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In recent years, there has been a growing focus on the development of medium-sized drugs based on peptides or nucleic acids owing to their potential therapeutic benefits. As some of these medium-sized drugs exert their therapeutic effects by adopting specific secondary structures, evaluating their conformational states is crucial to ensure the efficacy, quality, and safety of the drug products. It is important to assess the structural integrity of biomolecular therapeutics to guarantee their intended pharmacological activity and maintain the required standards for drug development and manufacturing. One widely utilized technique for quality evaluation is secondary structural analysis using circular dichroism (CD) spectroscopy. Given the higher production and quality control costs associated with medium-sized drugs compared with small-molecule drugs, developing analytical techniques that enable CD analysis with reduced sample volumes is highly desirable. Herein, we focused on a microsampling disk-type cell as a potential solution for reducing the required sample volume. We investigated whether CD spectral analysis using a microsampling disk could provide equivalent spectra compared with the standard cell (sample volume: approx. 300 µL). Our findings demonstrated that the microsampling disk (sample volume: 2-10 µL) could be successfully applied to CD spectral analysis of peptide and nucleic acid drugs, paving the way for more efficient and cost-effective quality evaluation processes.

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Bael (Aegle marmelos) beverage was pasteurized using continuous-microwave (MW) and traditional thermal processing and the activity of native enzymes, pulp-hydrolyzing enzymes, bioactive, physicochemical, and sensory properties were analyzed. First-order and linear biphasic models fitted well (R2 ≥ 0.90) for enzyme inactivation and bioactive alteration kinetics, respectively. For the most resistant enzyme, polyphenoloxidase (PPO), the inactivation target of ≥ 90 % was achieved at 90 °C TMW (final temperature under MW) and 95 °C for 5 min (conventional thermal). MW treatment displayed faster enzyme inactivation and better retention of TPC and AOC. MW treatment at 90 °C TMW showed 5.3 min D-value, 90% total carotenoid content, 3.42 crisp sensory score (out of 5), and no or minor change in physicochemical attributes. Thermal and MW treatment caused the loss of 14 and 10 bioactive compounds, respectively. The secondary and tertiary structural modifications of PPO enzyme-protein revealed MW's lethality primarily due to its thermal effects.

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Characterization and understanding of protein higher order structure (HOS) is essential at all stages of biologics development. Here, two folding variants of a bispecific monoclonal antibody, the correctly folded form and an alternative configuration with reduced potency, were characterized by several HOS characterization techniques. Specifically, differential scanning calorimetry (DSC), circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), Raman and Raman optical activity (ROA) spectroscopy were used together to elucidate the impacts of disulfide bond scrambling in the fused scFv domains on the structure and thermal stability of the antibody. This study illustrates the importance of selecting appropriate biophysical characterization techniques based on the nature and magnitude of the HOS change.

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Eukaryotic proteins often feature long stretches of amino acids that lack a well-defined three-dimensional structure and are referred to as intrinsically disordered proteins (IDPs) or regions (IDRs). Although these proteins challenge conventional structure-function paradigms, they play vital roles in cellular processes. Recent progress in experimental techniques, such as NMR spectroscopy, single molecule FRET, high speed AFM and SAXS, have provided valuable insights into the biophysical basis of IDP function. This review discusses the advancements made in these techniques particularly for the study of disordered regions in proteins. In NMR spectroscopy new strategies such as 13C detection, non-uniform sampling, segmental isotope labeling, and rapid data acquisition methods address the challenges posed by spectral overcrowding and low stability of IDPs. The importance of various NMR parameters, including chemical shifts, hydrogen exchange rates, and relaxation measurements, to reveal transient secondary structures within IDRs and IDPs are presented. Given the high flexibility of IDPs, the review outlines NMR methods for assessing their dynamics at both fast (ps-ns) and slow (µs-ms) timescales. IDPs exert their functions through interactions with other molecules such as proteins, DNA, or RNA. NMR-based titration experiments yield insights into the thermodynamics and kinetics of these interactions. Detailed study of IDPs requires multiple experimental techniques, and thus, several methods are described for studying disordered proteins, highlighting their respective advantages and limitations. The potential for integrating these complementary techniques, each offering unique perspectives, is explored to achieve a comprehensive understanding of IDPs.

