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At least 1,153 records · Page 64

Ionizing radiation induces heritable disruption of epithelial cell interactions

Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

Non-NASA Center↗

ChAdOx1 interacts with CAR and PF4 with implications for thrombosis with thrombocytopenia syndrome

Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

60 APPLIED LIFE SCIENCES↗

ftmsRanalysis: An R package for exploratory data analysis and interactive visualization of FT-MS data

The high-resolution and mass accuracy of Fourier transform mass spectrometry (FT-MS) has made it an increasingly popular technique for discerning the composition of soil, plant and aquatic samples containing complex mixtures of proteins, carbohydrates, lipids, lignins, hydrocarbons, phytochemicals and other compounds. Thus, there is a growing demand for informatics tools to analyze FT-MS data that will aid investigators seeking to understand the availability of carbon compounds to biotic and abiotic oxidation and to compare fundamental chemical properties of complex samples across groups. We present ftmsRanalysis, an R package which provides an extensive collection of data formatting and processing, filtering, visualization, and sample and group comparison functionalities. The package provides a suite of plotting methods and enables expedient, flexible and interactive visualization of complex datasets through functions which link to a powerful and interactive visualization user interface, Trelliscope. Example analysis using FT-MS data from a soil microbiology study demonstrates the core functionality of the package and highlights the capabilities for producing interactive visualizations.

97 MATHEMATICS AND COMPUTING↗

Mapping structural and dynamic divergence across the MBOAT family

Membrane-bound O-acyltransferases (MBOATs) are membrane-embedded enzymes that catalyze acyl chain transfer to a diverse group of substrates, including lipids, small molecules, and proteins. MBOATs share a conserved structural core, despite wide-ranging functional specificity across both prokaryotes and eukaryotes. The structural basis of catalytic specificity, regulation and interactions with the surrounding environment remain uncertain. Here, we combine comparative molecular dynamics (MD) simulations with bioinformatics to assess molecular and interactional divergence across the family. In simulations, MBOATs differentially distort the bilayer depending on their substrate type. Additionally, we identify lipid binding sites surrounding reactant gates in the surrounding membrane. Complementary bioinformatic analyses reveal a conserved role for re-entrant loop-2 in MBOAT fold stabilization and a key hydrogen bond bridging DGAT1 dimerization. Finally, we predict differences in MBOAT solvation and water gating properties. These data are pertinent to the design of MBOAT-specific inhibitors that encompass dynamic information within cellular mimetic environments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Interactive effects of growth hormone and exercise on muscle mass in suspended rats

Measures to attenuate muscle atrophy in rats in response to simulated microgravity (hindlimb suspension (HS)) have been only partially successful. In the present study, hypophysectomized rats were in HS for 7 days, and the effects of recombinant human growth hormone (GH), exercise (Ex), or GH+Ex on the weights, protein concentrations, and fiber cross-sectional areas (CSAs) of hindlimb muscles were determined. The weights of four extensor muscles, i.e., the soleus (Sol), medial (MG) and lateral (LG) gastrocnemius, and plantaris (Plt), and one adductor, i.e., the adductor longus (AL), were decreased by 10-22% after HS. Fiber CSAs were decreased by 34% in the Sol and by 1 17% in the MG after HS. In contrast, two flexors, i.e., the tibialis anterior (TA) and extensor digitorum longus (EDL), did not atrophy. In HS rats, GH treatment alone maintained the weights of the fast extensors (MG, LG, Plt) and flexors (TA, EDL) at or above those of control rats. This effect was not observed in the slow extensor (Sol) or AL. Exercise had no significant effect on the weight of any muscle in HS rats. A combination of GH and Ex treatments yielded a significant increase in the weights of the fast extensors and in the CSA of both fast and slow fibers of the MG and significantly increased Sol weight and CSA of the slow fibers of the Sol. The AL was not responsive to either GH or Ex treatments. Protein concentrations of the Sol and MG were higher only in the Sol of Ex and GH+Ex rats. These results suggest that while GH treatment or intermittent high intensity exercise alone have a minimal effect in maintaining the mass of unloaded muscle, there is a strong interactive effect of these two treatments.

