Biophysical Chemistry

GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size [~]7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

Alzheimers disease is a progressive degenerative condition that mainly affects cognition and memory. Recently, distinct clinical and neuropathological phenotypes have been identified in AD. Studies revealed that structural variation in A{beta} fibrillar aggregates correlates with distinct disease phenotypes. Moreover, environmental surroundings, including other biomolecules such as proteins and lipids, have been shown to interact and modulate A{beta} aggregation. Model membranes containing ganglioside (GM1) clusters are specifically known to promote A{beta} fibrillogenesis. This study unravels the modulatory effect of non-micellar GM1, a glycosphingolipid frequently released from the damaged neuronal membranes, on A{beta}42amyloid fibril formation. Using far-UV circular dichroism experiments, we observed a spontaneous change in the peptide secondary structure from random-coil to {beta}-turn with subsequent generation of predominantly {beta}-sheet-rich species upon interaction with GM1. Thioflavin-T (ThT) fluorescence assays further indicated that GM1 interacts with the amyloidogenic A{beta}42 primary nucleus leading to a possible formation of GM1-modified A{beta}42 fibril. Statistically, no significant difference in toxicity to RA-differentiated SH-SY5Y cells was observed between A{beta}42 fibrils and GM1-tweaked A{beta}42 aggregates. Moreover, GM1-modified A{beta}42 aggregates exhibited prion-like properties in catalyzing the amyloid fibril formation of both major isomers of A{beta}, A{beta}40, and A{beta}42.

Tauopathies are neurodegenerative disorders with increasing incidence and still without cure. The extensive time required for development and approval of novel therapeutics highlights the need for testing and repurposing known safe molecules. Since doxycycline impacts -synuclein aggregation and toxicity, herein we tested its effect on tau. We found that doxycycline reduces amyloid aggregation of the different isoforms of tau protein in a dose-dependent manner, remodeling the resultant species. Furthermore, doxycycline interacts with tau microtubule-binding domain preventing its aggregation. In a cell free system doxycycline also prevents tau seeding and in cell culture reduces toxicity of tau aggregates. Overall, our results expand the spectrum of action of doxycycline against aggregation-prone proteins, opening novel perspectives for its repurposing as a disease-modifying drug for tauopathies.

The microtubule-associated protein tau forms disease-specific filamentous aggregates in several different neurodegenerative diseases. In order to understand how tau undergoes misfolding into a specific filament type and to control this process for drug development purposes, it is crucial to study in vitro tau aggregation methods and investigate the structures of the obtained filaments at the atomic level. Here, we used the tau fragment dGAE, which aggregates spontaneously, to seed the formation of full-length tau filaments. The structures of dGAE and full-length tau filaments were investigated by solid-state MAS NMR, showing that dGAE allows propagation of a chronic traumatic encephalopathy (CTE)-like fold to the full-length tau. The obtained filaments efficiently seeded tau aggregation in HEK293T cells. This work demonstrates that in vitro preparation of disease-specific types of full-length tau filaments is feasible.

The search for peptides that can specifically bind to regulatory regions in DNA is a necessary step for creating drugs that can regulate gene expression. The study is dedicated to the peculiarities of binding of a model peptide, which carries an ionic self-complementary motif and can form amyloid-like fibrils [1], with model double-stranded DNAs. The stoichiometric ratios of the components of the complex were found using the retardation method in agarose gel. Using microscale thermophoresis, it was shown that the peptide in the amyloid-like state is capable of binding to model 45-bp double-stranded DNA, with a micromolar equilibrium dissociation constant. Using cryo-electron, transmission electron, and atomic force microscopy, the morphology of peptide-DNA complexes was studied. Using dynamic light scattering and nanoparticle tracking analysis, as well as small-angle neutron scattering, the spatial parameters of the resulting DNA-peptide complexes were characterized. Molecular dynamics simulations showed that the arginine side chains of the peptide are prone to interact with guanine nitrogenous bases. It was shown that the formation of peptide-dsDNA complexes interferes with the operation of restriction endonucleases that have guanine-cytosine pairs in the recognition center, which is consistent with the results of prediction of interaction sites obtained using computer modeling. The results of the work can be used in the development of peptides capable of interacting with functional regions of DNA, as well as in the development of new carriers for transfection of DNA constructs.