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Lung surfactant is a mixture of lipids and proteins and is essential for air breathing in mammals. The hydrophobic surfactant proteins B and C (SP-B and SP-C) assist in reducing surface tension in the lung alveoli by organizing the surfactant lipids. SP-B deficiency is life-threatening, and a lack of SP-C can lead to progressive interstitial lung disease. B-YL (41 amino acids) is a highly surface-active, sulfur-free peptide mimic of SP-B (79 amino acids) in which the four cysteine residues are replaced by tyrosine. Mammalian SP-C (35 amino acids) contains two cysteine-linked palmitoyl groups at positions 5 and 6 in the N-terminal region that override the ß-sheet propensities of the native sequence. Canine SP-C (34 amino acids) is exceptional because it has only one palmitoylated cysteine residue at position 4 and a phenylalanine at position 5. We developed canine SP-C constructs in which the palmitoylated cysteine residue at position 4 is replaced by phenylalanine (SP-Cff) or serine (SP-Csf) and a glutamic acid-lysine ion-lock was placed at sequence positions 20-24 of the hydrophobic helical domain to enhance its alpha helical propensity. AI modeling, molecular dynamics, circular dichroism spectroscopy, Fourier Transform InfraRed spectroscopy, and electron spin resonance studies showed that the secondary structure of canine SP-Cff ion-lock peptide was like that of native SP-C, suggesting that substitution of phenylalanine for cysteine has no apparent effect on the secondary structure of the peptide. Captive bubble surfactometry demonstrated higher surface activity for canine SP-Cff ion-lock peptide in combination with B-YL in surfactant lipids than with canine SP-Csf ion-lock peptide. These studies demonstrate the potential of canine SP-Cff ion-lock peptide to enhance the functionality of the SP-B peptide mimic B-YL in synthetic surfactant lipids.

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Prostate apoptosis response-4 (Par-4) tumor suppressor protein has gained attention as a potential therapeutic target owing to its unique ability to selectively induce apoptosis in cancer cells, sensitize them to chemotherapy and radiotherapy, and mitigate drug resistance. It has recently been reported that Par-4 interacts synergistically with cisplatin, a widely used anticancer drug. However, the mechanistic details underlying this relationship remain elusive. In this investigation, we employed an array of biophysical techniques, including circular dichroism spectroscopy, dynamic light scattering, and UV-vis absorption spectroscopy, to characterize the interaction between the active caspase-cleaved Par-4 (cl-Par-4) fragment and cisplatin. Additionally, elemental analysis was conducted to quantitatively assess the binding of cisplatin to the protein, utilizing inductively coupled plasma-optical emission spectroscopy and atomic absorption spectroscopy. Our findings provide evidence of direct interaction between cl-Par-4 and cisplatin, and reveal a binding stoichiometry of 1:1. This result provides insights that could be useful in enhancing the efficacy of cisplatin-based and tumor suppressor-based cancer therapies.

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Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.

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Circular Dichroism (CD) spectroscopy is a widely-used method for characterizing individual protein structures in solutions, membranes, films and macromolecular complexes, as well as for probing macromolecular interactions, conformational changes associated with binding substrates, and in different functionally-related environments. This paper describes a series of related computational and display tools that have been developed over many years to aid in those characterizations and functional interpretations. The new DichroPipeline described herein links a series of format-compatible data processing, analysis, and display tools to enable users to facilely produce the spectra, which can then be made available in the Protein Circular Dichroism Data Bank (https://pcddb.cryst.bbk.ac.uk/) resource, in which the CD spectral and associated metadata for each entry are linked to other structural and functional data bases including the Protein Data Bank (PDB), and the UniProt sequence data base, amongst others. These tools and resources thus provide the basis for a wide range of traceable structural characterizations of soluble, membrane and intrinsically-disordered proteins.

