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At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Influence of cellular redox reactions on the structure and function of light harvesting and photosystems

Photosynthesis enables the conversion of one of the most abundant and free forms of energy, sunlight, into chemical bonds through the utilization of highly tailored protein complexes. These enzymes work in unison to absorb, convert, and transform light into high-energy electrons which are used for various functions important to metabolism and cellular protection. Over the last ∼50 years, photosynthetic organisms, such as cyanobacteria, have been adapted and engineered to produce valuable compounds like hydrogen and ethylene, among others. Often this is performed by removing native and/or adding in exogenous energy utilization pathways so that light energy is re-directed towards the synthesis of desired compounds. However, the interplay between primary light capture, conversion reactions, and the downstream electron utilization sinks is not fully understood. Further complicating these strategies are the plethora of compensatory mechanisms that facilitate steady electron flow and the maintenance of photosynthesis under dynamic conditions. This manifests as structural and functional plasticity of the photosynthetic machinery, often seen in modulations of oligomeric compositions or changes in protein-protein interactions and coupling with redox enzymes. Understanding these mechanisms is crucial to biotechnology applications because re-engineering electron utilization sinks has profoundly different effects on the light capture and conversion reactions of photosynthesis. Optimization requires a molecular-level understanding of the functional interrelationships between electron sinks and photosynthetic components that influence photosynthetic efficiencies to realize potential improvements in product yields. Here, we aim to highlight how perturbation of reductive reactions is revealing the functional plasticity in key components of the photosynthetic energy transduction pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Cytoplasmic Receptor-like Kinase Contributes to Salinity Tolerance

Receptor-like cytoplasmic kinases (RLCKs) are receptor kinases that lack extracellular ligand-binding domains and have emerged as a major class of signaling proteins that regulate plant cellular activities in response to biotic/abiotic stresses and endogenous extracellular signaling molecules. We have identified a rice RLCK (OsRLCK311) that was significantly higher in transgenic pSARK-IPT rice (Oryza sativa) that exhibited enhanced growth under saline conditions. Overexpression of OsRLCK311 full-length protein (RLCK311FL) and the C-terminus of OsRLCK311 (ΔN) in Arabidopsis confirmed its role in salinity tolerance, both in seedlings and mature plants. Protein interaction assays indicated that OsRLCK311 and ΔN interacted in-vivo with the plasma membrane AQP AtPIP2;1. The RLCK311-PIP2;1 binding led to alterations in the stomata response to ABA, which was characterized by more open stomata of transgenic plants. Moreover, OsRLCK311-ΔN effect in mediating enhanced plant growth under saline conditions was also observed in the perennial grass Brachypodium sylvaticum, confirming its role in both dicots and monocots species. Lastly, OsRLCK311 interacted with the rice OsPIP2;1. We suggest that the rice OsRLCK311 play a role in regulating the plant growth response under saline conditions via the regulation of the stomata response to stress. This role seems to be independent of the RLCK311 kinase activity, since the overexpression of the RLCK311 C-terminus (ΔN), which lacks the kinase full domain, has a similar phenotype to RLCK311FL.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Model Membrane Systems Used to Study Plasma Membrane Lipid Asymmetry

It is well known that the lipid distribution in the bilayer leaflets of mammalian plasma membranes (PMs) is not symmetric. Despite this, model membrane studies have largely relied on chemically symmetric model membranes for the study of lipid–lipid and lipid–protein interactions. This is primarily due to the difficulty in preparing stable, asymmetric model membranes that are amenable to biophysical studies. However, in the last 20 years, efforts have been made in producing more biologically faithful model membranes. Here, we review several recently developed experimental and computational techniques for the robust generation of asymmetric model membranes and highlight a new and particularly promising technique to study membrane asymmetry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Bond-centric modular design of protein assemblies

Directional interactions that generate regular coordination geometries are a powerful means of guiding molecular and colloidal self-assembly, but implementing such high-level interactions with proteins remains challenging due to their complex shapes and intricate interface properties. Here we describe a modular approach to protein nanomaterial design inspired by the rich chemical diversity that can be generated from the small number of atomic valencies. We design protein building blocks using deep learning-based generative tools, incorporating regular coordination geometries and tailorable bonding interactions that enable the assembly of diverse closed and open architectures guided by simple geometric principles. Experimental characterization confirms the successful formation of more than 20 multicomponent polyhedral protein cages, two-dimensional arrays and three-dimensional protein lattices, with a high (10%–50%) success rate and electron microscopy data closely matching the corresponding design models. Due to modularity, individual building blocks can assemble with different partners to generate distinct regular assemblies, resulting in an economy of parts and enabling the construction of reconfigurable networks for designer nanomaterials.