Insulin amyloid aggregation is a key pathological and pharmaceutical concern, particularly in the context of Type-2 Diabetes (T2D), where amyloid deposition of protein can impair therapeutic efficacy and contribute to cell death leading to local tissue damage. Although gangliosides--glycosphingolipids containing sialic acid residues--are known to modulate amyloid formation in neurodegenerative disorders, their influence on insulin aggregation remains largely unexplored. In this study, we investigate the effects of gangliosides GM3 and GD3 on insulin aggregation. Using Thioflavin-T (ThT) based fluorescence kinetics, Fourier Transform Infrared (FTIR) spectroscopy, Circular Dichroism (CD) spectroscopy, Small Angle X-ray Scattering (SAXS), Nuclear Magnetic Resonance (NMR) spectroscopy, and Transmission Electron Microscopy (TEM), the aggregation pathway, changes in the secondary structure and morphology of insulin aggregates have been characterized. Our results show that both GM3 and GD3 lipids accelerated insulin aggregation in a concentration-dependent manner while steering the pathway away from classical fibril formation, producing short, beaded structures distinct from the extended fibrils observed under lipid-free conditions. CD and FTIR data analyses revealed that insulin in the presence of gangliosides formed non-fibrillar intermediates with distinct secondary structures: {beta}-sheet-rich globular clusters in presence of GD3 and -helical intermediates in GM3-treated samples. Cytotoxicity assays further demonstrated that ganglioside-induced aggregates are significantly less toxic to cells when compared to insulin-only aggregates. Furthermore, ganglioside-bound insulin oligomers retain seeding capacity, suggesting that they can nucleate further aggregation despite their non-fibrillar morphology. These findings underscore the role of gangliosides in modulating insulin amyloid polymorphism and toxicity, offering new insights into their potential impact on the pathology of T2D and treatment strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/703542v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@5bf40eorg.highwire.dtl.DTLVardef@f400ddorg.highwire.dtl.DTLVardef@164dcd8org.highwire.dtl.DTLVardef@def4e7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGangliosides GD3 and GM3 accelerate insulin aggregation, forming non-fibrillar assemblies. C_LIO_LIGanglioside-bound insulin aggregates are less cytotoxic than fibrillar aggregates. C_LIO_LIDespite altered morphology, ganglioside-bound aggregates retain seeding ability. C_LI

The SARS-CoV-2 envelope protein (E) is involved in a broad spectrum of functions in the cycle of the virus, including assembly, budding, envelope formation, and pathogenesis. To enable these activities, E is likely to be capable of changing its conformation depending on environmental cues. To investigate this issue, here we characterised the structural properties of the C-terminal domain of E (E-CTD), which has been reported to interact with host cell membranes. We first studied the conformation of the E-CTD in solution, finding characteristic features of a disordered protein. By contrast, in the presence of large unilamellar vesicles and micelles, which mimic cell membranes, the E-CTD was observed to become structured. The E-CTD was also found to display conformational changes with osmolytes. Furthermore, prolonged incubation of the E-CTD under physiological conditions resulted in amyloid-like fibril formation. Taken together, these findings indicate that the E-CTD can change its conformation depending on its environment, ranging from a disordered state, to a membrane-bound folded state, and an amyloid state. Our results thus provide insight into the structural basis of the role of E in the viral infection process. HighlightsO_LIThe E-CTD of SARS-CoV-2 is intrinsically disordered in solution C_LIO_LIThe E-CTD folds into an ordered structure in presence of membrane mimetics C_LIO_LIThe E-CTD displays conformational changes in the presence of osmolytes C_LIO_LIProlonged incubation of the E-CTD leads to its self-assembly into amyloid-like fibrils C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/424646v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a2d39aorg.highwire.dtl.DTLVardef@1f2581aorg.highwire.dtl.DTLVardef@17639eaorg.highwire.dtl.DTLVardef@d43ba1_HPS_FORMAT_FIGEXP M_FIG Structural heterogeneity of the E-CTD. The E-CTD shows a disordered secondary structure in an aqueous solution and converts into an ordered structure in the presence of membrane mimetics (neutral and negative lipids) and natural osmolytes (TMAO). Incubation at physiological condition shows typical amyloid-like fibrils. The yellow-colored structure represents a predicted structure of the E-CTD by PEP-FOLD. C_FIG

