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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Towards an Understanding of Oxidative Damage in an a-L-Arabinofuranosidase of Trichoderma reesei : a Molecular Dynamics Approach

Trichoderma reesei is a ‘workhorse’ fungus that produces glycosyl hydrolases (e.g., cellulases) at high titers for use in industrial bioprocessing. In this study, we focused on two side-chain hemicellulases, a-L-arabinofuranosidases and a-D-galactosidases, which are important for the treatment of lignocellulosic biomass, but susceptible to oxidative damage that can occur during industrial processing. The molecular details that render these enzymes inactive have not yet been identified. To approach this issue, we used proteomics to identify amino acid residues that were oxidized after a relevant oxidative treatment (Fenton reaction). These oxidative modifications were included in the 3D-protein structures, and using molecular dynamics simulations, we then studied the behaviors of non-modified and oxidized enzymes. These simulations showed significant alterations of the conformational stability of proteins when oxidized, as evidenced by changes in root mean square deviation (RMSD) and principal component analyses (PCA) trajectories. Likewise, enzyme-ligand interactions such as hydrogen bonds were greatly reduced in quantity and quality in the oxidized proteins. Finally, free energy landscape plots showed that there was a more rugged energy surface in the oxidized proteins, implying a less favorable reaction pathway. These results reveal the basis for loss of function in these carbohydrate active enzymes (CAZYs) in T. reesei.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Supercomputer-Based Ensemble Docking Drug Discovery Pipeline with Application to Covid-19

In this work, we present a supercomputer-driven pipeline for in silico drug discovery using enhanced sampling molecular dynamics (MD) and ensemble docking. Ensemble docking makes use of MD results by docking compound databases into representative protein binding-site conformations, thus taking into account the dynamic properties of the binding sites. We also describe preliminary results obtained for 24 systems involving eight proteins of the proteome of SARS-CoV-2. The MD involves temperature replica exchange enhanced sampling, making use of massively parallel supercomputing to quickly sample the configurational space of protein drug targets. Using the Summit supercomputer at the Oak Ridge National Laboratory, more than 1 ms of enhanced sampling MD can be generated per day. We have ensemble docked repurposing databases to 10 configurations of each of the 24 SARS-CoV-2 systems using AutoDock Vina. Comparison to experiment demonstrates remarkably high hit rates for the top scoring tranches of compounds identified by our ensemble approach. We also demonstrate that, using Autodock-GPU on Summit, it is possible to perform exhaustive docking of one billion compounds in under 24 h. Finally, we discuss preliminary results and planned improvements to the pipeline, including the use of quantum mechanical (QM), machine learning, and artificial intelligence (AI) methods to cluster MD trajectories and rescore docking poses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Residue Specific Characterization of Electrostatics, Conformational Heterogeneity, and Dynamics of Electron Transfer Proteins with Linear and Nonlinear Infrared Spectroscopy. Final Report

The goal of this research project was to further our understanding of biological electron transfer through the development and application of new methods for studying proteins and their complexes with high spatial and temporal detail. The approach was based on incorporation of reporter bonds with distinct vibrational frequencies at specific locations in proteins and their characterization through the interaction of the bonds with pulses of infrared light. The project focused on the proteins plastocyanin (Pc) and cytochrome (cyt) f, two electron transfer partners in photosynthesis. The first thrust of the project was the incorporation of carbon deuterium bonds as non-perturbative probes at a ligand to the copper ion of Pc and their use to detect changes in the bonding due to the number of electrons at the metal and how the bonding changes upon association with cyt f. Toward developing more advanced approaches to characterize proteins, we incorporated a stronger reporter, the cyano group of cyanophenylalanine, at several locations in Pc and measured the frequency dynamics in the absence and presence of cyt f. The acquired data improved the current model for the protein complex. Notably, the data indicated that the majority of the population is involved in a loosely bound complex, providing insight into the nature of the ensembles formed by electron transfer protein complexes that is not available via other approaches. In addition to learning about the protein complex, we made strides toward developing and improving the new experimental approach to study highly dynamic behaviors of proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

On the advantages of exploiting memory in Markov state models for biomolecular dynamics

Biomolecular dynamics play an important role in numerous biological processes. Markov State Models (MSMs) provide a powerful approach to study these dynamic processes by predicting long time scale dynamics based on many short molecular dynamics (MD) simulations. In an MSM, protein dynamics are modeled as a kinetic process consisting of a series of Markovian transitions between different conformational states at discrete time intervals (called “lag time”). To achieve this, a master equation must be constructed with a sufficiently long lag time to allow interstate transitions to become truly Markovian. This imposes a major challenge for MSM studies of proteins since the lag time is bound by the length of relatively short MD simulations available to estimate the frequency of transitions. Here, we show how one can employ the generalized master equation formalism to obtain an exact description of protein conformational dynamics both at short and long time scales without the time resolution restrictions imposed by the MSM lag time. Using a simple kinetic model, alanine dipeptide, and WW domain, we demonstrate that it is possible to construct these quasi-Markov State Models (qMSMs) using MD simulations that are 5–10 times shorter than those required by MSMs. These qMSMs only contain a handful of metastable states and, thus, can greatly facilitate the interpretation of mechanisms associated with protein dynamics. A qMSM opens the door to the study of conformational changes of complex biomolecules where a Markovian model with a few states is often difficult to construct due to the limited length of available MD simulations.