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Antibiotics are losing effectiveness as bacteria become resistant to conventional drugs. To find new alternatives, antimicrobial peptides (AMPs) are rationally designed with different lengths, charges, hydrophobicities (H), and hydrophobic moments (µH), containing only three types of amino acids: arginine, tryptophan, and valine. Six AMPs with low minimum inhibitory concentrations (MICs) and <25% toxicity to mammalian cells are selected for biophysical studies. Their secondary structures are determined using circular dichroism (CD), which finds that the % α-helicity of AMPs depends on composition of the lipid model membranes (LMMs): gram-negative (G(-)) inner membrane (IM) >gram-positive (G(+)) > Euk33 (eukaryotic with 33 mol% cholesterol). The two most effective peptides, E2-35 (16 amino acid [AA] residues) and E2-05 (22 AAs), are predominantly helical in G(-) IM and G(+) LMMs. AMP/membrane interactions such as membrane elasticity, chain order parameter, and location of the peptides in the membrane are investigated by low-angle and wide-angle X-ray diffuse scattering (XDS). It is found that headgroup location correlates with efficacy and toxicity. The membrane bending modulus KC displays nonmonotonic changes due to increasing concentrations of E2-35 and E2-05 in G(-) and G(+) LMMs, suggesting a bacterial killing mechanism where domain formation causes ion and water leakage.

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Intrinsically disordered proteins play important roles in cell signaling, and dysregulation of these proteins is associated with several diseases. Prostate apoptosis response-4 (Par-4), an approximately 40 kilodalton proapoptotic tumor suppressor, is a predominantly intrinsically disordered protein whose downregulation has been observed in various cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) is active and plays a role in tumor suppression by inhibiting cell survival pathways. Here, we employed site-directed mutagenesis to create a cl-Par-4 point mutant (D313K). The expressed and purified D313K protein was characterized using biophysical techniques, and the results were compared to that of the wild-type (WT). We have previously demonstrated that WT cl-Par-4 attains a stable, compact, and helical conformation in the presence of a high level of salt at physiological pH. Here, we show that the D313K protein attains a similar conformation as the WT in the presence of salt, but at an approximately two times lower salt concentration. This establishes that the substitution of a basic residue for an acidic residue at position 313 alleviates inter-helical charge repulsion between dimer partners and helps to stabilize the structural conformation.

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BACKGROUND: SIMR1281 is a potent anticancer lead candidate with multi- target activity against several proteins; however, its mechanism of action at the molecular level is not fully understood. Revealing the mechanism and the origin of multitarget activity is important for the rational identification and optimization of multitarget drugs. METHODS: We have used a variety of biophysical (circular dichroism, isothermal titration calorimetry, viscosity, and UV DNA melting), biochemical (topoisomerase I & II assays) and computational (molecular docking and MD simulations) methods to study the interaction of SIMR1281 with duplex DNA structures. RESULTS: The biophysical results revealed that SIMR1281 binds to dsDNA via an intercalation-binding mode with an average binding constant of 3.1 × 106 M-1. This binding mode was confirmed by the topoisomerases' inhibition assays and molecular modeling simulations, which showed the intercalation of the benzopyrane moiety between DNA base pairs, while the remaining moieties (thiazole and phenyl rings) sit in the minor groove and interact with the flanking base pairs adjacent to the intercalation site. CONCLUSIONS: The DNA binding characteristics of SIMR1281, which can disrupt/inhibit DNA function as confirmed by the topoisomerases' inhibition assays, indicate that the observed multi-target activity might originate from ligand intervention at nucleic acids level rather than due to direct interactions with multiple biological targets at the protein level. GENERAL SIGNIFICANCE: The findings of this study could be helpful to guide future optimization of benzopyrane-based ligands for therapeutic purposes.