Cross-seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation-prone proteins such as SN, which is central in the development of Parkinsons disease. This is particularly pertinent in view of the growing number of functional (i.e. benign and useful) amyloid proteins discovered in bacteria. Here we identify two functional amyloid proteins, Pr12 and Pr17, in fecal matter from Parkinsons disease transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid. Both proteins show robust aggregation into ThT-positive aggregates that contain higher-order {beta}-sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against formic acid, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected SN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross-seed fibrillation of SN. Our results support the use of proteomics and formic acid to identify amyloid protein in complex mixtures and indicates the existence of numerous functional amyloid proteins in microbiomes. IMPORTANCEThe bacterial microbiome in the gastrointestinal tract is increasingly seen as important for human health and disease. One area of particular interest is that of neurodegenerative diseases such as Parkinsons which involve pathological aggregation into amyloid of human proteins such as - synuclein (SN). Bacteria are known to form benign or functional amyloid, some of which may initiate unwanted aggregation of e.g. SN in the enteric nervous system through cross-seeding via contact with the microbiome. Here we show that the rat microbiome contains several proteins which form this type of amyloid aggregate both in vivo and in vitro. Although the two proteins we investigate in depth do not directly promote SN aggregation, our work shows that the microbiome potentially harbors a significant number of bacterial amyloid which could play a role in human physiology at various levels.

Here we used domain 3 of dengue virus serotype 3 envelope protein (D3ED3), a natively folded globular low-immunogenicity protein, to ask whether the biophysical nature of amorphous aggregates can affect immunogenicity. We prepared amorphous oligomers in five distinct ways. One oligomer type was produced using our SCP tag (Solubility Controlling Peptide) made of 5 Isoleucines (C5I). The others were prepared by miss-shuffling the SS bonds (Ms), heating (Ht), stirring (St), and freeze-thaw (FT). Dynamic light scattering showed that all five formulations contained oligomers of approximately identical sizes with hydrodynamic radii (Rh) between 30 and 55 nm. Circular dichroism (cd) indicated that the secondary structure content of oligomers formed by stirring and freeze-thaw was essentially identical to that of the native monomeric D3ED3. The secondary structure content of the Ms showed moderate changes, whereas the C5I and heat-induced (Ht) oligomers exhibited a significant change. Immunization in JcL:ICR mice showed that both C5I and Ms significantly increased the anti-D3ED3 IgG titer. Ht, St, and FT were barely immunogenic, similar to the monomeric D3ED3. Cell surface CD marker analysis by flow cytometry confirmed that immunization with Ms generated a strong central and effector T-cell memory. This result adds a new dimension to earlier studies where the strength of the immune response was associated solely with the presence and sizes of the oligomers. It also suggests that controlled oligomerization can provide a new, adjuvant-free method for increasing a proteins immunogenicity, yielding a potentially powerful platform for protein-based vaccines. SignificanceProtein aggregation is suspected to increase the immunogenicity of proteins. Here we show that the strength of the immune response depends not merely on the size of the oligomers/aggregates but also on their biophysical properties. Dengue virus 3 envelop protein domain 3 (D3ED3) was oligomerized/aggregated in five different ways. All five formulations contained oligomers with hydrodynamic radii between 30 and 55 nm. Two formulations, where D3 ED3 was natively folded, were not or poorly immunogenic. On the other hand, two others, where D3ED 3 was in a molten globule-like state, were strongly immunogenic. This result adds a new dimension to earlier studies where the strength of the immune response was associated solely with the presence and sizes of the oligomers.