Grindeland, Richard E.↗

A partially disordered region connects gene repression and activation functions of EZH2

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), which minimally requires two other subunits, EED and SUZ12, for enzymatic activity. EZH2 has been traditionally known to mediate histone H3K27 trimethylation, a hallmark of silent chromatin. Emerging evidence indicates that EZH2 also activates gene expression in cancer cells in a context distinct from canonical PRC2. The molecular mechanism underlying the functional conversion of EZH2 from a gene repressor to an activator is unclear. In this work, we show that EZH2 harbors a hidden, partially disordered transactivation domain (TAD) capable of interacting with components of active transcription machinery, mimicking archetypal acidic activators. The EZH2 TAD comprises the SRM (Stimulation-Responsive Motif) and SANT1 (SWI3, ADA2, N-CoR, and TFIIIB 1) regions that are normally involved in H3K27 methylation. The crystal structure of an EZH2–EED binary complex indicates that the EZH2 TAD mediates protein oligomerization in a noncanonical PRC2 context and is entirely sequestered. The EZH2 TAD can be unlocked by cancer-specific EZH2 phosphorylation events to undergo structural transitions that may enable subsequent transcriptional coactivator binding. The EZH2 TAD directly interacts with the transcriptional coactivator and histone acetyltransferase p300 and activates gene expression in a p300-dependent manner in cells. The corresponding TAD may also account for the gene activation function of EZH1, the paralog of EZH2. Distinct kinase signaling pathways that are known to abnormally convert EZH2 into a gene activator in cancer cells can now be understood in a common structural context of the EZH2 TAD.

59 BASIC BIOLOGICAL SCIENCES↗

Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation

The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

NASA Discipline Number 26-10↗

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization–Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

59 BASIC BIOLOGICAL SCIENCES↗

Quantum-enhanced photoprotection in neuroprotein architectures emerges from collective light-matter interactions

Background Superradiance is the phenomenon of many identical quantum systems absorbing and/or emitting photons collectively at a higher rate than any one system can individually. This phenomenon has been studied analytically in idealized distributions of electronic two-level systems (TLSs), each with a ground and excited state, as well as numerically in realistic photosynthetic nanotubes and cytoskeletal architectures. Methods Superradiant effects are studied here in idealized toy model systems and realistic biological mega-networks of tryptophan (Trp) molecules, which are strongly fluorescent amino acids found in many proteins. Each Trp molecule acts as a chromophore absorbing in the ultraviolet spectrum and can be treated approximately as a TLS, with its 1 L a excited singlet state; thus, organized Trp networks can exhibit superradiance. Such networks are found, for example, in microtubules, actin filaments, and amyloid fibrils. Microtubules and actin filaments are spiral-cylindrical protein polymers that play significant biological roles as primary constituents of the eukaryotic cytoskeleton, while amyloid fibrils have been targeted in a variety of neurodegenerative diseases. We treat these proteinaceous Trp networks as open quantum systems, using a non-Hermitian Hamiltonian to describe interactions of the chromophore network with the electromagnetic field. We numerically diagonalize the Hamiltonian to obtain its complex eigenvalues, where the real part is the energy and the imaginary part is its associated enhancement rate. We also consider multiple realizations of increasing static disorder in either the site energies or the decay rates. Results We obtained the energies and enhancement rates for realistic microtubules, actin filament bundles, and amyloid fibrils of differing lengths, and we use these values to calculate the quantum yield, which is the ratio of the number of photons emitted to the number of photons absorbed. We find that all three of these structures exhibit highly superradiant states near the low-energy portion of the spectrum, which enhances the magnitude and robustness of the quantum yield to static disorder and thermal noise. Conclusion The high quantum yield and stable superradiant states in these biological architectures may play a photoprotective rolein vivo, downconverting energetic ultraviolet photons—absorbed from those emitted by reactive free radical species—to longer, safer wavelengths and thereby mitigating biochemical stress and photophysical damage. Contrary to conventional assumptions that quantum effects cannot survive in large biosystems at high temperatures, our results suggest that macropolymeric collectives of TLSs in microtubules, actin filaments, and amyloid fibrils exhibit increasingly observable and robust effects with increasing length, up to the micron scale, due to quantum coherent interactions in the single-photon limit. Superradiant enhancement and high quantum yield exhibited in neuroprotein polymers could thus play a crucial role in information processing in the brain, the development of neurodegenerative diseases such as Alzheimer’s and related dementias, and a wide array of other pathologies characterized by anomalous protein aggregates.

