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At least 793 records · Page 44
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Investigating Electron Conductivity Regimes in the Bacterial Cytochrome Wire OmcS

The anaerobic bacterium Geobacter sulfurreducens produces extracellular, electronically conductive cytochrome polymer wires that are conductive over micron length scales. Structure models from cryo-electron microscopy data show OmcS wires form a linear chain of hemes along the protein wire axis, which is proposed as the structural basis supporting their electronic properties. However, the mechanism by which this heme arrangement supports long-range electronic conduction remains unknown. Structure models from cryo-electron microscopy data show these wires form a linear chain of hemes along the protein wire axis, which is proposed as the structural basis supporting their electronic properties. Existing computational models using static heme redox potentials and coupling energies fail to explain experimental observations, predicting conductances 10,000 to 100,000 times lower than measured values. Here, we investigate how dynamic disorder affects site energies, interheme coupling, and long-range electronic conductivity within these cytochrome wires. We introduce an approach to extract charge carrier site information directly from Kohn–Sham density functional theory, without employing projector schemes, and show that site and coupling energies are highly sensitive to changes in interheme geometry and the surrounding electrostatic environment. Unlike models that incorporate dynamic disorder as a thermally averaged quantity, our quantum charge carrier model incorporates proxies for dynamic disorder through decoherence corrections, yielding predicted diffusion coefficient closer to what is expected from experiment and comparable with other organic-based electronic materials. Based on these simulations, we propose that the instantaneous fluctuations of the local electrostatic environment can transiently lift energy degeneracies and delocalize charge carriers. Furthermore, these studies reveal how incorporating dynamic fluctuations associated with the environment resolves the discrepancy between theory and experiment in microbial cytochrome wires and highlight design principles for bioinspired, heme-based conductive materials.

Bioinorganic chemistry↗
DOE OSTI2023

Structural basis of agonist specificity of α 1A -adrenergic receptor

α 1 -adrenergic receptors (α 1 -ARs) play critical roles in the cardiovascular and nervous systems where they regulate blood pressure, cognition, and metabolism. However, the lack of specific agonists for all α 1 subtypes has limited our understanding of the physiological roles of different α 1 -AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α 1A -AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α 1A -AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α 1A -AR and α 1B -AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α 1A -AR mutants that are not responsive to A61603, and α 1B -AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α 1 -ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature

Abstract Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a challenge. Here, we report a straightforward liquid cell technique, originally developed for real-time visualization of dynamics at a liquid-gas interface using transmission electron microscopy, to image wet biological samples. Due to the scattering effects from the liquid phase, the micrographs display an amplitude contrast comparable to that observed in negatively stained samples. We succeed in resolving subunits within the protein complex GroEL imaged in a buffer solution at room temperature. Additionally, we capture various stages of virus cell entry, a process for which only sparse structural data exists due to their transient nature. To scrutinize the morphological details further, we used individual particle electron tomography for 3D reconstruction of each virus. These findings showcase this approach potential as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Integrative mapping reveals molecular features underlying the mechanism of nucleocytoplasmic transport

Nuclear pore complexes (NPCs) enable rapid, selective, and robust nucleocytoplasmic transport. To explain how transport emerges from the system components and their interactions, we used experimental data and theoretical information to construct an integrative Brownian dynamics model of transport through an NPC, coupled to a kinetic model of transport in the cell. The model recapitulates key aspects of transport for a wide range of molecular cargoes, including preribosomes and viral capsids. Our model quantifies how flexible phenylalanine-glycine (FG) repeat proteins create an entropic barrier to passive diffusion and how this barrier is selectively lowered in facilitated diffusion by the many transient interactions of nuclear transport receptors with the FG repeats. Selective transport is enhanced by “fuzzy” multivalent interactions, redundant FG repeat mass, coupling to the energy-dependent RanGTP concentration gradient, and exponential dependence of transport kinetics on the transport barrier. Our model will facilitate rational modulation of the NPC and its artificial mimics.

