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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Quantum-enhanced photoprotection in neuroprotein architectures emerges from collective light-matter interactions

Background Superradiance is the phenomenon of many identical quantum systems absorbing and/or emitting photons collectively at a higher rate than any one system can individually. This phenomenon has been studied analytically in idealized distributions of electronic two-level systems (TLSs), each with a ground and excited state, as well as numerically in realistic photosynthetic nanotubes and cytoskeletal architectures. Methods Superradiant effects are studied here in idealized toy model systems and realistic biological mega-networks of tryptophan (Trp) molecules, which are strongly fluorescent amino acids found in many proteins. Each Trp molecule acts as a chromophore absorbing in the ultraviolet spectrum and can be treated approximately as a TLS, with its 1 L a excited singlet state; thus, organized Trp networks can exhibit superradiance. Such networks are found, for example, in microtubules, actin filaments, and amyloid fibrils. Microtubules and actin filaments are spiral-cylindrical protein polymers that play significant biological roles as primary constituents of the eukaryotic cytoskeleton, while amyloid fibrils have been targeted in a variety of neurodegenerative diseases. We treat these proteinaceous Trp networks as open quantum systems, using a non-Hermitian Hamiltonian to describe interactions of the chromophore network with the electromagnetic field. We numerically diagonalize the Hamiltonian to obtain its complex eigenvalues, where the real part is the energy and the imaginary part is its associated enhancement rate. We also consider multiple realizations of increasing static disorder in either the site energies or the decay rates. Results We obtained the energies and enhancement rates for realistic microtubules, actin filament bundles, and amyloid fibrils of differing lengths, and we use these values to calculate the quantum yield, which is the ratio of the number of photons emitted to the number of photons absorbed. We find that all three of these structures exhibit highly superradiant states near the low-energy portion of the spectrum, which enhances the magnitude and robustness of the quantum yield to static disorder and thermal noise. Conclusion The high quantum yield and stable superradiant states in these biological architectures may play a photoprotective rolein vivo, downconverting energetic ultraviolet photons—absorbed from those emitted by reactive free radical species—to longer, safer wavelengths and thereby mitigating biochemical stress and photophysical damage. Contrary to conventional assumptions that quantum effects cannot survive in large biosystems at high temperatures, our results suggest that macropolymeric collectives of TLSs in microtubules, actin filaments, and amyloid fibrils exhibit increasingly observable and robust effects with increasing length, up to the micron scale, due to quantum coherent interactions in the single-photon limit. Superradiant enhancement and high quantum yield exhibited in neuroprotein polymers could thus play a crucial role in information processing in the brain, the development of neurodegenerative diseases such as Alzheimer’s and related dementias, and a wide array of other pathologies characterized by anomalous protein aggregates.

Physics↗
DOE OSTI2022

Putting Humpty Dumpty Back Together Again: What Does Protein Quantification Mean in Bottom-Up Proteomics?

Bottom-up proteomics provides peptide measurements and has been invaluable for moving proteomics into large-scale analyses. Commonly, a single quantitative value is reported for each protein-coding gene by aggregating peptide quantities into protein groups following protein inference or parsimony. However, given the complexity of both RNA splicing and post-translational protein modification, it is overly simplistic to assume that all peptides that map to a singular protein-coding gene will demonstrate the same quantitative response. Here, by assuming that all peptides from a protein-coding sequence are representative of the same protein, we may miss the discovery of important biological differences. To capture the contributions of existing proteoforms, we need to reconsider the practice of aggregating protein values to a single quantity per protein-coding gene.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Mesoscale fractal whey protein particles derived from microscale linear-shaped protein assemblies (Part 1): Manufacturing method and particle characteristics

