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All sources NASA NTRS DOE OSTI

DOE OSTI2022

Multi-head attention-based U-Nets for predicting protein domain boundaries using 1D sequence features and 2D distance maps

Abstract The information about the domain architecture of proteins is useful for studying protein structure and function. However, accurate prediction of protein domain boundaries (i.e., sequence regions separating two domains) from sequence remains a significant challenge. In this work, we develop a deep learning method based on multi-head U-Nets (called DistDom) to predict protein domain boundaries utilizing 1D sequence features and predicted 2D inter-residue distance map as input. The 1D features contain the evolutionary and physicochemical information of protein sequences, whereas the 2D distance map includes the structural information of proteins that was rarely used in domain boundary prediction before. The 1D and 2D features are processed by the 1D and 2D U-Nets respectively to generate hidden features. The hidden features are then used by the multi-head attention to predict the probability of each residue of a protein being in a domain boundary, leveraging both local and global information in the features. The residue-level domain boundary predictions can be used to classify proteins as single-domain or multi-domain proteins. It classifies the CASP14 single-domain and multi-domain targets at the accuracy of 75.9%, 13.28% more accurate than the state-of-the-art method. Tested on the CASP14 multi-domain protein targets with expert annotated domain boundaries, the average per-target F1 measure score of the domain boundary prediction by DistDom is 0.263, 29.56% higher than the state-of-the-art method.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Amantadine interactions with phase separated lipid membranes

Amantadine, a small amphilphic organic compound that consists of an adamantane backbone and an amino group, was first recognized as an antiviral in 1963 and received approval for prophylaxis against the type A influenza virus in 1976. Since then, it has also been used to treat Parkinson’s disease-related dyskinesia and is being considered as a treatment for corona viruses. Since amantadine usually targets membrane-bound proteins, its interactions with the membrane are also thought to be important. Biological membranes are now widely understood to be laterally heterogeneous and certain proteins are known to preferentially co-localize within specific lipid domains. Does amantadine, therefore, preferentially localize in certain lipid composition domains? To address this question, here, we studied amantadine’s interactions with phase separating membranes composed of cholesterol, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), as well as single-phase DPhPC (1,2-diphytanoyl-sn-glycero-3-phos-phocholine) membranes. From Langmuir trough and differential scanning calorimetry (DSC) measurements, we determined, respectively, that amantadine preferentially binds to disordered lipids, such as POPC, and lowers the phase transition temperature of POPC/DSPC/cholesterol mixtures, implying that amantadine increases membrane disorder. Further, using droplet interface bilayers (DIBs), we observed that amantadine disrupts DPhPC membranes, consistent with its disordering properties. Finally, we carried out molecular dynamics (MD) simulations on POPC/DSPC/cholesterol membranes with varying amounts of amantadine. Consistent with experiment, MD simulations showed that amantadine prefers to associate with disordered POPC-rich domains, domain boundaries, and lipid glycerol backbones. Since different proteins co-localize with different lipid domains, our results have possible implications as to which classes of proteins may be better targets for amantadine.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Structure-aware annotation of leucine-rich repeat domains

Protein domain annotation is typically done by predictive models such as HMMs trained on sequence motifs. However, sequence-based annotation methods are prone to error, particularly in calling domain boundaries and motifs within them. These methods are limited by a lack of structural information accessible to the model. With the advent of deep learning-based protein structure prediction, existing sequenced-based domain annotation methods can be improved by taking into account the geometry of protein structures. We develop dimensionality reduction methods to annotate repeat units of the Leucine Rich Repeat solenoid domain. The methods are able to correct mistakes made by existing machine learning-based annotation tools and enable the automated detection of hairpin loops and structural anomalies in the solenoid. The methods are applied to 127 predicted structures of LRR-containing intracellular innate immune proteins in the model plant Arabidopsis thaliana and validated against a benchmark dataset of 172 manually-annotated LRR domains.