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

Molecular-dynamics simulation methods for macromolecular crystallography

It is investigated whether molecular-dynamics (MD) simulations can be used to enhance macromolecular crystallography (MX) studies. Historically, protein crystal structures have been described using a single set of atomic coordinates. Because conformational variation is important for protein function, researchers now often build models that contain multiple structures. Methods for building such models can fail, however, in regions where the crystallographic density is difficult to interpret, for example at the protein–solvent interface. To address this limitation, a set of MD–MX methods that combine MD simulations of protein crystals with conventional modeling and refinement tools have been developed. In an application to a cyclic adenosine monophosphate-dependent protein kinase at room temperature, the procedure improved the interpretation of ambiguous density, yielding an alternative water model and a revised protein model including multiple conformations. The revised model provides mechanistic insights into the catalytic and regulatory interactions of the enzyme. The same methods may be used in other MX studies to seek mechanistic insights.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Aedes aegypti Odorant Binding Protein 22 selectively binds fatty acids through a conformational change in its C-terminal tail

Aedes aegypti is the primary vector for transmission of Dengue, Zika and chikungunya viruses. Previously it was shown that Dengue virus infection of the mosquito led to an in increased expression of the odorant binding protein 22 (AeOBP22) within the mosquito salivary gland and that siRNA mediated knockdown of AeOBP22 led to reduced mosquito feeding behaviors. Insect OBPs are implicated in the perception, storage and transport of chemosensory signaling molecules including air-borne odorants and pheromones. AeOBP22 is unusual as it is additionally expressed in multiple tissues, including the antenna, the male reproductive glands and is transferred to females during reproduction, indicating multiple roles in the mosquito life cycle. However, it is unclear what role it plays in these tissues and what ligands it interacts with. Here we present solution and X-ray crystallographic studies that indicate a potential role of AeOBP22 binding to fatty acids, and that the specifcity for longer chain fatty acids is regulated by a conformational change in the C-terminal tail that leads to creation of an enlarged binding cavity that enhances binding afnity. This study sheds light onto the native ligands for AeOBP22 and provides insight into its potential functions in diferent tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ 54 -dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL–NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph,Azotobacter vinelandii.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Ion-selective conformational stabilization of a disordered repeats-in-toxin protein domain

Ion-binding intrinsically disordered proteins (IDPs) recruit and bind to specific metal ions to perform critical biological functions. In proteins where ion binding and structural transitions are coupled, interactions with off-target toxic metals can dramatically disrupt protein structure and function, exemplified by lead and mercury poisoning. Understanding the complex mechanisms underlying how IDPs exclude or allow binding to different ionic species is crucial for addressing the origins of metal toxicity in biological systems. Here, we elucidate mechanisms of ion selectivity in an IDP that adopts a structure upon Ca 2+ binding. We probed ion-induced conformational changes of a repeats-in-toxin (RTX) protein domain in the presence of different ion ligands—Mg 2+ , Ca 2+ , Sr 2+ , and Ba 2+ —with chemical similarities but drastically different ionic radii. RTX adopts ion-selective conformations measured by x-ray crystallography, small-angle x-ray scattering, and circular dichroism. High-resolution x-ray structures reveal that Sr 2+ induces a nearly identical RTX structure as natively binding Ca 2+ , enabled by the intrinsic flexibility and disorder of the protein. Small-angle x-ray scattering and circular dichroism indicate that smaller Mg 2+ does not induce a significant conformational change in RTX, whereas larger Ba 2+ induces a partially folded structure. These results highlight the importance of geometric constraints imposed by protein structure in determining metal ion selectivity, yielding insights into how off-target ion binding may result in protein misfolding and malfunction.

Gudinas, Alana P. [Stanford Univ., CA (United Stat↗
DOE OSTI2025

Serial-femtosecond crystallography reveals how a phytochrome variant couples chromophore and protein structural changes

The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state. Thus, structural data for seven time frames were sorted into groups of molecular events along the reaction coordinate. They range from chromophore isomerization to the formation of Meta-F, the intermediate that precedes the functional relevant secondary structure transition of the tongue. Structural data for the early events were used to calculate the photoisomerization pathway to complement the experimental data. Late events allow identifying the molecular switch that is linked to the intramolecular proton transfer as a prerequisite for the following structural transitions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure, dynamics, and redox reactivity of an all-purpose flavodoxin

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial en- zymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld’s ability to work with diverse partners, we solved its crystal structure. We observed the canonical fla- vodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize part- ner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies of protein– protein associations and conformational consequences, we assigned the 19F NMR signals of all five tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3- 19 F-Tyr in place of Tyr has only a modest effect on Rp9Fld’s redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the 19 F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However, the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We pro- pose that the loops surrounding the flavin adopt altered con- formations upon binding to partners and may even participate actively in electron transfer.

19F NMR↗
DOE OSTI2022

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure–function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal β-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (K d ≤ 20 μM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

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

An automated liquid jet for fluorescence dosimetry and microsecond radiolytic labeling of proteins

X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method’s overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

De novo design of allosteric proteins

Methods and compositions for making and isolating allosteric DNA binding proteins that bind to one or more allosteric effectors to induce a conformation change in the proteins are provided.

Raman, Srivatsan↗
DOE OSTI2023

Allosterically coupled conformational dynamics in solution prepare the sterol transfer protein StarD4 to release its cargo upon interaction with target membranes

Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Design of stimulus-responsive two-state hinge proteins

In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here, in this work, we describe the design of “hinge” proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled.

59 BASIC BIOLOGICAL SCIENCES↗