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Ganirelix, a drug used in in vitro fertilization (IVF), prevents ovulation in women who are not ready to have children by inhibiting a gene that produces gonadotropin. Peptides are macromolecules that are able to preserve a predetermined shape while carrying out the structural and regulatory roles for which they were originally intended. Peptide structures can be altered in the production and storage processes. Therapeutic peptides' biological activity can be drastically altered by even small modifications in their primary and secondary structures. The molecules' secondary structures can be monitored by subjecting them to different processing or storage conditions. In our investigation, we used circular dichroism (CD) spectroscopy with two different software programs for secondary structure evaluation to look at how environmental factors like temperature and humidity affected the secondary structure of Ganirelix in an injectable formulation. The CD results revealed that the alpha-helical (regular and distorted), beta-sheet, beta-strands (regular and distorted), beta-turn, and random coil structures of temperature and humidity stressed generic drug products are comparable to reference-listed drug.

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Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such 'normalization' to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.

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Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled peptides stand out for their unique ability to stabilize highly frequent helical motifs, but they have failed to be systematically reported. Here, we exploit chemically diverse helix-inducing i, i + 4 constraints-lactam, hydrocarbon, triazole, double triazole and thioether-on two distinct short sequences derived from the N-terminal peptidase domain of hACE2 upon structural characterization and in silico alanine scan. Our overall objective was to provide a sequence-independent comparison of α-helix-inducing staples using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We identified a 9-mer lactam stapled peptide derived from the hACE2 sequence (His34-Gln42) capable of reaching its maximal helicity of 55% with antiviral activity in bioreporter- and pseudovirus-based inhibition assays. To the best of our knowledge, this study is the first comprehensive investigation comparing several cyclization methods with the goal of generating stapled peptides and correlating their secondary structures with PPI inhibitions using a highly topical model system (i.e., the interaction of SARS-CoV-2 Spike RBD with hACE2).

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Multiple heterohelicenes are a unique class of helical nonplanar scaffolds that have attracted great attention due to their appealing shapes, optical and electronic properties, and potential applications in chiral materials. This review describes the recent advances and challenges in the design and synthesis of representative multiple heterohelicenes with intriguing chiral properties. And the corresponding applications are also covered.

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The mutant p53Y220C (mutp53Y220C) is frequently observed in numerous tumors, including pancreatic cancer. The mutation creates a crevice in the DNA binding core domain and makes p53 a thermally unstable non-functional protein that assists tumor progression and confers resistance to chemotherapeutic drugs. Restoring mutp53 function to its wild type by selectively targeting this crevice with small molecules is a pivotal strategy to promote apoptosis. In this study, we have shown through different biophysical and cell-based studies that curcumin binds and rescues mutp53Y220C to an active wild-type conformation and restores its apoptotic transcription function in BxPC-3-pancreatic cancer cells. In addition, the curcumin-rescued-p53Y220C (CRp53) showed significant hyperphosphorylation at Ser15, Ser20, and acetylation at Lys382 with an 8-fold increase in transcription activity in the BxPC-3 cell lines. We also observed that the active CRp53 escapes Mdm2-mediated proteasomal degradation and the majority of the proteins were localized inside the nucleus with an increased half-life and transcription restoration compared to untreated BxPC-3 cells. By label-free proteomics analysis, we observed that upon curcumin treatment almost 227 proteins were dysregulated with the majority of them being transcriptional targets of p53. Based on our studies, it reflects that apoptosis in pancreatic cancer cells is mediated by curcumin-rescued mutant p53Y220C.

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Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These "toxic oligomers" have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AßO) and the oxidative stress elicited by AßO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer's disease in a powerful, synergistic manner. Examination of AßO structure by various biophysical tools shows that each stilbene candidate uniquely alters AßO conformation and toxicity, providing insight towards the future development of structural correctors for AßO. Correlations of AßO structural modulation and bioactivity displayed by each provides insights for future testing in vivo. The multi-target activity of these hybrid molecules represents a highly advantageous feature for disease modification in Alzheimer's, which displays a complex, multifactorial etiology. Importantly, these novel small molecules intervene with intraneuronal AßO, a necessary feature to counter the cycle of dysregulation, oxidative stress and inflammation triggered during the earliest stages of disease progression.