Xu, Boyan↗

DOE OSTI2023

Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries

Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1998

Advanced Protein Crystallization Facility (APCF)

This section of the Life and Microgravity Spacelab (LMS) publication contains articles entitled: (1) Crystallization of EGFR-EGF; (2) Crystallization of Apocrustacyanin C1; (3) Crystallization and X-ray Analysis of 5S rRNA and the 5S rRNA Domain A; (4) Growth of Lysozyme Crystals at Low Nucleation Density; (5) Comparative Analysis of Aspartyl tRNA-synthetase and Thaumatin Crystals Grown on Earth and In Microgravity; (6) Lysosome Crystal Growth in the Advanced Protein Crystallization Facility Monitored via Mach-Zehnder Interferometry and CCD Video; (7) Analysis of Thaumatin Crystals Grown on Earth and in Microgravity; (8) Crystallization of the Nucleosome Core Particle; (9) Crystallization of Photosystem I; (10) Mechanism of Membrane Protein Crystal Growth: Bacteriorhodopsin-mixed Micelle Packing at the Consolution Boundary, Stabilized in Microgravity; (11) Crystallization in a Microgravity Environment of CcdB, a Protein Involved in the Control of Cell Death; and (12) Crystallization of Sulfolobus Solfataricus

Source record↗

NASA NTRS2002

The M2 Proton Channel of Influenza Virus: How Does It Work?

The transport of protons across membranes is an essential process for both bioenergetics of modem cells and the origins of cellular life. All living systems make use of proton gradients across cell walls to convert environmental energy into a high-energy chemical compound, adenosine triphosphate (ATR), synthesized from adenosine diphosphate. ATR, in turn, is used as a source of energy to drive many cellular reactions. The ubiquity of this process in biology suggests that even the earliest cellular systems were relying on proton gradient for harvesting environmental energy needed to support their survival and growth. In contemporary cells, proton transfer is assisted by large, complex proteins embedded in membranes. The issue addressed in this study was: how the same process can be accomplished with the aid of similar, but much simpler molecules that could have existed in the protobiological milieu? The model system used in the study contained a bilayer membrane made of phospholipid, dimyristoylphosphatidylcholine (DMPC), which is a good model of the biological membranes focusing cellular boundaries. Both sides of the bilayer were surrounded by water which simulated the environment inside and outside the cell. Embedded in the membrane was a fragment of the Influenza-A M2 protein and enough sodium counterions to maintain system neutrality. This protein has been shown to exhibit remarkably high rates of proton transport and, therefore, is an excellent model to study the formation of proton gradients across membranes. The Influenza M2 protein is 97 amino acids in length, but a fragment 25 amino acids long, which contains a transmembrane domain of 19 amino acids flanked by 3 amino acids on each side, is sufficient to transport protons. Four identical protein fragments, each folded into a helix, aggregate to form small channels spanning the membrane. Protons are conducted through a narrow pore in the middle of the channel in response to applied voltage. This channel is large enough to contain water molecules, and is normally filled with water. In analogy to the mechanism of proton transfer in some other channels, it has been postulated that protons are translocated along the network of water molecules filling the pore of the channel. This mechanism, however, must involve an additional, important step because the channel contains four histidine amino acid residues, one from each of the helices, which are sufficiently large to occlude the pore and interrupt the water network. The histidine residues ensure channel selectivity by blocking transport of small such as sodium or potassium. They have been also implicated in gating protons due to the ability of each histidine to become positively charged by accepting an additional proton. Two mechanisms of gating have been proposed. In one mechanism, all four histidines acquire an additional proton and, due to repulsion between their positive charges, move away from one another, thus opening the channel. The alternative mechanism relies of the ability of protons to move between different atoms in a molecule (tautomerization). Thus, a proton is captured on one side of the gate while another proton is released from the opposite side, and the molecule returns to the initial state through tautomerization. The simulations were designed to test these two mechanisms. Large-scale, atomic-level molecular dynamics simulations of the channel, in which the histidine residues were in different protonation states revealed that all intermediate states of the system involved in the tautomerization mechanism are structurally stable and the arrangement of water molecules in the channel is conducive to the proton transport. In contrast, in the four-protonated state, postulated to exist in the gate-opening mechanism, the electrostatic repulsion between the histidine residues appears to be so large that the channel looses its structural integrity and one helix moves away from the remaining three. This result indicates that such a mechanism of proton transport is unlikely. The simulations revealed that translocation along a network of water molecules in the channel and tautomerization of the histidine residues in the M2 proteins in the most likely mechanism of proton transport. The results not only explain how a remarkably simple protein system can efficiently aid in the formation of proton gradients across cell walls, but also suggest how this system can be genetically re-engineered to become a directional, reversible proton pump. Such a pump can provide energy to laboratory-built models of simple cellular systems. If they were successfully constructed it would greatly advance our understanding of the beginnings of life and find important applications in medicine and pharmacology.