Physics↗

Chemo-mechanical-microstructural coupling in the tarsus exoskeleton of the scorpion Scorpio palmatus

The multiscale structure of biomaterials enables their exceptional mechanical robustness, yet the impact of each constituent at their relevant length scale remains elusive. Here, we used SAXD analysis to expose the intact chitin-fiber architecture within the exoskeleton on a scorpion's claw, revealing varying orientations, including Bouligand and unidirectional regions different from other arthropod species. We uncovered the contribution of individual components’ constituent behavior to its mechanical properties from the micro- to the nanoscale. At the microscale, in-situ micromechanical experiments were used to determine site-specific stiffness, strength, and failure of the biocomposite due to fiber orientation, while metal-crosslinking of proteins is characterized via fluorescence maps. At the constituent level, combined with FEA simulations, we uncovered the behavior of fiber-matrix deformation with fiber diameter <53.7 nm and protein modulus in the range 1.4–11 MPa. The unveiled microstructure-mechanics relationship sheds light on the evolved structural functionalities and constituents' interactions within the scorpion cuticle.

59 BASIC BIOLOGICAL SCIENCES↗

Adsorption of Non-Ionic Surfactant and Monoclonal Antibody on Siliconized Surface Studied by Neutron Reflectometry

The adsorption of monoclonal antibodies (mAbs) on hydrophobic surfaces is known to cause protein aggregation and degradation. Therefore, surfactants, such as Poloxamer 188, are widely used in therapeutic formulations to stabilize mAbs and protect mAbs from interacting with liquid-solid interfaces. Here, the adsorption of Poloxamer 188, one mAb and their competitive adsorption on a model hydrophobic siliconized surface is investigated with neutron scattering coupled with contrast variation to determine the molecular structure of adsorbed layers for each case. Small angle neutron scattering measurements of the affinity of Poloxamer 188 to this mAb indicate that there is negligible binding at these solution conditions. Neutron reflectometry measurements of the mAb show irreversible adsorption on the siliconized surface, which cannot be washed off with neat buffer. Poloxamer 188 can be adsorbed on the surface already occupied by mAb, which enables partial removal of some adsorbed mAb by washing with buffer. The adsorption of the surfactant introduces significant conformational changes for mAb molecules that remain on the surface. In contrast, if the siliconized surface is first saturated with the surfactant, no adsorption of mAb is observed. Competitive adsorption of mAb and Poloxamer 188 from solution leads to a surface dominantly occupied with surfactant molecules, whereas only a minor amount of mAb absorbs. These findings clearly indicate that Poloxamer 188 can protect against mAb adsorption as well as modify the adsorbed conformation of previously adsorbed mAb.

36 MATERIALS SCIENCE↗

The microbiome of a bacterivorous marine choanoflagellate contains a resource-demanding obligate bacterial associate

Abstract Microbial predators such as choanoflagellates are key players in ocean food webs. Choanoflagellates, which are the closest unicellular relatives of animals, consume bacteria and also exhibit marked biological transitions triggered by bacterial compounds, yet their native microbiomes remain uncharacterized. Here we report the discovery of a ubiquitous, uncultured bacterial lineage we name Candidatus Comchoanobacterales ord. nov., related to the human pathogen Coxiella and physically associated with the uncultured marine choanoflagellate Bicosta minor . We analyse complete ‘Comchoano’ genomes acquired after sorting single Bicosta cells, finding signatures of obligate host-dependence, including reduction of pathways encoding glycolysis, membrane components, amino acids and B-vitamins. Comchoano encode the necessary apparatus to import energy and other compounds from the host, proteins for host-cell associations and a type IV secretion system closest to Coxiella’s that is expressed in Pacific Ocean metatranscriptomes. Interactions between choanoflagellates and their microbiota could reshape the direction of energy and resource flow attributed to microbial predators, adding complexity and nuance to marine food webs.

59 BASIC BIOLOGICAL SCIENCES↗

Visualization of the HIV-1 Env glycan shield across scales

The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the “glycan shield,” is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure–function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.

