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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization

Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether “staples” into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with pchloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔT m ' = +27 °C) and temperature-induced heme loss (ΔT 50 = +30 °C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallographic analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

CRISPR–Cas9 bends and twists DNA to read its sequence

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CoarsenConf: Equivariant Coarsening with Aggregated Attention for Molecular Conformer Generation

Molecular conformer generation (MCG) is an important task in cheminformatics and drug discovery. The ability to efficiently generate low-energy 3D structures can avoid expensive quantum mechanical simulations, leading to accelerated virtual screenings and enhanced structural exploration. Several generative models have been developed for MCG, but many struggle to consistently produce high-quality conformers for meaningful downstream applications. To address these issues, we introduce CoarsenConf, which coarse-grains molecular graphs based on torsional angles and integrates them into an SE(3)-equivariant hierarchical variational autoencoder. Through equivariant coarse-graining, we aggregate the fine-grained atomic coordinates of subgraphs connected via rotatable bonds, creating a variable-length coarse-grained latent representation. Our model uses a novel aggregated attention mechanism to restore fine-grained coordinates from the coarse-grained latent representation, enabling efficient generation of accurate conformers. Furthermore, we evaluate the chemical and biochemical quality of our generated conformers on multiple downstream applications, including property prediction and large-scale oracle-based protein docking. Overall, CoarsenConf generates more accurate conformer ensembles compared to prior generative models.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Amino acid interacting network in the receptor-binding domain of SARS-CoV-2 spike protein

The relation between amino acid (AA) sequence and biologically active conformation controls the process of polypeptide chains folding into three-dimensional (3d) protein structures. The recent achievements in the resolution achieved in cryo-electron microscopy coupled with improvements in computational methodologies have accelerated the analysis of structures and properties of proteins. However, the detailed interaction between AAs has not been fully elucidated. Herein, we present a de novo method to evaluate inter-amino acid interactions based on the concept of accurately evaluating the amino acid bond pairs (AABP). The results obtained enabled the identification of complex 3d long-range interconnected AA interacting network in proteins. The method is applied to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. We show that although nearest-neighbor AAs in the primary sequence have large AABP, other nonlocal AAs make substantial contribution to AABP with significant participation of both covalent and hydrogen bonding. Detailed analysis of AABP in RBD reveals the pivotal role they play in sequence conservation with profound implications on residue mutations and for therapeutic drug design. This approach could be easily applied to many other proteins of biomedical interest in life sciences.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Angiotensin and biased analogs induce structurally distinct active conformations within a GPCR

Biased agonists of G protein–coupled receptors (GPCRs) preferentially activate a subset of downstream signaling pathways. In this work, we present crystal structures of angiotensin II type 1 receptor (AT1R) (2.7 to 2.9 angstroms) bound to three ligands with divergent bias profiles: the balanced endogenous agonist angiotensin II (AngII) and two strongly β-arrestin–biased analogs. Compared with other ligands, AngII promotes more-substantial rearrangements not only at the bottom of the ligand-binding pocket but also in a key polar network in the receptor core, which forms a sodium-binding site in most GPCRs. Divergences from the family consensus in this region, which appears to act as a biased signaling switch, may predispose the AT1R and certain other GPCRs (such as chemokine receptors) to adopt conformations that are capable of activating β-arrestin but not heterotrimeric G q protein signaling.

Wingler, Laura M. (ORCID:0000000236885088)↗
DOE OSTI2025

Using Multiple Isotope-Labeled Infrared Spectra for the Structural Characterization of an Intrinsically Disordered Peptide

Intrinsically disordered proteins (IDPs) rapidly interconvert between conformers, requiring an ensemble description. This complicates their experimental characterization, and force field limitations pose challenges for their simulation. Here, in this work, we use isotope-labeled and unlabeled infrared (IR) spectra to reweight simulated ensembles of the elastin-like peptide GVGVPGVG, a paradigmatic disordered peptide. By comparing the results obtained with different spectra, we explicitly show that the weights are underdetermined by the ensemble averaged data. We identify which labels and frequency regions maximize structural information while minimizing sensitivity to simulation error and show that these regions report on whether the peptide makes specific interactions. Our work shows the importance of incorporating simulations and simulated spectra at the planning stages of isotope-labeled IR experiments and more generally provides a framework for interpreting IR data for IDPs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Distinguishing Potassium Channel Resting State Conformations in Live Cells with Environment-Sensitive Fluorescence