Biomaterials – proteins↗
DOE OSTI2023

The Interaction between the DOCK7 Protein and the E2 Protein of Classical Swine Fever Virus Is Not Involved with Viral Replication or Pathogenicity

The classical swine fever virus (CSFV) particle consists of three glycoproteins, all of which have been shown to be important proteins involved in many virus functions, including interaction with several host proteins. One of these proteins, E2, has been shown to be directly involved with adsorption to the host cell and important for virus virulence. Using the yeast two-hybrid system, we have previously shown that CSFV E2 specifically interacts with the (DOCK7) dedicator of cytokinesis, a scaffolding protein. In this report, the interaction between E2 and DOCK7 was evaluated. To confirm the yeast two-hybrid results and to determine that DOCK7 interacts in swine cells with E2, we performed co-immunoprecipitation and proximity ligation assay (PLA). After demonstrating the protein interaction in swine cells, E2 amino acid residues Y65, V283, and T149 were determined to be critical for interaction with Dock7 by using a random mutated library of E2 and a reverse yeast two-hybrid approach. That disruption of these three residues with mutations Y65F, V283D, and T149A abrogated the Dock7-E2 protein interaction. These mutations were then introduced into a recombinant CSFV, E2DOCK7v, by a reverse genomics approach using the highly virulent CSFV Brescia isolate as a backbone. E2DOCKv was shown to have similar growth kinetics in swine primary macrophages and SK6 cell cultures to the parental Brescia strain. Similarly, E2DOCK7v demonstrated a similar level of virulence to the parental Brescia when inoculated in domestic pigs. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes indistinguishable from that produced by the parental strain. Therefore, interaction between CSFV E2 and host DOCK7 is not critically involved in the process of virus replication and disease production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Characterization of protein–ligand binding interactions of enoyl-ACP reductase ( FabI ) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

The enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. In this paper we describe the use of native MS to characterize the protein–ligand interactions of FabI with its coenzymes NAD + and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Non-TZF Protein AtC3H59/ZFWD3 Is Involved in Seed Germination, Seedling Development, and Seed Development, Interacting with PPPDE Family Protein Desi1 in Arabidopsis

Despite increasing reports on the function of CCCH zinc finger proteins in plant development and stress response, the functions and molecular aspects of many non-tandem CCCH zinc finger (non-TZF) proteins remain uncharacterized. AtC3H59/ZFWD3 is an Arabidopsis non-TZF protein and belongs to the ZFWD subfamily harboring a CCCH zinc finger motif and a WD40 domain. In this study, we characterized the biological and molecular functions of AtC3H59, which is subcellularly localized in the nucleus. The seeds of AtC3H59-overexpressing transgenic plants (OXs) germinated faster than those of wild type (WT), whereas atc3h59 mutant seeds germinated slower than WT seeds. AtC3H59 OX seedlings were larger and heavier than WT seedlings, whereas atc3h59 mutant seedlings were smaller and lighter than WT seedlings. Moreover, AtC3H59 OX seedlings had longer primary root length than WT seedlings, whereas atc3h59 mutant seedlings had shorter primary root length than WT seedlings, owing to altered cell division activity in the root meristem. During seed development, AtC3H59 OXs formed larger and heavier seeds than WT. Using yeast two-hybrid screening, we isolated Desi1, a PPPDE family protein, as an interacting partner of AtC3H59. AtC3H59 and Desi1 interacted via their WD40 domain and C-terminal region, respectively, in the nucleus. Taken together, our results indicate that AtC3H59 has pleiotropic effects on seed germination, seedling development, and seed development, and interacts with Desi1 in the nucleus via its entire WD40 domain. To our knowledge, this is the first report to describe the biological functions of the ZFWD protein and Desi1 in Arabidopsis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Evolutionary Variation in MADS Box Dimerization Affects Floral Development and Protein Abundance in Maize

Interactions between MADS-box transcription factors are critical in the regulation of floral development, and shifting MADS-box protein-protein interactions are predicted to have influenced floral evolution. However, precisely how evolutionary variation in protein-protein interactions affects MADS-box protein function remains unknown. To assess the impact of changing MADS-box protein-protein interactions on transcription factor function, we turned to the grasses, where interactions between B-class MADS-box proteins vary. We tested the functional consequences of this evolutionary variability using maize as an experimental system. We found that differential B-class dimerization was associated with subtle, quantitative differences in stamen shape. In contrast, differential dimerization resulted in large-scale changes to downstream gene expression. Differential dimerization also affected B-class complex composition and abundance, independent of transcript levels. This indicates that differential B-class dimerization affects protein degradation, revealing an important consequence for evolutionary variability in MADS-box interactions. Our results highlight complexity in the evolution of developmental gene networks - changing protein-protein interactions could affect not only the composition of transcription factor complexes, but also their degradation and persistence in developing flowers. Here, our results also show how coding change in a pleiotropic master regulator could have small, quantitative effects on development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Distinct Conformations of SARS-CoV-2 Omicron Spike Protein and Its Interaction with ACE2 and Antibody