Trichophyton rubrum is one of the leading causes of superficial skin infections worldwide. It is a keratinolytic fungus specialized in colonization of keratinized tissue of skin, hair and nails for long periods of time. The fungus encodes a wide repertoire of secreted proteases in its genome that not only aid in nutrient acquisition but also establishment of infection on the host. The proteases are synthesized in prepro form that requires removal of the prosegment for activation. In order to gain insights into the structural association of the pro domain with the catalytic domain, we investigate the structural features of the pro domain of the secreted sedolisin member Sub16 of T. rubrum. Our results show that the pro domain of Sub16 may have inherent flexibility in the absence of the associated catalytic domain which is stabilized in complex with catalytic domain. This is the first report of structural investigation on a stand-alone pro domain of sedolisin family of subtilases that will help in design of further structural studies of this protein.

Adenylate cyclase toxin (ACT) is one of the main virulence factors of Bordetella pertussis, with crucial role in colonization of human respiratory tract. ACT toxicity on target phagocytes results from translocation of its adenylate cyclase domain and production of high cAMP levels and from pore formation. Recently, we unveiled in ACT four cholesterol-recognition motifs involved in specific interaction with membrane cholesterol, which might stabilize membrane topology of critical helices for ACT activity. Here we explore an amphipathic peptide corresponding to ACT residues 454 to 487 containing one of such CRAC motifs. We show that P454-487 penetrates into DOPC vesicles as a long and tilted -helix, while in cholesterol presence experiments conformational changes that critically depend on the CRAC Phe-485 residue. Moreover, P454-487 is capable of blocking ACT toxicity on cells by outcompeting with the full-length toxin for membrane binding. We anticipate P454-487 may have potential clinical applicability in controlling Bordetella infection.

Calcium is crucial in insulin biology and Ca2+ sensor proteins enforced in insulin release and signalling. The neuronal calcium sensor proteins (NCS) such as NCS-1 and VILIP are shown to be involved in insulin secretion from {beta}-pancreatic cells. However, the expression of different NCS proteins in the pancreas and their functional significance and role in pathologies remained unexplored. The present work, through different biophysical methods, presented that NCS proteins interact with insulin. NCS-1, the founder member of NCS family proteins interacts with insulin in a Ca2+ independent manner and Ca2+ enhances the affinity of the interaction. The evolutionarily conserved cryptic EF-hand in NCS proteins was found to be an essential commodity for binding insulin. The presence of Ca2+ binding first EF-hand abolishes the interaction with insulin and suggests the significance of non-functional EF-hand. The fluorescence and circular dichroism (CD) spectroscopy show that insulin interaction induces structural changes in NCS-1, which is demonstrated by size exclusion chromatography and analytical ultra-centrifugation. The autism mutant NCS-1-R102Q relatively retained insulin binding properties but with a significant difference in binding thermodynamics. Considering substantial sequence similarity among different NCS proteins and localisation in the pancreas, we examined the insulin interaction with the neurocalcin delta (NCALD). The NCALD shows metal ion-independent insulin binding and contrary to NCS-1, the Ca2+ abolishes the insulin binding. This highlights the differential regulation of Ca2+ towards insulin interaction in NCS protein. Conclusively the present work highlight that NCS proteins interact with insulin and further investigation would aid to understand the significance of NCS proteins in insulin physiology/pathophysiology and possible new molecular targets in diabetes.

Incoherent neutron spectroscopy, in combination with dynamic light scattering was used to investigate the effect of ligand binding on the center-of-mass self-diffusion and internal diffusive dynamics of E.coli aspartate -decarboxylase (ADC). The X-ray crystal structure of the D-serine inhibitor complex with ADC was also determined, and molecular dynamics simulations used to further probe the structural rearrangements that occur as a result of ligand binding. These experiments reveal the existence of higher order oligomers of the ADC tetramer on ns-ms time-scales, and also show that ligand binding both affects the ADC internal diffusive dynamics and appears to further increase the size of the higher order oligomers.