Whey protein isolates (WPI) are widely used in processed foods for their versatile functional properties. Modifying the structural properties of proteins by assembling them into mesoscale or microscale particles may improve their functionality and broaden their applications. This study aims to manufacture and characterize mesoscale whey protein particles (WPP) derived from WPI. Two types of WPP, WPP1 (0.05 mL/min) and WPP2 (0.25 mL/min), were prepared through a multistep approach involving liquid antisolvent (LAS) precipitation, heat treatment, and microfluidization. Liquid antisolvent precipitation was performed by injecting a 20% (wt/vol) WPI dispersion (pH 7) into an ethanol-glycerol mixture (75:25, vol/vol) under laminar flow, followed by heat treatment at 80°C for 20 min as a particle hardening step. This process produced stable fiber- and ribbon-shaped whey protein assemblies (WPA), which served as precursors to WPP. Subsequent microfluidization (150 MPa, 6 passages) reduced the size of WPA, yielding mesoscale WPP with irregular morphologies and a more uniform size distribution, as revealed by microscopy and dynamic light scattering. ζ-Potential and fluorescence labeling indicated higher surface charge and surface hydrophobicity of WPP compared with untreated WPI. The WPP showed internal mass fractal and surface fractal structures at larger length scales, analyzed using small-angle X-ray scattering. Fourier transform infrared spectroscopy demonstrated an increased fraction of intermolecular β-sheets in WPP, suggesting that hydrogen bonding contributed to their formation. Gel electrophoresis confirmed that disulfide bonds served as the primary cross-links stabilizing the WPP structure. Furthermore, turbidity measurements showed that WPP exhibited superior colloidal phase stability compared with untreated WPI and maintained high colloidal stability under both acidic and neutral pH conditions.

Antisolvent precipitation↗
NASA NTRS1993

Characterization of auxin-binding proteins from zucchini plasma membrane

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

NASA Program Space Biology↗
NASA NTRS2006

Stress, and pathogen response gene expression in modeled microgravity

Purpose: Immune suppression in microgravity has been well documented. With the advent of human exploration and long-term space travel, the immune system of the astronaut must be optimally maintained. It is important to investigate the expression patterns of cytokine genes, because they are directly related to immune response. Heat shock proteins (HSPs), also called stress proteins, are a group of proteins that are present in the cells of every life form. These proteins are induced when a cell responds to stressors such as heat, cold and oxygen deprivation. Microgravity is another stressor that may regulate HSPs. Heat shock proteins trigger immune response through activities that occur both inside the cell (intracellular) and outside the cell (extracellular). Knowledge about these two gene groups could lead to establishment of a blueprint of the immune response and adaptation-related genes in the microgravity environment. Methods: Human peripheral blood cells were cultured in 1g (T flask) and modeled microgravity (MMG, rotating-wall vessel) for 24 and 72 hours. Cell samples were collected and subjected to gene array analysis using the Affymetrix HG_U95 array. Data was collected and subjected to a two-way analysis of variance. The genes related to immune and stress responses were analyzed. Results and Conclusions: HSP70 was up-regulated by more than two fold in microgravity culture, while HSP90 was significantly down-regulated. HSP70 is not typically expressed in all kinds of cells, but it is expressed at high levels in stress conditions. HSP70 participates in translation, protein translocation, proteolysis and protein folding, suppressing aggregation and reactivating denatured proteins. Increased serum HSP70 levels correlate with a better outcome for heat-stroke or severe trauma patients. At the same time, elevated serum levels of HSP70 have been detected in patients with peripheral or renal vascular disease. HSP90 has been identified in the cytosol, nucleus and endoplasmic reticulum, and exists in many tissue types. HSP90 associates with actin filaments in certain conditions and aids cell motility. The down-regulation of HSP90 could lead to deleterious effects in the lymphocytes, thereby contributing to suppressed immune function in microgravity. Interleukins such as IL 1 alpha, IL11 receptor chain alpha, IL7R, and IL4R were significantly down regulated in modeled microgravity. Further analysis of the genes involved in immune response at the protein level may provide a basis for prophylactic and countermeasure strategies to augment the human immune system for space exploration.

