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26 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Cryo-electron tomography related radiation-damage parameters for individual-molecule 3D structure determination

To understand the dynamic structure–function relationship of soft- and biomolecules, the determination of the three-dimensional (3D) structure of each individual molecule (nonaveraged structure) in its native state is sought-after. Cryo-electron tomography (cryo-ET) is a unique tool for imaging an individual object from a series of tilted views. However, due to radiation damage from the incident electron beam, the tolerable electron dose limits image contrast and the signal-to-noise ratio (SNR) of the data, preventing the 3D structure determination of individual molecules, especially at high-resolution. Although recently developed technologies and techniques, such as the direct electron detector, phase plate, and computational algorithms, can partially improve image contrast/SNR at the same electron dose, the high-resolution structure, such as tertiary structure of individual molecules, has not yet been resolved. Here, we review the cryo-electron microscopy (cryo-EM) and cryo-ET experimental parameters to discuss how these parameters affect the extent of radiation damage. This discussion can guide us in optimizing the experimental strategy to increase the imaging dose or improve image SNR without increasing the radiation damage. With a higher dose, a higher image contrast/SNR can be achieved, which is crucial for individual-molecule 3D structure. With 3D structures determined from an ensemble of individual molecules in different conformations, the molecular mechanism through their biochemical reactions, such as self-folding or synthesis, can be elucidated in a straightforward manner.

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

Early-time measure in eternal inflation

In a situation like eternal inflation, where our data is replicated at infinitely-many other space-time events, it is necessary to make a prior assumption about our location to extract predictions. The principle of mediocrity entails that we live at asymptotic late times, when the occupational probabilities of vacua has settled to a near-equilibrium distribution. In this paper we further develop the idea that we instead exist during the approach to equilibrium, much earlier than the exponentially-long mixing time. In this case we are most likely to reside in vacua that are easily accessed dynamically. Using first-passage statistics, we prove that vacua that maximize their space-time volume at early times have: 1. maximal ever-hitting probability; 2. minimal mean first-passage time; and 3. minimal decay rate. These requirements are succinctly captured by an early-time measure. The idea that we live at early times is a predictive guiding principle, with many phenomenological implications. First, our vacuum should lie deep in a funneled region, akin to folding energy landscapes of proteins. Second, optimal landscape regions are characterized by relatively short-lived vacua, with lifetime of order the de Sitter Page time. For our vacuum, this lifetime is ~ 10 130 years, which is consistent with the Standard Model estimate due to Higgs metastability. Third, the measure favors vacua with small, positive vacuum energy. This can address the cosmological constant problem, provided there are sufficiently many vacua in the entire ensemble of funnels. As a concrete example, we study the Bousso-Polchinski lattice of flux vacua, and find that the early-time measure favors lattices with the fewest number of flux dimensions. This favors compactifications with a large hierarchy between the lightest modulus and all other Kähler and complex structure moduli.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2023

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Further, our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Note on the Effects of Linear Topology Preservation in Monte Carlo Simulations of Knotted Proteins

Monte Carlo simulations are a powerful technique and are widely used in different fields. When applied to complex molecular systems with long chains, such as those in synthetic polymers and proteins, they have the advantage of providing a fast and computationally efficient way to sample equilibrium ensembles and calculate thermodynamic and structural properties under desired conditions. Conformational Monte Carlo techniques employ a move set to perform the transitions in the simulation Markov chain. While accepted conformations must preserve the sequential bonding of the protein chain model and excluded volume among its units, the moves themselves may take the chain across itself. We call this a break in linear topology preservation. In this manuscript, we show, using simple protein models, that there is no difference in equilibrium properties calculated with a move set that preserves linear topology and one that does not. However, for complex structures, such as those of deeply knotted proteins, the preservation of linear topology provides correct equilibrium results but only after long relaxation. In any case, to analyze folding pathways, knotting mechanisms and folding kinetics, the preservation of linear topology may be an unavoidable requirement.