The sequence 306VQIVYK311 is an aggregation prone region of the tau protein implicated in driving assembly of tau into paired helical filaments. These filaments accumulate as intraneuronal neurofibrillary tangles in Alzheimers disease and a range of tauopathies. Here, we demonstrate that VQIVYK forms highly ordered fibrillar samples after prolonged incubation at room temperature. Remarkably, aligned fibre bundles give rise to unusually detailed and highly oriented X-ray fibre diffraction patterns. Analysis of these patterns provide a model of the core protofilament structure that satisfies the experimental diffraction data. This structural model is consistent with data from X-ray crystallography of microcrystals and conforms to the cross-beta architecture that defines amyloid, but diffraction data analysis shows a highly twisted filamentous protofilament architecture. Analysis of individual fibril envelopes by 3D contact point reconstruction atomic force microscopy reveals a diverse polymorphous population with a major fibril morphology of apparent cylindrical fibrils, and morphological subpopulations of fibrils with clear left-hand twisting patterns, despite the commonality of the core structure indicated by the detailed diffraction pattern. Together, these data suggest that VQIVYK amyloid fibrils form a polymorphous amyloid population by assembly of highly ordered protofilaments and provides molecular information regarding amyloid fibril twist.

Similar aggregation and {beta}-sheet propensity of amino acids in globular proteins and amyloids, suggests comparable principles of their formation. Here we show that during the process of aggregation into amyloid-like fibers, these rules are not the same in an amino-acid-homopolymer (AAHP) polyglutamine (PolyGln). An aggregation kinetic analysis on nine-point mutants of a forty-six long PolyGln peptide was carried in physiological conditions. At the dynamic equilibrium state of aggregation, critical-concentration derived free-energy differences, signifying aggregation propensity of incorporated amino acids were obtained. None of the obtained propensities correlated with existing conventional aggregation and {beta}-sheet propensities of the amino acids in proteins and amyloids. Further, the differential aggregation behavior of all the peptides only correlated with van der Waals volume of the incorporated amino acid and not with any other physicochemical characteristic of amino acids. The new rules obtained from PolyGln AAHP provide an opportunity to explore physiological relevance of a mutation within AAHP in human proteome. Additionally, this study opens up new avenues for protein model design exploring folding and aggregation behavior of other amino-acid-homopolymer (AAHP) existing in the human proteome. SignificanceMutational analysis within PolyGln sequences adds to the knowledge of unique aggregation propensities of amino acids within PolyGln AAHP. This study highlights the importance of van der Waals volume in dictating stability-instability of an aggregation fold and in turn aggregation kinetics and thermodynamic stability of aggregates. The analysis signifies the role of Gln-Gln interlocking system within PolyGln folding motif and extent of disruption caused by van der Waals volume of an amino acid. The results can be taken as a starting point to evaluate the possible impact of amino acid insertions in PolyGln stretches of other proteins. It also opens opportunities to study the structural and functional relationship of other AAHPS for their unique folding and aggregation behavior. Learning outcome can be utilized as a bottom-up approach to design amyloid biomaterial with different strengths for biomedical applications.

The maintenance of long-term memory requires sustainable synaptic connections, mediated by the prion-like transformation of the translational regulator protein CPEB3 (Cytoplasmic Polyadenylation Element Binding protein isoform 3) in mammals. The N- terminal prion domain of CPEB3, composed of the two prion subdomains PRD1 and PRD2 has previously been demonstrated to perform a crucial role in imparting prion-like properties to the protein. We have already reported the amyloid-core of the first prion subdomain (PRD1) of the mouse CPEB3. Here, we have investigated the aggregation properties and the structural characteristics of the mouse PRD2 (mPRD2) in vitro. We found that the mPRD2 undergoes phase separation. Interestingly, the mPRD2 formed stable and amyloid-like solid condensates instead of the typical liquid condensate formation. Solid-state NMR and other biophysical studies revealed the existence of mixed secondary structures for mPRD2 in condensates. We propose that the distinct phase separation behaviour of the mPRD2 would be due to the conformational changes attributed to the pattern of the mPRD2 amino acid sequence, resulting in the formation of rigid and amyloid-like self-assembly.