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Preservation of the integrity of macromolecular higher-order structure is a tenet central to achieving biologic drug and vaccine product stability toward manufacturing, distribution, storage, handling, and administration. Given that mRNA lipid nanoparticles (mRNA-LNPs) are held together by an intricate ensemble of weak forces, there are some intriguing parallels to biologic drugs, at least at first glance. However, mRNA vaccines are not without unique formulation and stabilization challenges derived from the instability of unmodified mRNA and its limited history as a drug or vaccine. Since certain learning gained from biologic drug development may be applicable for the improvement of mRNA vaccines, we present a perspective on parallels and contrasts between the emerging role of higher-order structure pertaining to mRNA-LNPs compared to pharmaceutical proteins. In a recent publication, the location of mRNA encapsulated within lipid nanoparticles was identified, revealing new insights into the LNP structure, nanoheterogeneity, and microenvironment of the encapsulated mRNA molecules [Brader et al. Biophys. J. 2021, 120, 2766]. We extend those findings by considering the effect of encapsulation on mRNA thermal unfolding with the observation that encapsulation in LNPs increases mRNA unfolding temperatures.

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Multivalent complexes formed between the cancer-promoting transcriptional co-activator, Yes-associated protein (YAP), and proteins containing short linear motifs of type PPxY modulate cell proliferation and are attractive therapeutic targets. However, challenges producing PPxY polypeptides containing the full binding domain has limited understanding of the assembly process. Here, we successfully produced a polypeptide containing the complete set of three PPxY binding sites of Angiomotin-like 1 (AMOTL1), a scaffolding protein that regulates the nucleo-cytoplasmic shuttling of YAP via WW-PPxY interactions. Using an array of biophysical techniques including isothermal titration calorimetry, size-exclusion chromatography coupled to multi-angle light scattering, and solution nuclear magnetic resonance spectroscopy, we show that the AMOTL1 polypeptide is partially disordered, and binds the YAP WW domains to form an ensemble of complexes of varying stabilities. The binding process is initiated by the binding of one YAP WW domain to one AMOTL1 PPxY motif and is completed by transient interactions of the second YAP WW domain with a second AMOTL1 PPxY motif to form an equilibrating mixture composed of various species having two YAP sites bound to two conjugate AMOTL1 sites. We rationalize that the transient interactions fine-tune the stability of the complex for rapid assembly and disassembly in response to changes in the local cellular environment.

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The B-cell receptor (BCR), a complex comprised of a membrane-associated immunoglobulin and the Igα/ß heterodimer, is one of the most important immune receptors in humans and controls B-cell development, activity, selection, and death. BCR signaling plays key roles in autoimmune diseases and lymphoproliferative disorders, yet, despite the clinical significance of this protein complex, key regions (i.e., the transmembrane domains) have yet to be structurally characterized. The mechanism for BCR signaling also remains unclear and has been variously described by the mutually exclusive cross-linking and dissociation activation models. Common to these models is the significance of local plasma membrane composition, which implies that interactions between BCR transmembrane domains (TMDs) play a role in receptor functionality. Here we used an in vivo assay of TMD oligomerization called GALLEX alongside spectroscopic and computational methods to characterize the structures and interactions of human Igα and Igß TMDs in detergent micelles and natural membranes. We observed weak self-association of the Igß TMD and strong self-association of the Igα TMD, which scanning mutagenesis revealed was entirely stabilized by an E-X10-P motif. We also demonstrated strong heterotypic interactions between the Igα and Igß TMDs both in vitro and in vivo, which scanning mutagenesis and computational models suggest is multiconfigurational but can accommodate distinct interaction sites for self-interactions and heterotypic interactions of the Igα TMD. Taken together, these results demonstrate that the TMDs of the human BCR are sites of strong protein-protein interactions that may direct BCR assembly, endoplasmic reticulum retention, and immune signaling.

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