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At least 523 records · Page 29
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Programming Amphiphilic Peptoid Oligomers for Hierarchical Assembly and Inorganic Crystallization

Natural organisms make a wide variety of exquisitely complex, nano-, micro-, and macroscale structured materials in an energy-efficient and highly reproducible manner. During these processes, the information-carrying biomolecules (e.g., proteins, peptides, and carbohydrates) enable (1) hierarchical organization to assemble scaffold materials and execute high-level functions and (2) exquisite control over inorganic materials synthesis, generating biominerals whose properties are optimized for their functions. Inspired by nature, significant efforts have been devoted to developing functional materials that can rival those natural molecules by mimicking in vivo functions using engineered proteins, peptides, DNAs, sequence-defined synthetic molecules (e.g., peptoids), and other biomimetic polymers. Among them, peptoids, a new type of synthetic mimetics of peptides and proteins, have received particular attention because they combine the merits of both synthetic polymers (e.g., high chemical stability and efficient synthesis) and biomolecules (e.g., sequence programmability and biocompatibility). The lack of both chirality and hydrogen bonds in their backbone results in a highly designable peptoid-based system with reduced structural complexity and side chain-chemistry-dominated properties. Here in this Account, we present our recent efforts in this field by programming amphiphilic peptoid sequences for (1) the controlled self-assembly into different hierarchically structured nanomaterials with favorable properties and (2) manipulating inorganic (nano)crystal nucleation, growth, and assembly into superstructures. First, we designed a series of amphiphilic peptoids with controlled side chain chemistries that self-assembled into 1D highly stiff and dynamic nanotubes, 2D membrane-mimetic nanosheets, hexagonally patterned nanoribbons, and 3D nanoflowers. These crystalline nanostructures exhibited sequence-dependent properties and showed promise for different applications. The corresponding peptoid self-assembly pathways and mechanisms were also investigated by leveraging in situ atomic force microscopy studies and molecular dynamics simulations, which showed precise sequence dependency. Second, inspired by peptide- and protein-controlled formation of hierarchical inorganic nanostructures in nature, we developed peptoid-based biomimetic approaches for controlled synthesis of inorganic materials (e.g., noble metals and calcite), in which we took advantage of the substantial side chain chemistry of peptoids and investigated the relationship between the peptoid sequences and the morphology and growth kinetics of inorganic materials. For example, to overcome the challenges (e.g., complexity of protein- and peptide-folding, poor thermal and chemical stabilities) facing the area of protein- and peptide-controlled synthesis of inorganic materials, we recently reported the design of sequence-defined peptoids for controlled synthesis of highly branched plasmonic gold particles. Moreover, we developed a rule of thumb for designing peptoids that predictively enabled the morphological evolution from spherical to coral-shaped gold nanoparticles (NPs). With this Account, we hope to stimulate the research interest of chemists and materials scientists and promote the predictive synthesis of functional and robust materials through the design of sequence-defined synthetic molecules.

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

Structural Heterogeneity and Hydrodynamics of an Intrinsically Disordered Protein Condensate

Biology demonstrates precise control over the free-energy landscape through the selective partitioning of biomacromolecules into membraneless organelles, enabling essential functions such as biochemical transformations, signaling cascades, and mechanical reinforcement. Although the function of these condensates depends on their underlying structure and hydrodynamics, molecular-scale information on these systems remains sparse. Here, in this study, neutron scattering is used to probe the organization and dynamics of the intrinsically disordered N-terminal domain of Galectin-3, an extracellular lectin responsible for facilitating liquid–liquid phase separation on the cellular surface, in both dilute and condensed phases. Dilute solutions contain isolated protein chains in equilibrium with mesoscopic clusters, whereas the condensed phase adopts a bicontinuous, microemulsion-like morphology. The dilute phase behavior is quantitatively described by coarse-grained polymer models from soft-matter physics, demonstrating their predictive power for complex biological proteins. At elevated concentrations, the proteins self-assemble akin to block copolymers, microphase separating through the aggregation of hydrophobic domains along the protein contour. The resulting condensate remains fluid-like despite a 25-fold increase in concentration; its internal hydrodynamics slow by only a factor of 3 relative to dilute protein chains. These results provide a molecular-level framework for how disordered proteins achieve both the structural complexity and dynamic fluidity of biomolecular condensates.

