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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 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

VPS26 Moonlights as a β-Arrestin-like Adapter for a 7-Transmembrane RGS Protein in Arabidopsis thaliana

Extracellular signals perceived by 7-transmembrane (7TM)-spanning receptors initiate desensitization that involves the removal of these receptors from the plasma membrane. Agonist binding often evokes phosphorylation in the flexible C-terminal region and/or intracellular loop 3 of many 7TM G-protein-coupled receptors in animal cells, which consequently recruits a cytoplasmic intermediate adaptor, β-arrestin, resulting in clathrin-mediated endocytosis (CME) and downstream signaling such as transcriptional changes. Some 7TM receptors undergo CME without recruiting β-arrestin, but it is not clear how. Arrestins are not encoded in the Arabidopsis thaliana genome, yet Arabidopsis cells have a well-characterized signal-induced CME of a 7TM protein, designated Regulator of G Signaling 1 (AtRGS1). Here we show that a component of the retromer complex, Vacuolar Protein Sorting-Associated 26 (VPS26), binds the phosphorylated C-terminal region of AtRGS1 as a VPS26A/B heterodimer to form a complex that is required for downstream signaling. We propose that VPS26 moonlights as an arrestin-like adaptor in the CME of AtRGS1.

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

Machine Learning on a Robotic Platform for the Design of Polymer–Protein Hybrids

Polymer–protein hybrids are intriguing materials that can bolster protein stability in non-native environments, thereby enhancing their utility in diverse medicinal, commercial, and industrial applications. One stabilization strategy involves designing synthetic random copolymers with compositions attuned to the protein surface, but rational design is complicated by the vast chemical and composition space. Here, a strategy is reported to design protein-stabilizing copolymers based on active machine learning, facilitated by automated material synthesis and characterization platforms. The versatility and robustness of the approach is demonstrated by the successful identification of copolymers that preserve, or even enhance, the activity of three chemically distinct enzymes following exposure to thermal denaturing conditions. Although systematic screening results in mixed success, active learning appropriately identifies unique and effective copolymer chemistries for the stabilization of each enzyme. Overall, this work broadens the capabilities to design fit-for-purpose synthetic copolymers that promote or otherwise manipulate protein activity, with extensions toward the design of robust polymer–protein hybrid materials.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein’s function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε -acetyl- l -lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Artificial intelligence methods for protein structure and interaction prediction: Recent advances and challenges

Recent advances in artificial intelligence have introduced novel methods for high-accuracy prediction of protein tertiary structures, protein complex structures, and interactions between proteins and other biomolecules, such as small molecules and nucleic acids. Such advancements are accelerating biomedical research and the development of new protein design and bioengineering methods among many other important biotechnology applications. Here, in this review, we outline the recent advances in protein-centric biomolecular structure and interaction prediction, highlight some major challenges in the field, and discuss potential directions to address them.

Morehead, Alex [Lawrence Berkeley National Laborat↗
DOE OSTI2023

Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Peptides from human BNIP5 and PXT1 and non-native binders of pro-apoptotic BAK can directly activate or inhibit BAK-mediated membrane permeabilization

Apoptosis is important for development and tissue homeostasis, and its dysregulation can lead to diseases, including cancer. As an apoptotic effector, BAK undergoes conformational changes that promote mitochondrial outer membrane disruption, leading to cell death. This is termed “activation” and can be induced by peptides from the human proteins BID, BIM, and PUMA. To identify additional peptides that can regulate BAK, we used computational protein design, yeast surface display screening, and structure-based energy scoring to identify 10 diverse new binders. We discovered peptides from the human proteins BNIP5 and PXT1 and three non-native peptides that activate BAK in liposome assays and induce cytochrome c release from mitochondria. Crystal structures and binding studies reveal a high degree of similarity among peptide activators and inhibitors, ruling out a simple function-determining property. Our results shed light on the vast peptide sequence space that can regulate BAK function and will guide the design of BAK-modulating tools and therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Leveraging Triphenylphosphine-Containing Polymers to Explore Design Principles for Protein-Mimetic Catalysts

Complex interactions between noncoordinating residues are significant yet commonly overlooked components of macromolecular catalyst function. While these interactions have been demonstrated to impact binding affinities and catalytic rates in metalloenzymes, the roles of similar structural elements in synthetic polymeric catalysts remain underexplored. Using a model Suzuki–Miyuara cross-coupling reaction, we performed a series of systematic studies to probe the interconnected effects of metal–ligand cross-links, electrostatic interactions, and local rigidity in polymer catalysts. To achieve this, a novel bifunctional triphenylphosphine acrylamide (BisTPPAm) monomer was synthesized and evaluated alongside an analogous monofunctional triphenylphosphine acrylamide (TPPAm). In model copolymer catalysts, increased initial reaction rates were observed for copolymers untethered by Pd complexation (BisTPPAm-containing) compared to Pd-cross-linked catalysts (TPPAm-containing). Further, incorporating local rigidity through secondary structure-like and electrostatic interactions revealed nonmonotonic relationships between composition and the reaction rate, demonstrating the potential for tunable behavior through secondary-sphere interactions. Lastly, through rigorous cheminformatics featurization strategies and statistical modeling, we quantitated relationships between chemical descriptors of the substrate and reaction conditions on catalytic performance. Collectively, these results provide insights into relationships among the composition, structure, and function of protein-mimetic catalytic copolymers.