Sundaresan, Alamelu↗
DOE OSTI2022

Superoxide Dismutase 1 Folding Stability as a Target for Molecular Tweezers in SOD1-Related Amyotrophic Lateral Sclerosis

Protein misfolding and aggregation are hallmarks of many severe neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease. As a supramolecular ligand that binds to lysine and arginine residues, the molecular tweezer CLR01 was found to modify the aggregation pathway of disease-relevant proteins in vitro and in vivo with beneficial effects on toxicity. However, the molecular mechanisms of how tweezers exert these effects remain mainly unknown, hampering further drug development. Here, we investigate the modulation mechanism of unfolding and aggregation pathways of SOD1, which are involved in amyotrophic lateral sclerosis (ALS), by CLR01. Using a truncated version of the wildtype SOD1 protein, SOD1 bar , we show that CLR01 acts on the first step of the aggregation pathway, the unfolding of the SOD1 monomer. CLR01 increases, by ~10 °C, the melting temperatures of the A4V and G41D SOD1 mutants, which are commonly observed mutations in familial ALS. Molecular dynamics simulations and binding free energy calculations as well as native mass spectrometry and mutational studies allowed us to identify K61 and K92 as binding sites for the tweezers to mediate the stability increase. The data suggest that the modulation of SOD1 conformational stability is a promising target for future developments of supramolecular ligands against neurodegenerative diseases.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2002

Peptides at Membrane Surfaces and their Role in Prebiotic Evolution

Protocells had to transport ions and organic matter across membranes separating the interior of the cell from the environment, capture and utilize energy and transduce environmental signals. In a series of detailed, molecular-level computer simulations we show how these peptides in contact with membranes can acquire ordered structures and functions. We have investigated the stability of a simple alpha-helical peptide containing Leucine (L) and Serine (S) of the form (LSLLLSL)3 in a model membrane system. The parallel in-plane state is the most stable configuration. The transmembrane state is metastable, and about 15 kcal/mol is required to insert the peptide into the membrane. We investigated dimes of both (LSLLLSL)3 and glycophorin A, and show how the free energy of helix association can, at least partially, offset the free energy of insertion. We have also investigated the transmembrane pore of the influenza M2 protein. This aggregate of four identical alpha-helices, each built of 25 amino acids, forms an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism, which can involve strands of hydrogen-bonded water through the pore or proton transfer through tautomerization of protein residues. The channel can be re-engineered to act as a simple proton pump.

Pohorille, Andrew↗
NASA NTRS1981

Origins of the protein synthesis cycle

Largely derived from experiments in molecular evolution, a theory of protein synthesis cycles has been constructed. The sequence begins with ordered thermal proteins resulting from the self-sequencing of mixed amino acids. Ordered thermal proteins then aggregate to cell-like structures. When they contained proteinoids sufficiently rich in lysine, the structures were able to synthesize offspring peptides. Since lysine-rich proteinoid (LRP) also catalyzes the polymerization of nucleoside triphosphate to polynucleotides, the same microspheres containing LRP could have synthesized both original cellular proteins and cellular nucleic acids. The LRP within protocells would have provided proximity advantageous for the origin and evolution of the genetic code.

Fox, S. W.↗
DOE OSTI2023

Structural insights into the unique recognition module between α-synuclein peptide and nanobody

Nanobodies are single-domain fragments of antibodies with comparable specificity and affinity to antibodies. They are emerging as versatile tools in biology due to their relatively small size. Here, we report the crystal structure of a specific nanobody Nbα-syn01, bound to a 14 amino acid long peptide of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. The complex structure exhibits a unique binding pattern where the αSyn peptide replaces the N-terminal region of nanobody. Recognition is mediated principally by extended main chain interaction of the αSyn peptide and specificity of the interaction lies in the central 48–52 region of αSyn peptide. Structure-guided truncation of Nbα-syn01 shows tighter binding to αSyn peptide and improved inhibition of α-synuclein aggregation. The structure of the truncated complex was subsequently determined and was indistinguishable to full length complex as the full-length form had no visible electron density for the N-terminal end. These findings reveal the molecular basis for a previously unobserved binding mode for nanobody recognition of α-synuclein, providing an explanation for the enhanced binding, and potential for an alternate framework for structure-based protein engineering of nanobodies to develop better diagnostic and therapeutic tools.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Mixed lipid bilayers enable enhanced stability and activity retention of Lipase A in low-pH environments

