealed that TGW6 preferably interacted with indole compounds among the tested substrates, enzyme products and their analogs. Further analysis using 19F NMR with 1,134 fluorinated fragments emphasized the importance of indole fragments in recognition by TGW6. Finally, docking simulation analyses of the substrate and related fragments in the presence of TGW6 supported the interaction specificity for indole compounds. Herein, we describe the structure and substrate preference of TGW6 for interacting with indole fragments during substrate recognition. Uncovering the molecular details of TGW6 activity will stimulate the use of this enzyme for increasing crop yields and contributes to functional studies of IAA glycoconjugate hydrolases in auxin homeostasis.

Structural basis for the recognition of human hemoglobin by the heme-acquisition protein Shr from Streptococcus pyogenes.

In Gram-positive bacteria, sophisticated machineries to acquire the heme group of hemoglobin (Hb) have evolved to extract the precious iron atom contained in it. In the human pathogen Streptococcus pyogenes, the Shr protein is a key component of this machinery. Herein we present the crystal structure of hemoglobin-interacting domain 2 (HID2) of Shr bound to Hb. HID2 interacts with both, the protein and heme portions of Hb, explaining the specificity of HID2 for the heme-bound form of Hb, but not its heme-depleted form. Further mutational analysis shows little tolerance of HID2 to interfacial mutations, suggesting that its interaction surface with Hb could be a suitable candidate to develop efficient inhibitors abrogating the binding of Shr to Hb.

Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies.

Experimental modification in thermal stability of oligomers by alanine substitution and site saturation mutagenesis of interfacial residues.

For certain industrial applications, the stability of protein oligomers is important. In this study, we demonstrated an efficient method to improve the thermal stability of oligomers using the trimeric protein chloramphenicol acetyltransferase (CAT) as the model. We substituted all interfacial residues of CAT with alanine to detect residues critical for oligomer stability. Mutation of six of the forty-nine interfacial residues enhanced oligomer thermal stability. Site saturation mutagenesis was performed on these six residues to optimize the side chains. About 15% of mutations enhanced thermal stability by more than 0.5 °C and most did not disrupt activity of CAT. Certain combinations of mutations further improved thermal stability and resistance against heat treatment. The quadruple mutant, H17V/N34S/F134A/D157C, retained the same activity as the wild-type after heat treatment at 9 °C higher temperature than the wild-type CAT. Furthermore, combinations with only alanine substitutions also improved thermal stability, suggesting the method we developed can be used for rapid modification of industrially important proteins.

PRELP secreted from mural cells protects the function of blood brain barrier through regulation of endothelial cell-cell integrity.

Introduction: Proline/arginine-rich end leucine-rich repeat protein (PRELP), is a small secreted proteoglycan expressed by pericytes and vascular smooth muscle cells surrounding the brain vasculature of adult mouse. Methods: We utilised a Prelp knockout (Prelp -/-) mouse model to interrogate vasculature integrity in the brain alongside performing in vitro assays to characterise PRELP application to endothelial cells lines. Our findings were supplemented with RNA expression profiling to elucidate the mechanism of how PRELP maintains neurovasculature function. Results: Prelp -/- mice presented with neuroinflammation and reducedneurovasculature integrity, resulting in IgG and dextran leakage in the cerebellum and cortex. Histological analysis of Prelp -/- mice revealed reducedcell-cell integrity of the blood brain barrier, capillary attachment of pericytes andastrocyte end-feet. RNA-sequencing analysis found that cell-cell adhesion andinflammation are affected in Prelp -/- mice and gene ontology analysis as well as gene set enrichment analysis demonstrated that inflammation related processes and adhesion related processes such as epithelial-mesenchymal transition and apical junctions were significantly affected, suggesting PRELP is a regulator of cell-cell adhesion. Immunofluorescence analysis showed that adhesion junction protein expression levels of cadherin, claudin-5, and ZO-1, was suppressed in Prelp -/- mice neurovasculature. Additionally, in vitro studies revealed that PRELP application to endothelial cells enhances cell-cell integrity, induces mesenchymal-endothelial transition and inhibits TGF-ß mediated damage to cell-cell adhesion. Discussion: Our study indicates that PRELP is a novel endogenous secreted regulator of neurovasculature integrity and that PRELP application may be a potential treatment for diseases associated with neurovascular damage.

Conformational features and interaction mechanisms of VH H antibodies with ß-hairpin CDR3: A case of Nb8-HigB2 interaction.