The process of protein phase separation, particularly in the context of intrinsically disordered proteins, has been extensively studied for its implications in several neuro-degenerative diseases. Although the mechanism of protein phase separation and the involved molecular grammar have been well explored under in vitro conditions, the focus is now shifting towards developing more complex models of phase separation in order to mimic the biological systems more closely. Here, we studied the phase separation of alpha synuclein (-syn), an intrinsically disordered protein whose aggregation is implicated in the pathology of Parkinsons Disease, (PD) inside yeast cells (Saccharomyces cerevisiae). Using a positively charged polymer; polyethylenimine (PEI), which binds presumably at the negatively charged C-terminal domain of -syn, we find that the aggregation of -syn inside yeast can be modulated by at least two pathways: one involving phase separation and the second one without phase separation. We find further that these two pathways lead to varying fibril characteristics and toxicities. We believe that this model can be used as a quick and convenient system to screen novel and repurposed small molecules against toxic protein droplets.

Coronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.

Bicelles have been demonstrated to be a valuable tool for studying membrane protein interactions and structure in vitro. They are distinguished by a distinct lipid bilayer that mimics the plasma membrane of cells making it more native-like than its detergent micelle counter-part. Bicelles are typically comprised of a long-chain phospholipid such as dimyristoylphosphatidylcholine (DMPC) and a short-chain phospholipid such as dihexanoylphosphatidylcholine (DHPC). When mixed together in solution DMPC-DHPC bicelles assume a discoidal structure comprised of a heterogeneous arrangement where the short-chain lipids gather around the rim of the disk and the long-chain lipids form the flat, planar, bilayer region. In this study, the nonionic surfactant, C8E5, was used to prepare mixtures with DMPC to determine if it adopts properties similar to bicelles with a q [≥] 0.5. At q [≥] 0.5, DMPC-DHPC bicelles are bilayered and DMPC is sequestered from the detergent micelle-like DHPC. Mixtures of DMPC and C8E5 were prepared at various q values, a parameter used to describe the mole ratio of DMPC to DHPC in the preparation of bicelles. Employing biophysical methods like dynamic light scattering, 31P-NMR and analytical ultracentrifugation, properties of these lipid-detergent complexes are described. Interestingly they adopted a spherical-shaped micellar structure morphology and did not assume a discoidal shape typical of bicelles at q [≥] 0.5. However, they appear to retain bilayer-like properties that may prove beneficial for in vitro biophysical studies of membrane proteins.

In India, the breakthrough infections during second wave of COVID-19 pandemic was due to SARS-COV-2 delta variant (B.1.617.2). It was reported that majority of the infections were caused by the delta variant and only 9.8% percent cases required hospitalization whereas, only 0.4% fatality was observed. Sudden dropdown in COVID-19 infections was observed within a short timeframe, suggesting better host adaptation with evolved delta variant. Down regulation of host immune response against SARS-CoV-2 by ORF8 induced MHC-I degradation has been reported earlier. The Delta variant carried mutations (deletion) at Asp119 and Phe120 amino acids which are critical for ORF8 dimerization. The deletions of amino acids Asp119 and Phe120 in ORF8 of delta variant results in structural instability of ORF8 dimer caused by disruption of hydrogen bonding and salt bridges as revealed by structural analysis and MD simulation studies of ORF8 dimer. Further, flexible docking of wild type and mutant ORF8 dimer revealed reduced interaction of mutant ORF8 dimer with MHC-I as compared to wild type ORF8 dimer with MHC-1, thus implicating its possible role in MHC-I expression and host immune response against SARS-CoV-2. We thus propose that mutant ORF8 may not hindering the MHC-I expression thereby resulting in better immune response against SARS-CoV-2 delta variant, which partly explains the sudden drop of SARS-CoV-2 infection rate in the second wave of SARS-CoV-2 predominated by delta variant in India Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/457457v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@751eeaorg.highwire.dtl.DTLVardef@140b5b5org.highwire.dtl.DTLVardef@159a3a5org.highwire.dtl.DTLVardef@6c206_HPS_FORMAT_FIGEXP M_FIG C_FIG