Lipase A (LipA) from Bacillus subtilis is a versatile and industrially relevant enzyme, but its activity is compromised under acidic conditions due to aggregation and deactivation. In this study, we investigated the use of mixed lipid bilayers to stabilize and improve activity retention of LipA at low pH. Attachment to lipid bilayers, particularly those containing cationic lipids, greatly enhanced the long-term stability of LipA in acidic conditions while also leading to improvements in activity. Tethering to cationic bilayers not only shifted the apparent pH activity profile toward more acidic conditions, but also significantly enhanced activity retention upon incubation at pH 6. Notably, this protective effect persisted even without direct tethering, indicating that reversible, non-covalent interactions with the bilayer surface are sufficient for long-term stability. Circular dichroism further revealed that the secondary structure of LipA was retained, while dynamic light scattering suggested that activity loss in solution was primarily due to aggregation of the native state. Together, these findings support a model in which lipid bilayers mitigate aggregation and stabilize LipA through transient interactions and electrostatic modulation of the local surface environment. Furthermore, this approach exemplifies a simple, tether-free strategy for enhancing enzyme performance in acidic or destabilizing conditions, with implications for biocatalysis, biosensing, and therapeutic delivery.

Biocatalysis↗
DOE OSTI2023

Spatially confined protein assembly in hierarchical mesoporous metal-organic framework

Immobilization of biomolecules into porous materials could lead to significantly enhanced performance in terms of stability towards harsh reaction conditions and easier separation for their reuse. Metal-Organic Frameworks (MOFs), offering unique structural features, have emerged as a promising platform for immobilizing large biomolecules. Although many indirect methods have been used to investigate the immobilized biomolecules for diverse applications, understanding their spatial arrangement in the pores of MOFs is still preliminary due to the difficulties in directly monitoring their conformations. To gain insights into the spatial arrangement of biomolecules within the nanopores. We used in situ small-angle neutron scattering (SANS) to probe deuterated green fluorescent protein (d-GFP) entrapped in a mesoporous MOF. Our work revealed that GFP molecules are spatially arranged in adjacent nanosized cavities of MOF-919 to form “assembly” through adsorbate-adsorbate interactions across pore apertures. Our findings, therefore, lay a crucial foundation for the identification of proteins structural basics under confinement environment of MOFs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP’s propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Structural polymorphism of amyloid fibrils in ATTR amyloidosis revealed by cryo-electron microscopy

ATTR amyloidosis is caused by the deposition of transthyretin in the form of amyloid fibrils in virtually every organ of the body, including the heart. This systemic deposition leads to a phenotypic variability that has not been molecularly explained yet. In brain amyloid conditions, previous studies suggest an association between clinical phenotype and the molecular structures of their amyloid fibrils. Here we investigate whether there is such an association in ATTRv amyloidosis patients carrying the mutation I84S. Using cryo-electron microscopy, we determined the structures of cardiac fibrils extracted from three ATTR amyloidosis patients carrying the ATTRv-I84S mutation, associated with a consistent clinical phenotype. We found that in each ATTRv-I84S patient, the cardiac fibrils exhibited different local conformations, and these variations can co-exist within the same fibril. Our finding suggests that one amyloid disease may associate with multiple fibril structures in systemic amyloidoses, calling for further studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cryo-EM confirms a common fibril fold in the heart of four patients with ATTRwt amyloidosis

ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found unique structural variabilities in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structure’s impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Synthetic β-sheets mimicking fibrillar and oligomeric structures for evaluation of spectral X-ray scattering technique for biomarker quantification

Archetypical cross-β spines sharpen the boundary between functional and pathological proteins including β-amyloid, tau, α-synuclein and transthyretin are linked to many debilitating human neurodegenerative and non-neurodegenerative amyloidoses. An increased focus on development of pathogenic β-sheet specific fluid and imaging structural biomarkers and conformation-specific monoclonal antibodies in targeted therapies has been recently observed. Identification and quantification of pathogenic oligomers remain challenging for existing neuroimaging modalities. We propose two artificial β-sheets which can mimic the nanoscopic structural characteristics of pathogenic oligomers and fibrils for evaluating the performance of a label free, X-ray based biomarker detection and quantification technique. Highly similar structure with elliptical cross-section and parallel cross-β motif is observed among recombinant α-synuclein fibril, Aβ-42 fibril and artificial β-sheet fibrils. We then use these β-sheet models to assess the performance of spectral small angle X-ray scattering (sSAXS) technique for detecting β-sheet structures. sSAXS showed quantitatively accurate detection of antiparallel, cross-β artificial oligomers from a tissue mimicking environment and significant distinction between different oligomer packing densities such as diffuse and dense packings. The proposed synthetic β-sheet models mimicked the nanoscopic structural characteristics of β-sheets of fibrillar and oligomeric states of Aβ and α-synuclein based on the ATR-FTIR and SAXS data. The tunability of β-sheet proportions and shapes of structural motifs, and the low-cost of these β-sheet models can become useful test materials for evaluating β-sheet or amyloid specific biomarkers in a wide range of neurological diseases. By using the proposed synthetic β-sheet models, our study indicates that the sSAXS has potential to evaluate different stages of β-sheet-enriched structures including oligomers of pathogenic proteins.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1980

Physical chemistry and evolution of salt tolerance in halobacteria

The cellular constituents of extremely halophilic bacteria not only tolerate high salt concentration, but in many cases require it for optical functioning. The characteristics affected by salt include enzyme activity, stability, allosteric regulation, conformation and subunit association. The salt effects are of two major kinds: electrostatic shielding of negative charges by cations at low salt concentration, and hydrophobic stabilization by salting-out type salts at high salt concentration. The composition of halobacterial proteins shows an excess of acidic amino acids and a deficiency of nonpolar amino acids, which accounts for these effects. Since the cohesive forces are weaker and the repulsing forces are stronger in these proteins, preventing aggregation in salt, these structures are no longer suited for functioning in the absence of high salt concentrations. Unlike these nonspecific effects, ribosomes in halobacteria show marked preference for potassium over sodium ions. To ensure the proper intracellular ionic composition, powerful ion transport systems have evolved in the halobacteria, resulting in the extrusion of sodium ions and their replacement by potassium. It is likely that such membrane transport system for ionic movements is a necessary requisite for salt tolerance.

Lanyi, J. K.↗
NASA NTRS2002

Peptides at Membrane Surfaces and their Role in the Origin of Life

All ancestors of contemporary cells (protocells) had to transport ions and organic matter across membranous walls, capture and utilize energy and transduce environmental signals. In modern organisms, all these functions are preformed by membrane proteins. We make the parsimonious assumption that in the protobiological milieu the same functions were carried out by their simple analogs - peptides. This, however, required that simple peptides could self-organize into ordered, functional structures. In a series of detailed, molecular-level computer simulations we demonstrated how this is possible. One example is the peptide (LSLLLSL)3 which forms a trameric bundle capable of transporting protons across membranes. Another example is the transmembrane pore of the influenza M2 protein. This aggregate of four identical alpha-helices, each built of 25 amino acids, forms an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism in this channel. The channel can be re-engineered into a simple proton pump.

Pohorille, Andrew↗