60 APPLIED LIFE SCIENCES↗

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere “C-zone,” MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal that MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development and prompting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility in the C-zone? Globular domains critical to MyBP-C’s modulatory functions are absent from MyBP-H, suggesting that MyBP-H may be functionally silent. However, our results suggest an active role. In vitro motility experiments indicate MyBP-H shares MyBP-C’s capacity as a molecular “brake.” These results provide new insights and raise questions about the role of the C-zone during muscle development.

59 BASIC BIOLOGICAL SCIENCES↗

tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating > 200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G–U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G–C base pairs (G3:C70 and G5:C68).

59 BASIC BIOLOGICAL SCIENCES↗

Crystal structure of dihydrofolate reductase from the filarial nematode W. bancrofti in complex with NADPH and folate

Lymphatic filariasis is a debilitating illness with an estimated 50 million cases as of 2018. The majority of cases are caused by the parasitic worm W. bancrofti and additional cases by the worms B. malayi and B. timori. Dihydrofolate reductase (DHFR) is an established target in the treatment of cancer, bacterial, and protozoal infections and may be a potential target for drugs targeting parasitic worm infections, including filariasis. Recent studies have shown that known antifolate compounds, including methotrexate, inhibit the activity of W. bancrofti DHFR (WbDHFR). However, the absence of structural information for filarial DHFRs has limited the study of more in-depth structure-function relationships. We report the structure of WbDHFR complexed with NADPH and folate using X-ray diffraction data measured to 2.47 Å resolution. The structure of WbDHFR reveals the usual DHFR fold and is currently only the second nematode DHFR structure in the Protein Data Bank. The equilibrium dissociation constants for NADPH (90 ± 29 nM) and folate (23 ± 4 nM) were determined by equilibrium titrations. The interactions of known antifolates with WbDHFR were analyzed using molecular docking programs and molecular dynamics simulations. Antifolates with a hydrophobic core and extended linker formed favorable interactions with WbDHFR. These combined data should now facilitate the rational design of filarial DHFR inhibitors, which in turn can be used to determine whether DHFR is a viable drug target for filariasis and whether existing antifolates may be repurposed for its treatment.

60 APPLIED LIFE SCIENCES↗

Reversible domain closure modulates GlnBP ligand binding affinity

Glutamine binding protein (GlnBP) is an Escherichia Coli periplasmic binding protein, which binds and carries glutamine to the inner membrane ATP-binding cassette (ABC) transporter. GlnBP binds the ligand with affinity around 0.1μM measured by isothermal titration calorimetry (ITC) and ligand binding stabilizes protein structure shown by its increase in thermodynamic stability. However, the molecular determinant of GlnBP ligand binding is not known. Electrostatic and hydrophobic interaction between GlnBP and glutamine are critical factors. We propose that the freedome of closure movement is also vital for ligand binding. In order to approve this hypothesis, we generate a series of mutants with different linker length that has different magnitude of domain closure. Mutants show different ligand binding affinity, which indicates that the propensity of domain closure determines the ligand binding affinity. Ligand binding triggers gradual ensemble conformational change. Structural changes upon ligand binding are monitored by combination of small angle x-ray scattering (SAXS) and NMR spectroscopy. Detailed structure characterization of GlnBP contributes to a better understanding of ligand binding and provides the structural basis for biosensor design.

59 BASIC BIOLOGICAL SCIENCES↗

In Vitro Evolution and Computational Approaches to Predict, Prevent and Control Future Pandemics

The natural tendency of virus to mutate and the under-sampling of the environment makes it difficult to become aware of the emergence of new viral strains with pandemic potential. Being able to predict what mutations make a virus more infective might allow to spot such strains with minimal sampling and potentially allow to predict/prevent the next pandemic. The team attempted to mimic natural viral mutations and recombination through computational and experimental methods producing a variety of mutants of a SARS-COV-2 protein (receptor binding domain, RBD, of spike protein) responsible for viral entry in mammalian cells. The library of mutants was then interrogated for ability and lack-there-of to interact with the host cell receptor ushering viral entry, Angiotensin-converting enzyme 2 (ACE2). The negative and positive data set is intended to “teach the rules” of virus-host receptor interaction. Additionally, the positive clones were used to screen a set of antibody mutants designed to widen the breadth of viral mutants recognition, to demonstrate that this kind of libraries could also be a tool to produce antibody therapeutics impervious to viral mutation, even before a pandemic strain is discovered.

59 BASIC BIOLOGICAL SCIENCES↗