Catalysts↗
DOE OSTI2022

Discrete Orientations of Interfacial Waters Direct Crystallization of Mica-Binding Proteins

Understanding the basis of templated molecular assembly on a solid surface requires a fundamental comprehension of both short- and long-range aqueous response to the surface under a variety of solution conditions. Herein we provide a detailed picture of how the molecular-scale response to different mica surfaces yields distinct solvent orientations that produce quasi-static directional potentials onto which macromolecules can adsorb. We connect this directionality to observed (a)symmetric epitaxial alignment of designed proteins onto these surfaces, corroborate our findings with 3D atomic force microscopy experiments, and identify slight differences in surface structure as the origin of this effect. Furthermore, our work provides a detailed picture of the intrinsic electrolyte response in the vicinity of mineral interfaces, with clear predictions for experiment, and highlights the role of solvent on the predictive assembly of hierarchical materials on mineral surfaces.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Nanomolar Sensitivity Chirality Transfer from Designed Helical Repeat Proteins to Achiral CdS Nanorods

Bridging chirality across length scales with inorganic− organic hybrid materials is a rapidly expanding area of research. Here, we establish asymmetry at CdS nanorod (NR) interfaces using a designed helical repeat protein bearing four cysteine residues (DHR- 4Cys). Hydrophobic NRs are transferred into water with glycine, and then glycine is displaced by DHR-4Cys, leveraging the thiophilicity of cadmium. Circular dichroism (CD) in the visible, coincident with CdS electronic transitions, reveals a chiral DHR-4Cys:CdS interface. Here, the dissymmetry factor [g-factor = 4.5 × 10 −4 (short NRs) and 5.0 × 10 −4 (long NRs)] is weakly dependent on the NR length, and CD persists at nanomolar protein loadings. Additionally, control experiments demonstrate that DHR-4Cys:CdS NR chirality is dictated by the local coordination of Cys with no significant contribution from the chiral secondary structure of the protein (g-factors of short and long Cys:CdS NRs are 4.8 × 10 −4 and 4.0 × 10 −4 , respectively). Together with far-UV CD and transmission electron microscopy, which provide evidence of preserved protein structure, these results provide the first demonstration that a structurally defined protein can induce chirality in CdS nanocrystals while maintaining protein structure at biologically relevant concentrations.

Cadmium sulfide↗
DOE OSTI2024

De novo design of modular protein hydrogels with programmable intra- and extracellular viscoelasticity

Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Opportunities and Challenges for Machine Learning-Assisted Enzyme Engineering

Enzymes can be engineered at the level of their amino acid sequences to optimize key properties such as expression, stability, substrate range, and catalytic efficiency or even to unlock new catalytic activities not found in nature. Because the search space of possible proteins is vast, enzyme engineering usually involves discovering an enzyme starting point that has some level of the desired activity followed by directed evolution to improve its “fitness” for a desired application. Recently, machine learning (ML) has emerged as a powerful tool to complement this empirical process. ML models can contribute to (1) starting point discovery by functional annotation of known protein sequences or generating novel protein sequences with desired functions and (2) navigating protein fitness landscapes for fitness optimization by learning mappings between protein sequences and their associated fitness values. In this Outlook, we explain how ML complements enzyme engineering and discuss its future potential to unlock improved engineering outcomes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Engineering a disulfide-gated switch in streptavidin enables reversible binding without sacrificing binding affinity

Although high affinity binding between streptavidin and biotin is widely exploited, the accompanying low rate of dissociation prevents its use in many applications where rapid ligand release is also required. To combine extremely tight and reversible binding, we have introduced disulfide bonds into opposite sides of a flexible loop critical for biotin binding, creating streptavidin muteins (M88 and M112) with novel disulfide-switchable binding properties. Crystal structures reveal how each disulfide exerts opposing effects on structure and function. Whereas the disulfide in M112 disrupts the closed conformation to increase k off , the disulfide in M88 stabilizes the closed conformation, decreasing k off 260-fold relative to streptavidin. The simple and efficient reduction of this disulfide increases k off 19,000-fold, thus creating a reversible redox-dependent switch with 70-fold faster dissociation kinetics than streptavidin. The facile control of disulfide formation in M88 will enable the development of many new applications requiring high affinity and reversible binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT

Abstract Organisms have a myriad of strategies for sensing, responding to, and combating reactive oxygen species, which are unavoidable consequences of aerobic life. In the heterocystous cyanobacterium Nostoc sp. PCC 7120, one such strategy is the use of an ArsR-SmtB transcriptional regulator RexT that senses H 2 O 2 and upregulates expression of thioredoxin to maintain cellular redox homeostasis. Different from many other members of the ArsR-SmtB family which bind metal ions, RexT has been proposed to use disulfide bond formation as a trigger to bind and release DNA. Here, we present high-resolution crystal structures of RexT in the reduced and H 2 O 2 -treated states. These structures reveal that RexT showcases the ArsR-SmtB winged-helix-turn-helix fold and forms a vicinal disulfide bond to orchestrate a response to H 2 O 2 . The importance of the disulfide-forming Cys residues was corroborated using site-directed mutagenesis, mass spectrometry, and H 2 O 2 -consumption assays. Furthermore, an entrance channel for H 2 O 2 was identified and key residues implicated in H 2 O 2 activation were pinpointed. Finally, bioinformatics analysis of the ArsR-SmtB family indicates that the vicinal disulfide “redox switch” is a unique feature of cyanobacteria in the Nostocales order, presenting an interesting case where an ArsR-SmtB protein scaffold has been evolved to showcase peroxidatic activity and facilitate redox-based regulation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. We report experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

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

Arabidopsis ACYL CARRIER PROTEIN4 and RHOMBOID LIKE10 act independently in chloroplast phosphatidate synthesis

Abstract ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently.

59 BASIC BIOLOGICAL SCIENCES↗