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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
NASA NTRS2006

Computer Modeling of Protocellular Functions: Peptide Insertion in Membranes

Lipid vesicles became the precursors to protocells by acquiring the capabilities needed to survive and reproduce. These include transport of ions, nutrients and waste products across cell walls and capture of energy and its conversion into a chemically usable form. In modem organisms these functions are carried out by membrane-bound proteins (about 30% of the genome codes for this kind of proteins). A number of properties of alpha-helical peptides suggest that their associations are excellent candidates for protobiological precursors of proteins. In particular, some simple a-helical peptides can aggregate spontaneously and form functional channels. This process can be described conceptually by a three-step thermodynamic cycle: 1 - folding of helices at the water-membrane interface, 2 - helix insertion into the lipid bilayer and 3 - specific interactions of these helices that result in functional tertiary structures. Although a crucial step, helix insertion has not been adequately studied because of the insolubility and aggregation of hydrophobic peptides. In this work, we use computer simulation methods (Molecular Dynamics) to characterize the energetics of helix insertion and we discuss its importance in an evolutionary context. Specifically, helices could self-assemble only if their interactions were sufficiently strong to compensate the unfavorable Free Energy of insertion of individual helices into membranes, providing a selection mechanism for protobiological evolution.

Rodriquez-Gomez, D.↗
NASA NTRS2017

Prebiotic Peptide (Amide) Bond Synthesis Accelerated by Glycerol and Bicarbonate Under Neutral to Alkaline Dry-Down Conditions

Past studies of prebiotic peptide bond synthesis have generally been carried out in the acidic to neutral pH range [1, 2]. Here we report a new process for peptide bond (amide) synthesis in the neutral to alkaline pH range that involves simple dry-down heating of amino acids in the presence of glycerol and bicarbonate. Glycerol was included in the reaction mixture as a solvent and to provide hydroxyl groups for possible formation of ester intermediates previously implicated in peptide bond synthesis under acidic to neutral conditions [1]. Bicarbonate was added to raise the reaction pH to 8-9.

Forsythe, J. G.↗
DOE OSTI2024

Peptide inhibitors of CRISPR-Cas9

Specific and broad-spectrum inhibitory peptides to CRISPR Cas9 variants (SpyCas9, SauCas9, and CjeCas9) are disclosed. A method of making and using these peptides with therapeutic CRISPR Cas9 (to improve desired targeting) or against harmful CRISPR Cas9 components (already active in a patient) is also disclosed. The peptides combined with a delivery system is also disclosed.

Harmon, Brooke Nicole↗
NASA NTRS1991

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.

Endothelium, Vascular/chemistry↗
DOE OSTI2025

X-ray absorption spectroscopy of lanmodulin-derived peptides bound to rare earth elements

A sustainable and robust supply chain of rare earth elements (REEs) is necessary to meet our consumer, national security and clean energy goals. However, current intra-REE separation technologies (e.g. solvent extraction) are costly and carry a heavy environmental burden. Therefore, the development of new aqueous based ligands that are selective for individual REEs will be integral in future REE production systems. To develop these ligands, an understanding of how ligand coordination structure relates to selectivity is imperative. We used X-ray absorption spectroscopy (XAS) to observe the local structure around four lanthanide (Ln) ions (La, Ce, Pr and Nd) complexed by water and several relevant chelating ligands [lanmodulin EF-hand 1 peptides (LanM1), ethyl­enedi­amine­tetra­acetic acid (EDTA), amino­tris­(methyl­ene­phospho­nic acid) (ATMP) and citric acid]. To collect these liquid-phase XAS spectra, we developed a new flow cell that prevents bubble interference and beam damage to the samples. In the X-ray absorption near-edge structure (XANES), we observed energy shifts in the white line, white line broadening and differences in the white line intensity of different Ln–ligand complexes between ligands. In the extended X-ray absorption fine structure (EXAFS), we distinguished differences in peak intensity and distance between coordinating ligands. Differences in the local coordination structure between Ln–LanM1 peptide complexes were more subtle compared with the other ligands (La–water, La–EDTA, La–ATMP and La–citric acid complexes). Further XANES and EXAFS studies, in combination with modelling and other techniques, could greatly improve our structural knowledge of how these aqueous ligands bind Ln ions and how they can be used to design more selective ligands for more efficient and sustainable REE separations.