Carrick, Brian R. [Massachusetts Inst. of Technolo↗
DOE OSTI2020

Substrate Channeling via a Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase

Molecular dynamics studies showed that LDH and GAPDH tetramers can form a complex which can breakdown when the enzymes are saturated with NADH molecules. The interaction sites are conserved between LDH isozymes from heart and muscle cells, and between GAPDH molecules from rabbit and yeast cells. Positive electric fields between the NAD(H) binding sites showed that NAD(H) channeling within LDH-GAPDH complex can be an extension of NAD(H) channeling between the adjacent subunits within LDH and GAPDH tetramers. Analytical ultracentrifugation and enzyme kinetics studies indicated that LDH and GAPDH molecules can form a transient channeling complex. The transient complex can support the channeled and the diffusive paths in parallel, the relative contribution from each path depends on the off-rates for (GAPDH-NADH)-LDH complex and GAPDH-NADH complex. Negative cooperativity in NADH binding to different subunits in GAPDH tetramers can provide allosteric regulation of NADH channeling from GAPDH to LDH.

INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMI↗
DOE OSTI2024

Biomolecular condensates form spatially inhomogeneous network fluids

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Nuclear–Electronic Orbital QM/MM Approach: Geometry Optimizations and Molecular Dynamics

Hybrid quantum mechanical/molecular mechanical (QM/MM) methods allow simulations of chemical reactions in atomistic solvent and heterogeneous environments such as proteins. Herein, the nuclear–electronic orbital (NEO) QM/MM approach is introduced to enable the quantization of specified nuclei, typically protons, in the QM region using a method such as NEO-density functional theory (NEO-DFT). This approach includes proton delocalization, polarization, anharmonicity, and zero-point energy in geometry optimizations and dynamics. Expressions for the energies and analytical gradients associated with the NEO-QM/MM method, as well as the previously developed polarizable continuum model (NEO-PCM), are provided. Geometry optimizations of small organic molecules hydrogen bonded to water in either dielectric continuum solvent or explicit atomistic solvent illustrate that aqueous solvation can strengthen hydrogen-bonding interactions for the systems studied, as indicated by shorter intermolecular distances at the hydrogen-bond interface. We then performed a real-time direct dynamics simulation of a phenol molecule in explicit water using the NEO-QM/MM method. Furthermore, these developments and initial examples provide the foundation for future studies of nuclear–electronic quantum dynamics in complex chemical and biological environments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Dissipation Pathways in a Photosynthetic Complex

Determining how energy flows within and between molecules is crucial for understanding chemical reactions, material properties, and even vital processes such as photosynthesis. While the general principles of energy transfer are well established, elucidating the specific molecular pathways by which energy is funneled remains challenging, as it requires tracking energy flow in complex molecular environments. Here, we demonstrate how photon excitation energy is partially dissipated in the light-harvesting Fenna–Matthews–Olson (FMO) complex, mediating the excitation energy transfer from light-harvesting chlorosomes to the photosynthetic reaction center in green sulfur bacteria. Specifically, we isolate the contribution of the protein and specific vibrational modes of the pigment molecules to the energy dynamics. For this, we introduce an efficient computational implementation of a recently proposed theory of dissipation pathways for open quantum systems, based on second-order perturbation theory in the electronic couplings. Using it and a state-of-the-art FMO model with highly structured and chromophore-specific spectral densities, we demonstrate that energy dissipation is dominated by low-frequency modes (<800 cm –1 ) as their energy range is near-resonance with the energy gaps between electronic states of the pigments. We identify the most important modes for dissipation to be in-plane breathing modes (∼200 cm –1 ) of the bacteriochlorophylls in the complex. Conversely, far-detuned intramolecular vibrations with higher frequencies (>800 cm –1 ) play no role in dissipation. Interestingly, the FMO complex first needs to borrow energy from the environment to release excess photonic energy, indicating that the energy exchange between the system and thermal environment is not strictly unidirectional in time but involves a transient thermally activated step. Beyond their fundamental value, these insights can guide the development of artificial light-harvesting devices and, more broadly, engineer environments for chemical and quantum control tasks.

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

Molecular mechanism of biased signaling at the kappa opioid receptor

The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Extended experimental inferential structure determination method in determining the structural ensembles of disordered protein states

Proteins with intrinsic or unfolded state disorder comprise a new frontier in structural biology, requiring the characterization of diverse and dynamic structural ensembles. Here we introduce a comprehensive Bayesian framework, the Extended Experimental Inferential Structure Determination (X-EISD) method, which calculates the maximum log-likelihood of a disordered protein ensemble. X-EISD accounts for the uncertainties of a range of experimental data and back-calculation models from structures, including NMR chemical shifts, J-couplings, Nuclear Overhauser Effects (NOEs), paramagnetic relaxation enhancements (PREs), residual dipolar couplings (RDCs), hydrodynamic radii (R h ), single molecule fluorescence Förster resonance energy transfer (smFRET) and small angle X-ray scattering (SAXS). We apply X-EISD to the joint optimization against experimental data for the unfolded drkN SH3 domain and find that combining a local data type, such as chemical shifts or J-couplings, paired with long-ranged restraints such as NOEs, PREs or smFRET, yields structural ensembles in good agreement with all other data types if combined with representative IDP conformers.

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

Xanthophyll-cycle based model of the rapid photoprotection of Nannochloropsis in response to regular and irregular light/dark sequences

We explore the photoprotection dynamics of Nannochloropsis oceanica using time-correlated single photon counting under regular and irregular actinic light sequences. The varying light sequences mimic natural conditions, allowing us to probe the real-time response of non-photochemical quenching (NPQ) pathways. Durations of fluctuating light exposure during a fixed total experimental time and prior light exposure of the algae are both found to have a profound effect on NPQ. These observations are rationalized with a quantitative model based on the xanthophyll cycle and the protonation of LHCX1. The model is able to accurately describe the dynamics of non-photochemical quenching across a variety of light sequences. The combined model and observations suggest that the accumulation of a quenching complex, likely zeaxanthin bound to a protonated LHCX1, is responsible for the gradual rise in NPQ. Additionally, the model makes specific predictions for the light sequence dependence of xanthophyll concentrations that are in reasonable agreement with independent chromatography measurements taken during a specific light/dark sequence.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2003

Science Issues Associated with the Use of a Microfluidic Chip Designed Specifically for Protein Crystallization

The Iterative Biological Crystallization team in partnership with Caliper Technologies has produced a prototype microfluidic chip for batch crystallization that has been designed and tested. The chip is designed for the mixing and dispensing of up to five solutions with possible variation of the recipe being delivered to two growth wells. Developments that have led to the successful on-chip crystallization of a few model proteins have required investigative insight into many different areas, including fluid mixing dynamics, surface treatments, quantification and fidelity of reagent delivery. This presentation will encompass the ongoing studies and data accumulated toward these efforts.

Holmes, Anna M.↗
NASA NTRS2003

Plasma membrane disruption: repair, prevention, adaptation

Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

NASA Discipline Cell Biology↗
NASA NTRS2003

Dissipating Step Bunches during Crystallization under Transport Control

In studies of crystal formation by the generation and spreading of layers, equidistant step trains are considered unstable---bunches and other spatiotemporal patterns of the growth steps are viewed as ubiquitous. We provide an example to the opposite. We monitor the spatiotemporal dynamics of steps and the resulting step patterns during crystallization of the proteins ferritin and apoferritin using the atomic force microscope. The variations in step velocity and density are not correlated, indicating the lack of a long-range attraction between the steps. We show that (i) because of its coupling to bulk transport, nucleation of new layers is chaotic and occurs at the facet edges, where the interfacial supersaturation is higher; (ii) step bunches self-organize via the competition for supply from the solution; and, (iii) bunches of weakly interacting steps decay as they move along the face. Tests by numerical modeling support the conclusions about the mechanisms underlying our observations. The results from these systems suggest that during crystallization controlled by transport, with weakly or noninteracting growth steps, the stable kinetic state of the surface is an equidistant step train, and step bunches only arise during nucleation of new layers. Since nucleation only occurs at a few sites on the surface, the surface morphology may be controllably patterned or smoothened by locally controlling nucleation.

Lin, Hong↗
DOE OSTI2024

Dynamic Implications of Noncovalent Interactions in Amphiphilic Single-Chain Polymer Nanoparticles

Single-chain polymer nanoparticles (SCNPs) combine the chemical diversity of synthetic polymers with the intricate structure of biopolymers, generating versatile biomimetic materials. The mobility of polymer chain segments at length scales similar to secondary structural elements in proteins is critical to SCNP structure and thus function. However, the influence of noncovalent interactions used to form SCNPs (e.g., hydrogen-bonding and biomimetic secondary-like structure) on these conformational dynamics is challenging to quantitatively assess. To isolate the effects of noncovalent interactions on SCNP structure and conformational dynamics, we synthesized a series of amphiphilic copolymers containing dimethylacrylamide and monomers capable of forming these different interactions: (1) di(phenylalanine) acrylamide that forms intramolecular β-sheet-like cross-links, (2) phenylalanine acrylamide that forms hydrogen-bonds but lacks a defined local structure, and (3) benzyl acrylamide that has the lowest propensity for hydrogen-bonding. Each SCNP formed folded structures comparable to those of intrinsically disordered proteins, as observed by size exclusion chromatography and small angle neutron scattering. The dynamics of these polymers, as characterized by a combination of dynamic light scattering and neutron spin echo spectroscopy, was well described using the Zimm with internal friction (ZIF) model, highlighting the role of each noncovalent interaction to additively restrict the internal relaxations of SCNPs. These results demonstrate the utility of local scale interactions to control SCNP polymer dynamics, guiding the design of functional biomimetic materials with refined binding sites and tunable kinetics.