Ion channels are polymorphic membrane proteins whose high-resolution structures offer images of individual conformations, giving us starting points for identifying the complex and transient allosteric changes that give rise to channel physiology. Here, we report live-cell imaging of voltage-dependent structural changes of voltage-gated Kv2.1 channels using peptidyl tarantula toxins labeled with an environment-sensitive fluorophore, whose spectral shifts enable identification of voltage-dependent conformation changes in the resting voltage sensing domain (VSD) of the channel. We synthesize a new environment-sensitive, far-red fluorophore, julolidine phenoxazone (JP) azide, and conjugate it to tarantula toxin GxTX to characterize Kv2.1 VSD allostery during membrane depolarization. JP has an inherent response to the polarity of its immediate surroundings, offering site-specific structural insight into each channel conformation. Using voltage-clamp spectroscopy to collect emission spectra as a function of membrane potential, we find that they vary with toxin labeling site, the presence of Kv2 channels, and changes in membrane potential. With a high-affinity conjugate in which the fluorophore itself interacts closely with the channel, the emission shift midpoint is 50 mV more negative than the Kv2.1 gating current midpoint. This suggests that substantial conformational changes at the toxin-channel interface are associated with early gating charge transitions and these are not concerted with VSD motions at more depolarized potentials. These fluorescent probes enable study of conformational changes that can be correlated with electrophysiology, putting channel structures and models into a context of live-cell membranes and physiological states.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

De novo design of high-affinity binders of bioactive helical peptides

Many peptide hormones form an α-helix on binding their receptors, and sensitive methods for their detection could contribute to better clinical management of disease. De novo protein design can now generate binders with high affinity and specificity to structured proteins. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Polyampholyte physics: Liquid–liquid phase separation and biological condensates

Here we review the current understanding of molecular interactions that govern liquid-liquid phase separation (LLPS) of biological condensates. The connection between sequence, chain conformation, and phase separation of intrinsically disordered proteins (IDPs) and their model polyampholytes is discussed. In particular, we highlight how the charge pattern influences the conformation and phase behavior of natural IDPs. We then describe recent results from theoretical treatments of polyampholytes implementing random phase approximation, field-theoretic simulations, and transfer matrix theory that show an increase in charge segregation results in an increased tendency to phase separate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Implication of rare genetic variants of NODAL and ACVR1B in congenital heart disease patients from Indian population

Highlights: • Screening of 300 CHD patients revealed two rare missense variants (p.P51L & p.D327E) in NODAL. • A novel missense variation p.M345I was identified in ACVR1B. • Bioinformatic analyses suggested that these variants could have deleterious role. • Three-dimensional modelling suggested conformation changes in the mutated proteins compared to wild-type. • In vitro studies demonstrated that missense variants affect the function of NODAL and ACVR1B. NODAL signaling plays an essential role in vertebrate embryonic patterning and heart development. Accumulating evidences suggest that genetic mutations in TGF-β/NODAL signaling pathway can cause congenital heart disease in humans. To investigate the implication of NODAL signaling in isolated cardiovascular malformation, we have screened 300 non-syndromic CHD cases and 200 controls for NODAL and ACVR1B by Sanger sequencing and identified two rare missense (c.152C > T; p.P51L and c.981 T > A; p.D327E) variants in NODAL and a novel missense variant c.1035G > A; p.M345I in ACVR1B. All these variants are absent in 200 controls. Three-dimensional protein-modelling demonstrates that both p.P51L and p.D327E variations of NODAL and p.M345I mutation of ACVR1B, affect the tertiary structure of respective proteins. Variants of NODAL (p.P51L and p.D327E) and ACVR1B (p.M345I), significantly reduce the transactivation of AR3-Luc, (CAGA){sub 12}-Luc and (SBE){sub 4}-Luc promoters. Moreover, qRT-PCR results have also deciphered a reduction in the expression of cardiac-enriched transcription factors namely Gata4, Nkx2-5, and Tbx5 in both the mutants of NODAL. Decreased expression of, Gata4, Nkx2-5, Tbx5, and lefty is observed in p.M345I mutant of ACVR1B as well. Additionally, reduced phosphorylation of SMAD2/3 in response to these variants, suggests impaired NODAL signaling and possibly responsible for defective cell fate decision and differentiation of cardiomyocytes leading to CHD phenotype.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Crystal structures of SARS-CoV-2 ADP-ribose phosphatase: from the apo form to ligand complexes

Among 15 nonstructural proteins (Nsps), the newly emerging Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) encodes a large, multidomain Nsp3. One of its units is the ADP-ribose phosphatase domain (ADRP; also known as the macrodomain, MacroD), which is believed to interfere with the host immune response. Such a function appears to be linked to the ability of the protein to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remain unknown. Here, five high-resolution (1.07–2.01 Å) crystal structures corresponding to the apo form of the protein and its complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADP-ribose have been determined. The protein is shown to undergo conformational changes to adapt to the ligand in the manner previously observed in close homologues from other viruses. A conserved water molecule is also identified that may participate in hydrolysis. This work builds foundations for future structure-based research on ADRP, including the search for potential antiviral therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Sensitive detection of structural dynamics using a statistical framework for comparative crystallography