The ß-hairpin conformation is regarded as an important basic motif to form and regulate protein-protein interactions. Single-domain VH H antibodies are potential therapeutic and diagnostic tools, and the third complementarity-determining regions of the heavy chains (CDR3s) of these antibodies are critical for antigen recognition. Although the sequences and conformations of the CDR3s are diverse, CDR3s sometimes adopt ß-hairpin conformations. However, characteristic features and interaction mechanisms of ß-hairpin CDR3s remain to be fully elucidated. In this study, we investigated the molecular recognition of the anti-HigB2 VH H antibody Nb8, which has a CDR3 that forms a ß-hairpin conformation. The interaction was analyzed by evaluation of alanine-scanning mutants, molecular dynamics simulations, and hydrogen/deuterium exchange mass spectrometry. These experiments demonstrated that positions 93 and 94 (Chothia numbering) in framework region 3, which is right outside CDR3 by definition, play pivotal roles in maintaining structural stability and binding properties of Nb8. These findings will facilitate the design and optimization of single-domain antibodies.

High-throughput analysis system of interaction kinetics for data-driven antibody design.

Surface plasmon resonance (SPR) is widely used for antigen-antibody interaction kinetics analysis. However, it has not been used in the screening phase because of the low throughput of measurement and analysis. Herein, we proposed a high-throughput SPR analysis system named "BreviA" using the Brevibacillus expression system. Brevibacillus was transformed using a plasmid library containing various antibody sequences, and single colonies were cultured in 96-well plates. Sequence analysis was performed using bacterial cells, and recombinant antibodies secreted in the supernatant were immobilized on a sensor chip to analyze their interactions with antigens using high-throughput SPR. Using this system, the process from the transformation to 384 interaction analyses can be performed within a week. This system utility was tested using an interspecies specificity design of an anti-human programmed cell death protein 1 (PD-1) antibody. A plasmid library containing alanine and tyrosine mutants of all complementarity-determining region residues was generated. A high-throughput SPR analysis was performed against human and mouse PD-1, showing that the mutation in the specific region enhanced the affinity for mouse PD-1. Furthermore, deep mutational scanning of the region revealed two mutants with > 100-fold increased affinity for mouse PD-1, demonstrating the potential efficacy of antibody design using data-driven approach.

Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coated allergen nanoparticles.

Allergen immunotherapy (AIT) is the only curative treatment for allergic diseases. However, AIT has many disadvantages related to efficiency, safety, long-term duration, and patient compliance. Dendritic cells (DCs) have an important role in antigen-specific tolerance induction; thus, DC-targeting strategies to treat allergies such as glutaraldehyde crosslinked antigen to mannoprotein (MAN) have been established. However, glutaraldehyde crosslinking may reduce the antigen presentation efficiency of DCs. To overcome this, we developed a MAN-coated ovalbumin (OVA) nanoparticle (MDO), which uses intermolecular disulfide bond to crosslink OVA and MAN. MDO effectively targeted DCs resulting in tolerogenic DCs, and promoted higher antigen presentation efficiency by DCs compared with OVA or glutaraldehyde crosslinked nanoparticles. In vitro and in vivo experiments showed that DCs exposed to MDO induced Treg cells. Moreover, MDO had low reactivity with anti-OVA antibodies and did not induce anaphylaxis in allergic mice, demonstrating its high safety profile. In a mouse model of allergic asthma, MDO had significant preventative and therapeutic effects when administered orally or subcutaneously. Therefore, MDO represents a promising new approach for the efficient and safe treatment of allergies.

Enhancing thermal stability in the CH2 domain to suppress aggregation through the introduction of simultaneous disulfide bonds in Pichia pastoris.

Protein aggregations decrease production yields and impair the efficacy of therapeutics. The CH2 domain is a crucial part of the constant region of human IgG. But, it is also the least stable domain in IgG, which can result in antibody instability and aggregation problems. We created a novel mutant of the CH2 domain (T250C/L314C, mut10) by introducing a disulfide bond and expressed it using Pichia pastoris. The mut10 variant exhibited enhanced thermal stability, resistance to enzymatic degradation, and reduced aggregation in comparison to the original CH2 domain. However, it was less stable than mut20 (L242C/K334C), which is the variant prepared in a previous study (Gong et al., J. Biol. Chem., 2009). A more advanced mutant, mut25, was created by combining mut10 and mut20. Mut25 artificially contains two disulfide bonds. The new mutant, mut25, showed enhanced thermal stability, increased resistance to enzymatic digestion, and reduced aggregation in comparison to mut20. According to our knowledge, mut25 achieves an unprecedented level of stability among the humanized whole CH2 domains that have been reported so far. Mut25 has the potential to serve as a new platform for antibody therapeutics due to its ability to reduce immunogenicity by decreasing aggregation.