Especial, João N. C. (ORCID:0000000254765370)↗
DOE OSTI2020

Dueling Backbones: Comparing Peptoid and Peptide Analogues of a Mussel Adhesive Protein

Ensembles of amino acid side chains often dominate the interfacial interactions of intrinsically disordered proteins; however, backbone contributions are far from negligible. Using a combination of nanoscale force measurements and molecular dynamics simulations, we demonstrated with analogous mussel-mimetic adhesive peptides and peptoids both 34 residues long that highly divergent adhesive/cohesive outcomes can be achieved on mica surfaces by altering backbone chemistry only. The Phe, Tyr, and Dopa containing peptoid variants used in this study deposited as dehydrated and incompressible films that facilitated analysis of peptoid side chain contributions to adhesion and cohesion. For example, whereas Phe and Dopa peptoids exhibited similar cohesion, Dopa peptoids were ~3 times more adhesive than Phe peptoids on mica. Compared with the peptides, Phe peptoid achieved only ~20% of Phe containing peptide adhesion, but the Dopa peptoids were >2-fold more adhesive than the Dopa peptides. Cation-π interactions accounted for some but not all of the cohesive interactions. Finally, our results were corroborated by molecular dynamics simulations and highlight the importance of backbone chemistry and the potential of peptoids or peptoid/peptide hybrids as wet adhesives and primers.

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

Structural switching dynamically controls the doubly pseudoknotted Rous sarcoma virus–programmed ribosomal frameshifting element

A hallmark of retrovirus replication is the translation of two different polyproteins from one RNA through programmed –1 frameshifting. This is a mechanism in which the actively translating ribosome is induced to slip in the 5′ direction at a defined codon and then continues translating in the new reading frame. Programmed frameshifting controls the stoichiometry of viral proteins and is therefore under stringent evolutionary selection. Forty years ago, the first frameshifting stimulatory element was discovered in the Rous sarcoma virus. The ~120 nt RNA segment was predicted to contain a pseudoknot, but its 3D structure has remained elusive. Now, we have determined cryoEM and X-ray crystallographic structures of this classic retroviral element, finding that it adopts a butterfly-like double-pseudoknot fold. One “wing” contains a dynamic pyrimidine-rich helix, observed crystallographically in two conformations and in a third conformation via cryoEM. The other wing encompasses the predicted pseudoknot, which interacts with a second unexpected pseudoknot through a toggle residue, A2546. This key purine switches conformations between structural states and tunes the stability of interacting residues in the two wings. We find that its mutation can modulate frameshifting by as much as 50-fold, likely by altering the relative abundance of different structural states in the conformational ensemble of the RNA. Taken together, our structure–function analyses reveal how a dynamic double pseudoknot junction stimulates frameshifting by taking advantage of conformational heterogeneity, supporting a multistate model in which high Shannon entropy enhances frameshifting efficiency.

Science & Technology - Other Topics↗
NASA NTRS1998

Folding and translocation of the undecamer of poly-L-leucine across the water-hexane interface. A molecular dynamics study

The undecamer of poly-L-leucine at the water-hexane interface is studied by molecular dynamics simulations. This represents a simple model relevant to folding and insertion of hydrophobic peptides into membranes. The peptide, initially placed in a random coil conformation on the aqueous side of the system, rapidly translocates toward the hexane phase and undergoes interfacial folding into an alpha-helix in the subsequent 36 ns. Folding is nonsequential and highly dynamic. The initially formed helical segment at the N-terminus of the undecamer becomes transiently broken and, subsequently, reforms before the remainder of the peptide folds from the C-terminus. The formation of intramolecular hydrogen bonds during the folding of the peptide is preceded by a dehydration of the participating polar groups, as they become immersed in hexane. Folding proceeds through a short-lived intermediate, a 3(10)-helix, which rapidly interconverts to an alpha-helix. Both helices contribute to the equilibrium ensemble of folded structures. The helical peptide is largely buried in hexane, yet remains adsorbed at the interface. Its preferred orientation is parallel to the interface, although the perpendicular arrangement with the N-terminus immersed in hexane is only slightly less favorable. In contrast, the reversed orientation is highly unfavorable, because it would require dehydration of C-terminus carbonyl groups that do not participate in intramolecular hydrogen bonding. For the same reason, the transfer of the undecamer from the interface to the bulk hexane is also unfavorable. The results suggest that hydrophobic peptides fold in the interfacial region and, simultaneously, translocate into the nonpolar side of the interface. It is further implied that peptide insertion into the membrane is accomplished by rotating from the parallel to the perpendicular orientation, most likely in such a way that the N-terminus penetrates the bilayer.

NASA Discipline Exobiology↗
DOE OSTI2021

Evolutionary Diversification of Host-Targeted Bartonella Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure–function analyses of the highly versatile FIC domains.

59 BASIC BIOLOGICAL SCIENCES↗