Pohorille, Andrew↗

NASA NTRS2001

Proton Transport

The transport of protons across membranes is an essential process for both bioenergetics of modern cells and the origins of cellular life. All living systems make use of proton gradients across cell walls to convert environmental energy into a high-energy chemical compound, adenosine triphosphate (ATP), synthesized from adenosine diphosphate. ATP, in turn, is used as a source of energy to drive many cellular reactions. The ubiquity of this process in biology suggests that even the earliest cellular systems were relying on proton gradient for harvesting environmental energy needed to support their survival and growth. In contemporary cells, proton transfer is assisted by large, complex proteins embedded in membranes. The issue addressed in this Study was: how the same process can be accomplished with the aid of similar but much simpler molecules that could have existed in the protobiological milieu? The model system used in the study contained a bilayer membrane made of phospholipid, dimyristoylphosphatidylcholine (DMPC) which is a good model of the biological membranes forming cellular boundaries. Both sides of the bilayer were surrounded by water which simulated the environment inside and outside the cell. Embedded in the membrane was a fragment of the Influenza-A M$_2$ protein and enough sodium counterions to maintain system neutrality. This protein has been shown to exhibit remarkably high rates of proton transport and, therefore, is an excellent model to study the formation of proton gradients across membranes. The Influenza M$_2$ protein is 97 amino acids in length, but a fragment 25 amino acids long. which contains a transmembrane domain of 19 amino acids flanked by three amino acids on each side. is sufficient to transport protons. Four identical protein fragments, each folded into a helix, aggregate to form small channels spanning the membrane. Protons are conducted through a narrow pore in the middle of the channel in response to applied voltage. This channel is large enough to contain water molecules. and is normally filled with water. In analogy to the mechanism of proton transfer in some other channels, it has been postulated that protons are translocated along the network of water molecules filling the pore of the channel. This mechanism, however, must involve an additional important step because the channel contains four histidine amino acid residues, one from each of the helices, which are sufficiently large to occlude the pore and interrupt the water network. The histidine residues ensure channel selectivity by blocking transport of small ions, such as sodium or potassium. They have been also implicated in gating protons due to the ability of each histidine to become positively charged by accepting an additional proton. Two mechanisms of gating have been proposed. In one mechanism, all four histidines acquire an additional proton and, due to repulsion between their positive charges, move away from one another, thus opening the channel. The alternative mechanism relies of the ability of protons to move between different atoms in a molecule (tautomerization). Thus, a proton is captured on one side of the gate while another proton is released from the opposite side, and the molecule returns to the initial state through tautomerization. The simulations were designed to test these two mechanisms. Large-scale, atomic-level molecular dynamics simulations of the channel with the histidine residues in different protonation states revealed that all intermediate states of the system involved in the tautomerization mechanism are structurally stable and the arrangement of water molecules in the channel is conducive to the proton transport. In contrast, in the four-protonated state, postulated to exist in the gate-opening mechanism, the electrostatic repulsion between the histidine residues appears to be so large that the channel loses its structural integrity and one helix moves away from the remaining three. Additional information is contained within the original extended abstract.

Pohorille, Andrew↗

DOE OSTI2023

High-Throughput Directed Evolution of Marine Microalgae and Phototrophic Consortia for Improved Biomass Yields (Final Report)

Primary project achievements include using selective pressures (O 2 , light, temperature) and developing culturing regimes for the diatom Nitzschia inconspicua str. hildebrandi to attain enrichments with an ~90% increase in areal biomass productivity relative to the parental strain under pond-mimicking conditions with high O 2 stress in laboratory bioreactors. The resulting strain (GAI-337) was tested further for dilution time, culture density, CO 2 supplementation, pH, temperature, and dissolved O 2 concentration under outdoor pond-mimicking conditions to improve areal productivities. These experiments yielded an optimum harvest and dilution time just after sunset, ~0.45 g AFDW L -1 initial culture density for maximal productivities, no requirement for CO 2 supplementation or pH control, maximal performance under a diel temperature curve going from 24 °C at night to 36 °C during the day, and benefits from some O 2 removal from the culture by bubbling with air. Using pond-mimicking laboratory bioreactors, N. inconspicua GAI-337 achieved ~42 g AFDW m -2 d -1 . Nutrient limitation experiments resulted in a biomass composition that equated to ~160 Gallons of Gasoline Equivalent energy per ton AFDW, highlighting the potential of GAI-337 as a promising renewable fuel feedstock strain. Genome resequencing has revealed genome alterations potentially contributing to the improved growth of GAI-337 in the laboratory. Based on the comparative analyses of the GAI-337 and GAI-229 (reference) strains, we identified 144 single nucleotide substitutions that resulted in amino acid change, 7 single nucleotide substitutions that resulted in protein truncation; 5 deletions; and 1 frameshift mutation. From the mutations that potentially affect expression of functionally annotated genes, particular interest was noted for an interferon-induced 6-16 family protein that may be involved in the host immune response against microbe invasion; the chaperone protein DnaK, which may function to protect the folding of proteins within the cell; and SPRY domain protein that is found in many eukaryotic proteins important in cell signaling pathways. Transcriptome analysis revealed over 1000 genes with increased transcript levels. Many of these and many of the genes with mutations are not yet functionally annotated and an increased bioinformatics effort is necessary to more completely analyze the Nitzschia inconspicua genome. Adaptive laboratory evolution (ALE) was performed for over 300 days using consecutive 0.5°C temperature increases in a constant temperature incubator to attain greater thermal tolerance in Nitzschia inconspicua. The adapted strain was able to grow at a constant temperature of 37.5°C; whereas this constant temperature was lethal to the parental control, which had an upper temperature boundary of 35.5°C prior to adaptive evolution. Several high-temperature clonal isolates were obtained from the evolved population following ALE, and increased temperature tolerance was observed in clonal adapted cultures. The final temperature adaptation was maintained through cryopreservation and was observed in multiple clonal isolates, including multiple clonal isolates with significantly increased cell size, indicating the potential occurrence of a sexual cycle during the clonal isolation process. A survey of Nannochloropsis strains was conducted for tolerances to high pH and high bicarbonate media. Nannochloropsis granulata showed promising growth in diel bioreactors and was successfully grown at the GAI Kauai farm site in long-term growth campaigns. Co-culturing using Nitzschia inconspicua, Nannochloropsis and a cyanobacterium were assembled in the laboratory to determine if productivity synergies could be attained. Although all strains grew well in the laboratory high-bicarbonate media individually, the cyanobacterium quickly outgrew the other strains in the laboratory consortium pushing the co-culture away from a diverse (and potentially synergistic assemblage) phototroph culture towards a monoculture dominated by the cyanobacterium. Several outdoor growth campaigns were conducted, with productivities ranging between 10-20 g/m 2 /d of biomass. The best performing strain in the laboratory (GAI-337) did not outperform reference strains at the Kauai farm under the conditions used. Addition growth campaigns are necessary under conditions that result in higher biomass (>20 g/m 2 /d) and that attain higher O 2 levels are likely necessary. Initial data indicate that the thermally adapted strain did slightly better than the control strain at higher temperatures; however, additional campaigns are necessary to establish statistical significance. In summary, Nitzschia inconspicua is able to attain exemplary biomass and lipid yields in the laboratory bioreactors. Strain evolution to both O 2 and temperature resulted in targeted strain improvements. Additional outdoor campaigns are necessary to determine if laboratory improvements translate to the field.

09 BIOMASS FUELS↗

DOE OSTI2024

Tailoring Hierarchical Structure and Rare Earth Affinity of Compositionally Identical Polymers via Sequence Control

Macromolecule sequence, structure, and function are inherently intertwined. While well-established relationships exist in proteins, they are more challenging to define for synthetic polymer nanoparticles due to their molecular weight, sequence, and conformational dispersities. Furthermore, to explore the impact of sequence on nanoparticle structure, we synthesized a set of 16 compositionally identical, sequence-controlled polymers with distinct monomer patterning of dimethyl acrylamide and a bioinspired, structure-driving di(phenylalanine) acrylamide (FF). Sequence control was achieved through multiblock polymerizations, yielding unique ensembles of polymer sequences which were simulated by kinetic Monte Carlo simulations. Systematic analysis of the global (tertiary- and quaternary-like) structure in this amphiphilic copolymer series revealed the effect of multiple sequence descriptors: the number of domains, the hydropathy of terminal domains, and the patchiness (density) of FF within a domain, each of which impacted both chain collapse and the distribution of single- and multichain assemblies. Furthermore, both the conformational freedom of chain segments and local-scale, β-sheet-like interactions were sensitive to the patchiness of FF. To connect sequence, structure, and target function, we evaluated an additional series of nine sequence-controlled copolymers as sequestrants for rare earth elements (REEs) by incorporating a functional acrylic acid monomer into select polymer scaffolds. We identified key sequence variables that influence the binding affinity, capacity, and selectivity of the polymers for REEs. Collectively, these results highlight the potential of and boundaries of sequence control via multiblock polymerizations to drive primary sequence ensembles hierarchical structures, and ultimately the functionality of compositionally identical polymeric materials.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