Raveh, Barak [School of Computer Science and Engin↗
DOE OSTI2024

High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2021

Viruses infecting a warm water picoeukaryote shed light on spatial co-occurrence dynamics of marine viruses and their hosts

The marine picoeukaryote Bathycoccus prasinos has been considered a cosmopolitan alga, although recent studies indicate two ecotypes exist, Clade BI (B. prasinos) and Clade BII. Viruses that infect Bathycoccus Clade BI are known (BpVs), but not that infect BII. We isolated three dsDNA prasinoviruses from the Sargasso Sea against Clade BII isolate RCC716. The BII-Vs do not infect BI, and two (BII-V2 and BII-V3) have larger genomes (~210 kb) than BI-Viruses and BII-V1. BII-Vs share ~90% of their proteins, and between 65% to 83% of their proteins with sequenced BpVs. Phylogenomic reconstructions and PolB analyses establish close-relatedness of BII-V2 and BII-V3, yet BII-V2 has 10-fold higher infectivity and induces greater mortality on host isolate RCC716. BII-V1 is more distant, has a shorter latent period, and infects both available BII isolates, RCC716 and RCC715, while BII-V2 and BII-V3 do not exhibit productive infection of the latter in our experiments. Global metagenome analyses show Clade BI and BII algal relative abundances correlate positively with their respective viruses. The distributions delineate BI/BpVs as occupying lower temperature mesotrophic and coastal systems, whereas BII/BII-Vs occupy warmer temperature, higher salinity ecosystems. Accordingly, with molecular diagnostic support, we name Clade BII Bathycoccus calidus sp. nov. and propose that molecular diversity within this new species likely connects to the differentiated host-virus dynamics observed in our time course experiments. Overall, the tightly linked biogeography of Bathycoccus host and virus clades observed herein supports species-level host specificity, with strain-level variations in infection parameters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cell-Free Screening, Production and Animal Testing of a STI-Related Chlamydial Major Outer Membrane Protein Supported in Nanolipoproteins

Background: Vaccine development against Chlamydia, a prevalent sexually transmied infection (STI), is imperative due to its global public health impact. However, significant challenges arise in the production of effective subunit vaccines based on recombinant protein antigens, particularly with membrane proteins like the Major Outer Membrane Protein (MOMP). Methods: Cellfree protein synthesis (CFPS) technology is an aractive approach to address these challenges as a method of high-throughput membrane protein and protein complex production coupled with nanolipoprotein particles (NLPs). NLPs provide a supporting scaffold while allowing easy adjuvant addition during formulation. Over the last decade, we have been working toward the production and characterization of MOMP-NLP complexes for vaccine testing. Results: The work presented here highlights the expression and biophysical analyses, including transmission electron microscopy (TEM) and dynamic light scaering (DLS), which confirm the formation and functionality of MOMP-NLP complexes for use in animal studies. Moreover, immunization studies in preclinical models compare the past and present protective efficacy of MOMP-NLP formulations, particularly when co-adjuvanted with CpG and FSL1. Conclusion: Ex vivo assessments further highlight the immunomodulatory effects of MOMP-NLP vaccinations, emphasizing their potential to elicitrobust immune responses. However, further research is warranted to optimize vaccine formulations further, validate efficacy against Chlamydia trachomatis, and beer understand the underlying mechanisms of immune response.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Direct Observation of the Dynamics of Single-Cell Metabolic Activity during Microbial Diauxic Growth

Understanding how genetic information is realized as the behavior of individual cells is a long-term goal of biology but represents a significant technological challenge. In clonal microbial populations, variation in gene regulation is often interpreted as metabolic heterogeneity. This follows the central dogma of biology, in which information flows from DNA to RNA to protein and ultimately manifests as activity. At present, DNA and RNA can be characterized in single cells, but the abundance and activity of proteins cannot. Inferences about metabolic activity usually therefore rely on the assumption that transcription reflects activity. By tracking the atoms from which they build their biomass, we make direct observations of growth rate and substrate specialization in individual cells throughout a period of growth in a changing environment. This approach allows the flow of information from DNA to be constrained from the distal end of the regulatory cascade and will become an essential tool in the rapidly advancing field of single-cell metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A monomeric mycobacteriophage immunity repressor utilizes two domains to recognize an asymmetric DNA sequence

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

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↗
DOE OSTI2024

Multitiered computational methodology for extracting three-dimensional rotational diffusion coefficients from x-ray photon correlation spectroscopy data without structural information