Specific peptide conjugation to a therapeutic antibody leads to enhanced therapeutic potency and thermal stability by reduced Fc dynamics.

Antibody-drug conjugates are powerful tools for combatting a wide array of cancers. Drug conjugation to a therapeutic antibody often alters molecular characteristics, such as hydrophobicity and effector function, resulting in quality deterioration. To develop a drug conjugation methodology that maintains the molecular characteristics of the antibody, we engineered a specific peptide for conjugation to the Fc region. We used trastuzumab and the chelator (DOTA) as model antibody and payload, respectively. Interestingly, peptide/DOTA-conjugated trastuzumab exhibited enhanced antibody-dependent cellular cytotoxicity (ADCC) and increased thermal stability. Detailed structural and thermodynamic analysis clarified that the conjugated peptide blocks the Fc dynamics like a "wedge." We revealed that (1) decreased molecular entropy results in enhanced ADCC, and (2) blockade of Fc denaturation results in increased thermal stability. Thus, we believe that our methodology is superior not only for drug conjugation but also as for reinforcing therapeutic antibodies to enhance ADCC and thermal stability.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex.

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes.

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VHHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis. Furthermore, we determined that the VHHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

Quantitative analysis of antibody aggregates by combination of pinched-flow fractionation and coulter method.

For the pharmaceutical development of proteins, multiple methods of analysis are recommended for evaluating aggregation, and the development of more quantitative and simpler analytical techniques for subvisible particles is expected. This study introduces the Pinched-Flow Fractionation (PFF)-Coulter method, which combines the Pinched-flow fractionation (PFF) and Coulter methods to analyze the concentration of submicron-sized particles. The PFF method separates the particles by size. Separated particles were individually detected using the Coulter method. We have utilized the PFF-Coulter method to quantitatively analyze particle concentrations using standard particles, evaluate detection limits, variability, and correlation between theoretical and measured values, and analyze mixtures of different particle sizes. The PFF-Coulter method allows for quantitatively analyzing of particle sizes from 0.2 to 2.0 µm. The quantifiable weight concentration range was 2.5 × 10-2 - 50 µg/mL, and the number concentration range was 104-1010 particles/mL. The sample volume was small (<10 µL). The PFF-Coulter method is capable of quantitative analysis that complements data from conventional measurement techniques, and when used in conjunction with existing submicron-size particle analysis techniques, will enable more accurate particle analysis.

Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5'-phosphate (PLP) via Schiff base formation.

D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5'-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 µM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine ß-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.

Megalin is involved in angiotensinogen-induced, angiotensin II-mediated ERK1/2 signaling to activate Na + -H + exchanger 3 in proximal tubules.

BACKGROUND: Kidney angiotensin (Ang) II is produced mainly from liver-derived, glomerular-filtered angiotensinogen (AGT). Podocyte injury has been reported to increase the kidney Ang II content and induce Na + retention depending on the function of megalin, a proximal tubular endocytosis receptor. However, how megalin regulates the renal content and action of Ang II remains elusive. METHODS: We used a mass spectrometry-based, parallel reaction-monitoring assay to quantitate Ang II in plasma, urine, and kidney homogenate of kidney-specific conditional megalin knockout (MegKO) and control (Ctl) mice. We also evaluated the pathophysiological changes in both mouse genotypes under the basal condition and under the condition of increased glomerular filtration of AGT induced by administration of recombinant mouse AGT (rec-mAGT). RESULTS: Under the basal condition, plasma and kidney Ang II levels were comparable in the two mouse groups. Ang II was detected abundantly in fresh spot urine in conditional MegKO mice. Megalin was also found to mediate the uptake of intravenously administered fluorescent Ang II by PTECs. Administration of rec-mAGT increased kidney Ang II, exerted renal extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, activated proximal tubular Na + -H + exchanger 3 (NHE3), and decreased urinary Na + excretion in Ctl mice, whereas these changes were suppressed but urinary Ang II was increased in conditional MegKO mice. CONCLUSION: Increased glomerular filtration of AGT is likely to augment Ang II production in the proximal tubular lumen. Thus, megalin-dependent Ang II uptake should be involved in the ERK1/2 signaling that activates proximal tubular NHE3 in vivo , thereby causing Na + retention.

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling activity through epitope selection.

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

Ferguson plot analysis of multiple intermediate species of thermally unfolded bovine serum albumin.

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of ∼68 °C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 °C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 °C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded.