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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 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

In silico λ-dynamics predicts protein binding specificities to modified RNAs

Abstract RNA modifications shape gene expression through a variety of chemical changes to canonical RNA bases. Although numbering in the hundreds, only a few RNA modifications are well characterized, in part due to the absence of methods to identify modification sites. Antibodies remain a common tool to identify modified RNA and infer modification sites through straightforward applications. However, specificity issues can result in off-target binding and confound conclusions. This work utilizes in silico λ-dynamics to efficiently estimate binding free energy differences of modification-targeting antibodies between a variety of naturally occurring RNA modifications. Crystal structures of inosine and N6-methyladenosine (m6A) targeting antibodies bound to their modified ribonucleosides were determined and served as structural starting points. λ-Dynamics was utilized to predict RNA modifications that permit or inhibit binding to these antibodies. In vitro RNA-antibody binding assays supported the accuracy of these in silico results. High agreement between experimental and computed binding propensities demonstrated that λ-dynamics can serve as a predictive screen for antibody specificity against libraries of RNA modifications. More importantly, this strategy is an innovative way to elucidate how hundreds of known RNA modifications interact with biological molecules without the limitations imposed by in vitro or in vivo methodologies.

Biochemistry & Molecular Biology↗
DOE OSTI2023

PTM‐Psi : A python package to facilitate the computational investigation of p ost‐ t ranslational m odification on p rotein s tructures and their i mpacts on dynamics and functions

Abstract Post‐translational modification (PTM) of a protein occurs after it has been synthesized from its genetic template, and involves chemical modifications of the protein's specific amino acid residues. Despite of the central role played by PTM in regulating molecular interactions, particularly those driven by reversible redox reactions, it remains challenging to interpret PTMs in terms of protein dynamics and function because there are numerous combinatorially enormous means for modifying amino acids in response to changes in the protein environment. In this study, we provide a workflow that allows users to interpret how perturbations caused by PTMs affect a protein's properties, dynamics, and interactions with its binding partners based on inferred or experimentally determined protein structure. This Python‐based workflow, called PTM‐Psi , integrates several established open‐source software packages, thereby enabling the user to infer protein structure from sequence, develop force fields for non‐standard amino acids using quantum mechanics, calculate free energy perturbations through molecular dynamics simulations, and score the bound complexes via docking algorithms. Using the S ‐nitrosylation of several cysteines on the GAP2 protein as an example, we demonstrated the utility of PTM‐Psi for interpreting sequence–structure–function relationships derived from thiol redox proteomics data. We demonstrate that the S ‐nitrosylated cysteine that is exposed to the solvent indirectly affects the catalytic reaction of another buried cysteine over a distance in GAP2 protein through the movement of the two ligands. Our workflow tracks the PTMs on residues that are responsive to changes in the redox environment and lays the foundation for the automation of molecular and systems biology modeling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Temporal dynamics of protein and post-translational modification abundances in Populus leaf across a diurnal period

Populus spp. are dedicated woody biomass feedstocks for advanced biofuels and bioproducts. Proper growth and fitness of poplar as a sustainable feedstock depends on timely perception and response to environmental signals (e.g., light, temperature, water). Poplar leaves, like other C3 photosynthesis plants, have evolved oscillating or circadian rhythms that play important roles in synchronizing biological processes with external cues. To characterize this phenomenon at a molecular level, we employed bottom-up proteomics using high-resolution mass spectrometry and de novo-assisted database searching to identify abundance changes in proteins and post-translational modifications in poplar leaf tissue sampled across a 12/12-hour light/dark diurnal period.

09 BIOMASS FUELS↗
DOE OSTI2022

Dynamics retrieval from stochastically weighted incomplete data by low-pass spectral analysis

Time-resolved serial femtosecond crystallography (TR-SFX) provides access to protein dynamics on sub-picosecond timescales, and with atomic resolution. Due to the nature of the experiment, these datasets are often highly incomplete and the measured diffracted intensities are affected by partiality. To tackle these issues, one established procedure is that of splitting the data into time bins, and averaging the multiple measurements of equivalent reflections within each bin. This binning and averaging often involve a loss of information. Here, we propose an alternative approach, which we call low-pass spectral analysis (LPSA). In this method, the data are projected onto the subspace defined by a set of trigonometric functions, with frequencies up to a certain cutoff. This approach attenuates undesirable high-frequency features and facilitates retrieving the underlying dynamics. A time-lagged embedding step can be included prior to subspace projection to improve the stability of the results with respect to the parameters involved. Subsequent modal decomposition allows to produce a low-rank description of the system's evolution. Using a synthetic time-evolving model with incomplete and partial observations, we analyze the LPSA results in terms of quality of the retrieved signal, as a function of the parameters involved. We compare the performance of LPSA to that of a range of other sophisticated data analysis techniques. We show that LPSA allows to achieve excellent dynamics reconstruction at modest computational cost. Finally, we demonstrate the superiority of dynamics retrieval by LPSA compared to time binning and merging, which is, to date, the most commonly used method to extract dynamical information from TR-SFX data.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Orthogonal Degron System for Controlled Protein Degradation in Cyanobacteria

Synechococcus elongatus PCC 7942 is a model cyanobacterium for study of the circadian clock, photosynthesis, and bioproduction of chemicals, yet nearly 40% of its gene identities and functions remain unknown, in part due to limitations of the existing genetic toolkit. While classical techniques for the study of genes (e.g., deletion or mutagenesis) can yield valuable information about the absence of a gene and its associated protein, there are limits to these approaches, particularly in the study of essential genes. In this research, we developed a tool for inducible degradation of target proteins in S. elongatus by adapting a method using degron tags from the Mesoplasma florum transfer-messenger RNA (tmRNA) system. We observed that M. florum lon protease can rapidly degrade exogenous and native proteins tagged with the cognate sequence within hours of induction. We used this system to inducibly degrade the essential cell division factor, FtsZ, as well as shell protein components of the carboxysome. Our results have implications for carboxysome biogenesis and the rate of carboxysome turnover during cell growth. Lon protease control of proteins offers an alternative approach for the study of essential proteins and protein dynamics in cyanobacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Design of light- and chemically responsive protein assemblies through host-guest interactions

Host-guest (HG) interactions have been widely used to build responsive materials and molecular machines owing to their inherently dynamic nature, interaction specificity, and responsiveness to diverse stimuli. Here, in this work, we have set out to exploit these advantages of HG chemistry in the design of dynamic protein assemblies, using a C 4 symmetric protein, C98 RhuA, as a building block. We show that a C98 RhuA variant individually modified with β-cyclodextrin (βCD) (host) or azobenzene (guest) functionalities can specifically pair with each other to form highly ordered 1D and 2D assemblies. Association and dissociation of βCD RhuA- azo RhuA assemblies can be controlled by UV and visible light as well as by small-molecule modulators of βCD-azobenzene interactions. Kinetics analyses reveal that βCD RhuA- azo RhuA nanotubes assemble without a nucleation barrier, a highly unusual occurrence for helical supramolecular systems. Taken together, our findings provide a compelling example for achieving complex structural and dynamic outcomes in protein assembly through simple chemical design.

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

A conserved chaperone protein is required for the formation of a noncanonical type VI secretion system spike tip complex

Type VI secretion systems (T6SSs) are dynamic protein nanomachines found in Gram-negative bacteria that deliver toxic effector proteins into target cells in a contact-dependent manner. Prior to secretion, many T6SS effector proteins require chaperones and/or accessory proteins for proper loading onto the structural components of the T6SS apparatus. However, despite their established importance, the precise molecular function of several T6SS accessory protein families remains unclear. In this study, we set out to characterize the DUF2169 family of T6SS accessory proteins. Using gene co-occurrence analyses, we find that DUF2169-encoding genes strictly co-occur with genes encoding T6SS spike complexes formed by valine-glycine repeat protein G (VgrG) and DUF4150 domains. Although structurally similar to Pro-Ala-Ala-Arg (PAAR) domains, “PAAR-like” DUF4150 domains lack PAAR motifs and instead contain a conserved PIPY motif, leading us to designate them PIPY domains. Next, we present both genetic and biochemical evidence that PIPY domains require a cognate DUF2169 protein to form a functional T6SS spike complex with VgrG. This contrasts with canonical PAAR proteins, which bind VgrG on their own to form functional spike complexes. By solving the first crystal structure of a DUF2169 protein, we show that this T6SS accessory protein adopts a novel protein fold. Furthermore, biophysical and structural modeling data suggest that DUF2169 contains a dynamic loop that physically interacts with a hydrophobic patch on the surface of its cognate PIPY domain. Based on these findings, we propose a model whereby DUF2169 proteins function as molecular chaperones that maintain VgrG–PIPY spike complexes in a secretion-competent state prior to their export by the T6SS apparatus.

DUF2169↗
DOE OSTI2026

Towards time-resolved MicroED grid preparation using mix-and-inject gas dynamic virtual nozzles

Recent progress in gas dynamic virtual nozzle (GDVN) technologies in combination with high-brilliance synchrotron and X-ray free-electron lasers (XFELs) has allowed the visualization of protein dynamics in crystallo by mixing macromolecular protein crystals with a substrate using tunable mixing times on the order of milliseconds to seconds prior to serial X-ray diffraction data collection. This has become the method of choice for high-resolution structure determination of intermediate states. However, such experiments require large counts of crystals of proper sizes for high-resolution data collection, and premium beam times for screening efforts. Cryogenic microcrystal electron diffraction (MicroED) represents a complementary technique that may be a more accessible avenue for time-resolved nanocrystallography compared with serial X-ray diffraction experiments. MicroED can produce full diffraction datasets from just a few submicrometre-thick crystals, and the approach is more readily accessible, requiring standard cryogenic transmission electron microscopy (TEM) equipment available at many universities and institutes. Cryogenic MicroED, like other forms of cryo-EM, begins with rapidly freezing biological material on electron microscopy grids. In the case of MicroED, micro- to nano-crystals (<500 nm thick) are deposited onto electron microscopy grids and plunge-frozen for subsequent electron diffraction data collection. Here, we have incorporated GDVN technology developed originally for XFEL experiments into the freezing process as a first step towards time-resolved studies. We describe the limited deposition efficiency of the model MicroED protein proteinase K on TEM grids using GDVNs, preceding sample vitrification and successful MicroED data collection. We discuss both the initial results from such experiments and the methodological challenges in developing this approach into a reliable workflow for millisecond-to-second time-resolved structural studies of macromolecules. Our results promise a strategy to deposit crystals on grids using GDVNs and determine high-resolution structures by MicroED, constituting a first step towards development of time-resolved MicroED experiments.

MicroED↗
DOE OSTI2022

Conserved conformational dynamics determine enzyme activity

Homologous enzymes often exhibit different catalytic rates despite a fully conserved active site. The canonical view is that an enzyme sequence defines its structure and function and, more recently, that intrinsic protein dynamics at different time scales enable and/or promote catalytic activity. Here, we show that, using the protein tyrosine phosphatase PTP1B, residues surrounding the PTP1B active site promote dynamically coordinated chemistry necessary for PTP1B function. However, residues distant to the active site also undergo distinct intermediate time scale dynamics and these dynamics are correlated with its catalytic activity and thus allow for different catalytic rates in this enzyme family. We identify these previously undetected motions using coevolutionary coupling analysis and nuclear magnetic resonance spectroscopy. Our findings strongly indicate that conserved dynamics drives the enzymatic activity of the PTP family. Characterization of these conserved dynamics allows for the identification of novel regulatory elements (therapeutic binding pockets) that can be leveraged for the control of enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Solvent organization in the ultrahigh-resolution crystal structure of crambin at room temperature

Ultrahigh-resolution structures provide unprecedented details about protein dynamics, hydrogen bonding and solvent networks. The reported 0.70 Å, room-temperature crystal structure of crambin is the highest-resolution ambient-temperature structure of a protein achieved to date. Sufficient data were collected to enable unrestrained refinement of the protein and associated solvent networks using SHELXL . Dynamic solvent networks resulting from alternative side-chain conformations and shifts in water positions are revealed, demonstrating that polypeptide flexibility and formation of clathrate-type structures at hydrophobic surfaces are the key features endowing crambin crystals with extraordinary diffraction power.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