X-ray photon correlation spectroscopy (XPCS) is a powerful technique for analyzing particle systems by investigating their dynamics in suspensions across a broad range of temporal and spatial scales. This is done by illuminating samples with coherent x-ray beams and calculating the correlation function of the obtained x-ray scattering images. XPCS is uniquely suited for studying Brownian dynamics, consisting of translational and rotational diffusion. While traditional XPCS image analysis techniques can extract translational diffusion components, they are unable to estimate rotational diffusion coefficients. Here, we introduce a methodology that combines the angular-temporal cross-correlation analysis and a algorithmic framework called Multi-Tiered Estimation for Correlation Spectroscopy in 3D for estimating three-dimensional rotational diffusion coefficients from XPCS images of three-dimensional particle systems. We demonstrate our methodology for extracting rotational diffusion coefficients from XPCS data by applying it to simulated noisy x-ray images of systems of crossing nanotubes and proteins that evolve under translational and rotational Brownian motion for different diffusion rates. Furthermore, our results show that our approach determines rotational diffusion coefficients within a few percent error.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2020

Mechanisms of activation and desensitization of full-length glycine receptor in lipid nanodiscs

Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Lanthanide binding peptide surfactants at air–aqueous interfaces for interfacial separation of rare earth elements

Rare earth elements (REEs) are critical materials to modern technologies. They are obtained by selective separation from mining feedstocks consisting of mixtures of their trivalent cation. We are developing an all-aqueous, bioinspired, interfacial separation using peptides as amphiphilic molecular extractants. Lanthanide binding tags (LBTs) are amphiphilic peptide sequences based on the EF-hand metal binding loops of calcium-binding proteins which complex selectively REEs. We study LBTs optimized for coordination to Tb 3+ using luminescence spectroscopy, surface tensiometry, X-ray reflectivity, and X-ray fluorescence near total reflection, and find that these LBTs capture Tb 3+ in bulk and adsorb the complex to the interface. Molecular dynamics show that the binding pocket remains intact upon adsorption. We find that, if the net negative charge on the peptide results in a negatively charged complex, excess cations are recruited to the interface by nonselective Coulombic interactions that compromise selective REE capture. If, however, the net negative charge on the peptide is −3, resulting in a neutral complex, a 1:1 surface ratio of cation to peptide is achieved. Surface adsorption of the neutral peptide complexes from an equimolar mixture of Tb 3+ and La 3+ demonstrates a switchable platform dictated by bulk and interfacial effects. The adsorption layer becomes enriched in the favored Tb 3+ when the bulk peptide is saturated, but selective to La 3+ for undersaturation due to a higher surface activity of the La 3+ complex.

Ortuno Macias, Luis E. (ORCID:0000000284342192)↗
DOE OSTI2020

Microscale thermophoresis as a powerful tool for screening glycosyltransferases involved in cell wall biosynthesis

Background: Identification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of glycosyltransferases are very low throughput and acceptor substrates are generally not available. Results: We optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein-ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of glycosyltransferase family GT61 in sorghum for substrate screening and function prediction. Conclusions: This method is rapid and sensitive to allow determination of both donor and acceptor substrates of glycosyltransferases. MST enables high throughput screening of glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of glycosyltransferase function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cyclic Peptides for Lanthanide Binding

Lanthanide ions are difficult to separate from one another due to their similar chemical properties. The discovery of lanthanide-binding peptides and proteins in nature has led to an increased interest in the possibility of utilizing the strong binding of peptides to lanthanide ions for their separations; as such, there has been an effort to identify or design peptides with improved lanthanide binding and selectivity toward particular lanthanide ions. Here, in this study, we designed and characterized lanthanide-binding cyclic peptides (LBCPs) with molecular dynamics simulations, electronic structure calculations, and emission spectroscopy. Luminescent decay measurements were done to determine the number of water molecules coordinated to the Eu 3+ ion in Eu-LBCP complexes and compare to the predicted number of water molecules by computation to assess the lanthanide-binding affinity of LBCPs. Measured stability constants show binding of the LBCPs to the Eu 3+ ion with stronger than micromolar affinity. We were able to identify multiple peptides that selectively bind to middle lanthanides. We describe the structural basis of the lanthanide-binding selectivity trend with strongest binding to the middle lanthanides, followed by the heavier lanthanides, and finally to the lighter ions.