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

Enhanced Binding of SARS-CoV-2 Spike Protein to Receptor by Distal Polybasic Cleavage Sites

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a crucial role in binding the human cell receptor ACE2 that is required for viral entry. Many studies have been conducted to target the structures of RBD–ACE2 binding and to design RBD-targeting vaccines and drugs. Nevertheless, mutations distal from the SARS-CoV-2 RBD also impact its transmissibility and antibody can target non-RBD regions, suggesting the incomplete role of the RBD region in the spike protein–ACE2 binding. Here, in order to elucidate distant binding mechanisms, we analyze complexes of ACE2 with the wild-type spike protein and with key mutants via large-scale all-atom explicit solvent molecular dynamics simulations. We find that though distributed approximately 10 nm away from the RBD, the SARS-CoV-2 polybasic cleavage sites enhance, via electrostatic interactions and hydration, the RBD–ACE2 binding affinity. A negatively charged tetrapeptide (GluGluLeuGlu) is then designed to neutralize the positively charged arginine on the polybasic cleavage sites. We find that the tetrapeptide GluGluLeuGlu binds to one of the three polybasic cleavage sites of the SARS-CoV-2 spike protein lessening by 34% the RBD–ACE2 binding strength. This significant binding energy reduction demonstrates the feasibility to neutralize RBD–ACE2 binding by targeting this specific polybasic cleavage site. Our work enhances understanding of the binding mechanism of SARS-CoV-2 to ACE2, which may aid the design of therapeutics for COVID-19 infection.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1997

Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells

Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentrations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis.

NASA Discipline Cell Biology↗
DOE OSTI2020

Assessment of software methods for estimating protein-protein relative binding affinities

A growing number of computational tools have been developed to accurately and rapidly predict the impact of amino acid mutations on protein-protein relative binding affinities. Such tools have many applications, for example, designing new drugs and studying evolutionary mechanisms. In the search for accuracy, many of these methods employ expensive yet rigorous molecular dynamics simulations. By contrast, non-rigorous methods use less exhaustive statistical mechanics, allowing for more efficient calculations. However, it is unclear if such methods retain enough accuracy to replace rigorous methods in binding affinity calculations. This trade-off between accuracy and computational expense makes it difficult to determine the best method for a particular system or study. Here, eight non-rigorous computational methods were assessed using eight antibody-antigen and eight non-antibody-antigen complexes for their ability to accurately predict relative binding affinities (ΔΔG) for 654 single mutations. In addition to assessing accuracy, we analyzed the CPU cost and performance for each method using a variety of physico-chemical structural features. This allowed us to posit scenarios in which each method may be best utilized. Most methods performed worse when applied to antibody-antigen complexes compared to non-antibody-antigen complexes. Rosetta-based JayZ and EasyE methods classified mutations as destabilizing (ΔΔG < -0.5 kcal/mol) with high (83–98%) accuracy and a relatively low computational cost for non-antibody-antigen complexes. Some of the most accurate results for antibody-antigen systems came from combining molecular dynamics with FoldX with a correlation coefficient (r) of 0.46, but this was also the most computationally expensive method. Overall, our results suggest these methods can be used to quickly and accurately predict stabilizing versus destabilizing mutations but are less accurate at predicting actual binding affinities. This study highlights the need for continued development of reliable, accessible, and reproducible methods for predicting binding affinities in antibody-antigen proteins and provides a recipe for using current methods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Covalent Drug Binding in Live Cells Monitored by Mid-Infrared Quantum Cascade Laser Spectroscopy: Photoactive Yellow Protein as a Model System

The detection of drug-target interactions in live cells enables analysis of therapeutic compounds in a native cellular environment. Recent advances in spectroscopy and molecular biology have facilitated the development of genetically encoded vibrational probes like nitriles that can sensitively report on molecular interactions. Nitriles are powerful tools for measuring electrostatic environments within condensed media like proteins, but such measurements in live cells have been hindered by low signal-to-noise ratios. In this study, we design a spectrometer based on a double-beam quantum cascade laser (QCL)-based transmission infrared (IR) source with balanced detection that can significantly enhance sensitivity to nitrile vibrational probes embedded in proteins within cells compared to a conventional FTIR spectrometer. Here, using this approach, we detect small-molecule binding in Escherichia coli, with particular focus on the interaction between para-Coumaric acid (pCA) and nitrile-incorporated photoactive yellow protein (PYP). This system effectively serves as a model for investigating covalent drug binding in a cellular environment. Notably, we observe large spectral shifts of up to 15 cm –1 for nitriles embedded in PYP between the unbound and drug-bound states directly within bacteria, in agreement with observations for purified proteins. Such large spectral shifts are ascribed to the changes in the hydrogen-bonding environment around the local environment of nitriles, accurately modeled through high-level molecular dynamics simulations using the AMOEBA force field. Our findings underscore the QCL spectrometer’s ability to enhance sensitivity for monitoring drug–protein interactions, offering new opportunities for advanced methodologies in drug development and biochemical research.

chromophores↗