Since November 2021, Omicron has been the dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant that causes the coronavirus disease 2019 (COVID-19) and has continuously impacted human health. Omicron sublineages are still increasing and cause increased transmission and infection rates. The additional 15 mutations on the receptor binding domain (RBD) of Omicron spike proteins change the protein conformation, enabling the Omicron variant to evade neutralizing antibodies. For this reason, many efforts have been made to design new antigenic variants to induce effective antibodies in SARS-CoV-2 vaccine development. However, understanding the different states of Omicron spike proteins with and without external molecules has not yet been addressed. In this review, we analyze the structures of the spike protein in the presence and absence of angiotensin-converting enzyme 2 (ACE2) and antibodies. Compared to previously determined structures for the wildtype spike protein and other variants such as alpha, beta, delta, and gamma, the Omicron spike protein adopts a partially open form. The open-form spike protein with one RBD up is dominant, followed by the open-form spike protein with two RBD up, and the closed-form spike protein with the RBD down. It is suggested that the competition between antibodies and ACE2 induces interactions between adjacent RBDs of the spike protein, which lead to a partially open form of the Omicron spike protein. The comprehensive structural information of Omicron spike proteins could be helpful for the efficient design of vaccines against the Omicron variant.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Barcoded protein array for multiplex single-molecule interaction profiling

Methods for attaching barcodes to polypeptides are provided. Methods for detecting molecular interactions at the single molecule level are provided. Embodiments of the invention are directed to a ONA barcoded protein array technology for parallel protein interaction profiling on a single molecule basis. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Novel methods are described herein that measure protein interactions based on the statistical analysis of co-localized polonies arising from barcoding DNAs of interacting proteins.

Church, George M.↗
DOE OSTI2023

Resolving protein-mineral interfacial interactions during in vitro mineralization by atom probe tomography

Organic macromolecules exert remarkable control over the nucleation and growth of inorganic crystallites during (bio)mineralization, as exemplified during enamel formation where the protein amelogenin regulates the formation of hydroxyapatite (HAP). However, it is poorly understood how fundamental processes at the organic-inorganic interface, such as protein adsorption and/or incorporation into minerals, regulates nucleation and crystal growth due to technical challenges in observing and characterizing mineral-bound organics at high-resolution. Here, atom probe tomography techniques were developed and applied to characterize amelogenin-mineralized HAP particles in vitro, revealing distinct organic-inorganic interfacial structures and processes at the nanoscale. Specifically, visualization of amelogenin across the mineralized particulate demonstrates protein can become entrapped during HAP crystal aggregation and fusion. Identification of protein signatures and structural interpretations were further supported by standards analyses, i.e., defined HAP surfaces with and without amelogenin adsorbed. These findings represent a significant advance in the characterization of interfacial structures and, more so, interpretation of fundamental organic-inorganic processes and mechanisms influencing crystal growth. Ultimately, this approach can be broadly applied to inform how potentially unique and diverse organic-inorganic interactions at different stages regulates the growth and evolution of various biominerals.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Dynamic entity formed by protein and its hydration water

The interaction between protein and water plays a pivotal role in shaping the structure, dynamics, and function of biomacromolecules. A comprehensive understanding of this intricate interplay necessitates a systematic evaluation of interaction strength and its consequential impact on the dynamics of proteins and water across diverse protein systems. Despite numerous works on understanding the dynamics of water and proteins and the coupling between them, there are still unanswered questions. Here, we combine neutron scattering and isotope labeling to probe the dynamics of proteins and their hydration water in a variety of protein systems. We consider proteins of different structures and varying thermostability as well as proteins within living cells with distinct growth temperatures. Simultaneous characterization of protein and hydration water dynamics across diverse systems was achieved. Moreover, we performed water sorption isothermal measurements on three representative proteins to correlate the observed dynamics with the strength of the interaction energies governing each system. The experimental results underscore that proteins manifesting stronger attractive interactions with water display diffusionlike dynamics with higher flexibility upon hydration, concomitant with a reduced mobility in hydration water. Significantly, our findings suggest that, in fact, it is the interaction between protein and its hydration water that facilitates the transfer of mobility from water to protein, with stronger interactions correlating to greater protein flexibility and slower hydration water diffusion. Published by the American Physical Society 2024

Ye, Yongfeng (ORCID:0009000792374198)↗
DOE OSTI2024

Anisotropic interactions for continuum modeling of protein–membrane systems

In this work, a model for anisotropic interactions between proteins and cellular membranes is proposed for large-scale continuum simulations. The framework of the model is based on dynamic density functional theory, which provides a formalism to describe the lipid densities within the membrane as continuum fields while still maintaining the fidelity of the underlying molecular interactions. Within this framework, we extend recent results to include the anisotropic effects of protein–lipid interactions. As applications, we consider two membrane proteins of biological interest: a RAS–RAF complex tethered to the membrane and a membrane embedded G protein-coupled receptor. A strong qualitative and quantitative agreement is found between the numerical results and the corresponding molecular dynamics simulations. Combining the scope of continuum level simulations with the details from molecular level particle simulations enables research into protein–membrane behaviors at a more biologically relevant scale, which crucially can also be accessed via experiment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Accurate prediction of protein structures and interactions using a three-track neural network

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo–electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. Here, we make the method available to the scientific community to speed biological research.

59 BASIC BIOLOGICAL SCIENCES↗