ions↗
DOE OSTI2022

Interplay between LHCSR proteins and state transitions governs the NPQ response in Chlamydomonas during light fluctuations

Abstract Photosynthetic organisms use sunlight as the primary energy source to fix CO 2 . However, in nature, light energy is highly variable, reaching levels of saturation for periods ranging from milliseconds to hours. In the green microalga Chlamydomonas reinhardtii , safe dissipation of excess light energy by nonphotochemical quenching (NPQ) is mediated by light‐harvesting complex stress‐related (LHCSR) proteins and redistribution of light‐harvesting antennae between the photosystems (state transition). Although each component underlying NPQ has been documented, their relative contributions to NPQ under fluctuating light conditions remain unknown. Here, by monitoring NPQ in intact cells throughout high light/dark cycles of various illumination periods, we find that the dynamics of NPQ depend on the timescales of light fluctuations. We show that LHCSRs play a major role during the light phases of light fluctuations and describe their role in growth under rapid light fluctuations. We further reveal an activation of NPQ during the dark phases of all high light/dark cycles and show that this phenomenon arises from state transition. Finally, we show that LHCSRs and state transition synergistically cooperate to enable NPQ response during light fluctuations. These results highlight the dynamic functioning of photoprotection under light fluctuations and open a new way to systematically characterize the photosynthetic response to an ever‐changing light environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Pathways and Mechanism of Caffeine Binding to Human Adenosine A 2A Receptor

Caffeine (CFF) is a common antagonist to the four subtypes of adenosine G-protein-coupled receptors (GPCRs), which are critical drug targets for treating heart failure, cancer, and neurological diseases. However, the pathways and mechanism of CFF binding to the target receptors remain unclear. In this study, we have performed all-atom-enhanced sampling simulations using a robust Gaussian-accelerated molecular dynamics (GaMD) method to elucidate the binding mechanism of CFF to human adenosine A 2A receptor (A 2A AR). Multiple 500–1,000 ns GaMD simulations captured both binding and dissociation of CFF in the A 2A AR. The GaMD-predicted binding poses of CFF were highly consistent with the x-ray crystal conformations with a characteristic hydrogen bond formed between CFF and residue N6.55 in the receptor. In addition, a low-energy intermediate binding conformation was revealed for CFF at the receptor extracellular mouth between ECL2 and TM1. While the ligand-binding pathways of the A 2A AR were found similar to those of other class A GPCRs identified from previous studies, the ECL2 with high sequence divergence serves as an attractive target site for designing allosteric modulators as selective drugs of the A 2A AR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Modeling bacterial microcompartment architectures for enhanced cyanobacterial carbon fixation

The carboxysome is a bacterial microcompartment (BMC) which plays a central role in the cyanobacterial CO 2 -concentrating mechanism. These proteinaceous structures consist of an outer protein shell that partitions Rubisco and carbonic anhydrase from the rest of the cytosol, thereby providing a favorable microenvironment that enhances carbon fixation. The modular nature of carboxysomal architectures makes them attractive for a variety of biotechnological applications such as carbon capture and utilization. In silico approaches, such as molecular dynamics (MD) simulations, can support future carboxysome redesign efforts by providing new spatio-temporal insights on their structure and function beyond in vivo experimental limitations. However, specific computational studies on carboxysomes are limited. Fortunately, all BMC (including the carboxysome) are highly structurally conserved which allows for practical inferences to be made between classes. Here, we review simulations on BMC architectures which shed light on (1) permeation events through the shell and (2) assembly pathways. These models predict the biophysical properties surrounding the central pore in BMC-H shell subunits, which in turn dictate the efficiency of substrate diffusion. Meanwhile, simulations on BMC assembly demonstrate that assembly pathway is largely dictated kinetically by cargo interactions while final morphology is dependent on shell factors. Overall, these findings are contextualized within the wider experimental BMC literature and framed within the opportunities for carboxysome redesign for biomanufacturing and enhanced carbon fixation.

59 BASIC BIOLOGICAL SCIENCES↗