Chemical and conformational changes are crucial to protein function and its pharmacological control. X-ray crystallography can reveal these changes in atomic detail, but standard analysis methods, which refine separate datasets, often overlook differences that are subtle or arise in only a subset of molecules. Direct comparison of crystallographic datasets is, in principle, more powerful, but systematic errors (“scales”) often mask changes in the crystallographic observables (“structure factors”). Machine learning algorithms that jointly estimate scales and structure factors can address this limitation. Here, we augment this approach with multivariate, structured priors derived from crystallographic theory, implemented in the variational deep learning framework Careless. Doing so strongly improves the detection of protein dynamics, element-specific anomalous signals, and the binding of drug candidates, offering a robust approach to comparative crystallography and, potentially, to detection of protein dynamics by other structure determination methods.

Hekstra, Doeke R. [Harvard Univ., Cambridge, MA (U↗
DOE OSTI2021

Characterization of cellular oxidative stress response by stoichiometric redox proteomics

The thiol redox proteome refers to all proteins whose cysteine thiols are subjected to various redox-dependent posttranslational modifications (PTMs) including S-glutathionylation (SSG), S-nitrosylation (SNO), S-sulfenylation (SOH), and S-sulfhydration (SSH). These modifications can impact various aspects of protein function, including activity, binding, conformation, localization, and interactions with other molecules. It is highly desirable to have robust redox proteomics methods that can provide global, site-specific, and stoichiometric quantification of redox PTMs in order to elucidate the complex redox landscape and identify novel redox regulators in signaling and regulation. Mass spectrometry-based redox proteomics has emerged as the primary platform for enabling the broad characterization of thiol PTMs in cells and tissues. Herein we review recent advances in MS-based redox proteomics approaches for quantitative profiling of redox PTMs in cells and tissues at physiological or oxidative stress conditions and highlight some recent applications using these enabling techniques. Considering the relative maturity of available methods, a primary emphasis will be on two types of modifications: 1) total oxidation (i.e., all reversible thiol modifications), the level of which represents the overall redox state, and 2) S-glutathionylation, a major form of reversible thiol oxidation. We also discuss the significance of stoichiometric measurements of thiol PTMs as well as future perspectives towards a better understanding of cellular redox regulatory networks in cells and tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Deciphering the structure of Arabidopsis thaliana 5-enol-pyruvyl-shikimate-3-phosphate synthase: An essential step toward the discovery of novel inhibitors to supersede glyphosate

Glyphosate interferes with plant aromatic metabolism through the inhibition of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase [EPSPS, EC 2.5.1.19]. For this reason, EPSPS has been extensively studied in a vast array of organisms. This notwithstanding, up to date, the crystal structure of the protein has been solved exclusively in a few prokaryotes, while that of the plant enzyme has been only deduced in silico by similarity. This study aimed at determining the structure of EPSPS from the plant model species Arabidopsis thaliana, which has been cloned, heterologously expressed and affinity-purified. The kinetic properties of the enzyme have been determined, as well as its susceptibility to the inhibition brought about by glyphosate. The crystal structure of the protein has been resolved at high resolution (1.4 Å), showing open conformation of the enzyme, which is the state ready for substrate/inhibitor binding. This provides a framework for the structure-based design of novel EPSPS inhibitors. Surface regions near the active-site cleft entrance or at the interdomain hinge appear promising for inhibitor selectivity, while bound chloride near the active site is a potential placeholder for anionic moieties of future herbicides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Zymogen and activated protein C have similar structural architecture

Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule Förster resonance energy transfer and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca 2+ to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid EGF domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing EGF domains, such as factors VII, IX, and X.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Phosphatidic acid: an emerging versatile class of cellular mediators

Abstract Lipids function not only as the major structural components of cell membranes, but also as molecular messengers that transduce signals to trigger downstream signaling events in the cell. Phosphatidic acid (PA), the simplest and a minor class of glycerophospholipids, is a key intermediate for the synthesis of membrane and storage lipids, and also plays important roles in mediating diverse cellular and physiological processes in eukaryotes ranging from microbes to mammals and higher plants. PA comprises different molecular species that can act differently, and is found in virtually all organisms, tissues, and organellar membranes, with variations in total content and molecular species composition. The cellular levels of PA are highly dynamic in response to stimuli and multiple enzymatic reactions can mediate its production and degradation. Moreover, its unique physicochemical properties compared with other glycerophospholipids allow PA to influence membrane structure and dynamics, and interact with various proteins. PA has emerged as a class of new lipid mediators modulating various signaling and cellular processes via its versatile effects, such as membrane tethering, conformational changes, and enzymatic activities of target proteins, and vesicular trafficking.

Biochemistry & Molecular Biology↗