EDTA↗
DOE OSTI2026

Characterization of Oxidative Modifications to Short Peptides Using Low Dose Rate X-Rays

The method of X-ray footprinting and mass spectrometry (XFMS) using high flux synchrotron X-ray sources has become an established method in structural biology and is based on the radiolytic production of hydroxyl radicals, which oxidatively modify protein sidechains. While other methods of producing hydroxyl radicals are available, one benefit of using high flux density sources is that hydroxyl radical scavenging reactions can be minimized, and exposure times kept short to minimize secondary reactions. Here we present an application of the XFMS method using low dose rate X-rays from a commercial instrument. We demonstrate the feasibility of the approach using short peptides, characterizing the oxidative modifications +14, +16, and +32 Da under both aerobic and low oxygen conditions, and we additionally quantify the hydrogen peroxide production for various doses using the low dose rate source. These results provide fundamental information on the oxidative damage to peptides due to hydroxyl radicals using a low dose rate X-ray source.

X-ray methods↗
DOE OSTI2025

A Route to Design Novel Functional Peptides by Applying a Denoising Diffusional Model to mRNA Display Libraries

In vitro directed evolution techniques, such as mRNA display, enable peptide ligand discovery and optimization. However, physical libraries that rely on a genetic code can only search a small fraction of sequence space due to inherent biases in the genetic code and experimental limitations. To address this challenge, denoising diffusion implicit models (DDIMs) are applied to generate novel peptide ligands against B‐cell lymphoma extra‐large (Bcl‐x L ), a key cancer target. Starting with high‐throughput sequencing data from previous selections, a DDIM is trained to produce novel sequences with high affinity binding. Experimental validation confirms that most generated sequences are functionally equivalent to the original library members for Bcl‐x L binding and demonstrated comparable binding kinetics and affinity relative to the wildtype and nearest original neighbors. Importantly, this approach generated rare sequences not easily accessible via mutation and directed evolution. These results indicate that DDIMs can complement and expand directed evolution data, efficiently exploring underrepresented regions of sequence space. This approach provides a broadly applicable framework for accelerating ligand discovery and optimizing molecular properties across diverse targets.

Qi, Pearl [Mork Family Department of Chemical Engi↗
DOE OSTI2025

An Antibody Cocktail-Based Immunoaffinity-LC-MS Method Enabled Ultra-Sensitive and Robust Quantification of Circulating Proinsulin Proteoforms and C-Peptide

Accurately measuring circulating proinsulin proteoforms is crucial for clinical investigation of diabetes, but was previously not feasible owing to limited assay specificity/sensitivity. Here, in this study, we devised a highly sensitive LC-MS-based strategy to quantify intact proinsulin, des-31,32 and des-64,65 proinsulin, and C-peptide in circulation. The method involves: (i) quantitative, robust affinity capture using an optimized antibody cocktail, eliminating the severe quantitative bias across multiple proteoforms typically introduced when using a single antibody; (ii) Lys-C digestion producing unique signature peptides for each proteoform, and (iii) trapping-nano-LC coupled with FAIMS/dCV-MS for an ultrasensitive analysis. The selective trapping/delivery ensured sensitive/selective analysis of the targets while achieving excellent analytical robustness that is critical for clinical assays, and the FAIMS/dCV substantially reduces baseline noise/interferences, further enhancing S/N. The assay achieved exceptional sensitivity, with serum LOQs of 1.7, 2.3, and 3.6 pg/mL respectively for intact-proinsulin, des-31,32 and des-64,65, representing the first assay capable of sensitively quantifying these major circulating proinsulin proteoforms. We applied this assay to 78 subjects, including autoantibody positive (n = 20) and new-onset type 1 diabetes (T1D, n = 19) with respective age/sex/BMI-matched controls, enabling the first accurate profiling of proinsulin proteoforms in clinical groups. The assay results demonstrated a clear separation of control and new-onset T1D groups that a parallel total-proinsulin ELISA assay fails to capture. Furthermore, distinct expression patterns in relative abundance ratios among proteoforms were observed across clinical groups. This assay may provide valuable insights into the β-cell functions and the onset/progression of diabetes and other associated conditions. Moreover, the strategy is broadly applicable to targeted measurement of other biomarker proteoforms.

Shen, Qingqing [University at Buffalo, NY (United ↗
DOE OSTI2026

Revealing Hidden Quinones Through Diagnostic MS² Fragmentation of Peptide–Quinone Adducts

Quinones are redox-active components of natural organic matter that mediate electron transfer and influence biogeochemical processes, but many quinones in pyrogenic organic matter (PyOM) remain unresolved because they ionize poorly by mass spectrometry. Here, we present a peptide-tagging approach to improve detection of cysteine-reactive electrophiles in PyOM, with quinones expected to be a dominant subset based on reaction chemistry and selectivity experiments. A cysteine-containing peptide was used to form Michael-addition adducts, enhancing electrospray ionization and enabling untargeted screening by high-performance liquid chromatography-high-resolution tandem mass spectrometry. The method was benchmarked with five quinone standards and applied to extracts from charred plant material as a discovery-level screen for cysteine-reactive targets. We identified 98 quinone-candidate adducts (mean neutral mass ~603 Da), of which more than 70% were not detectable in native MS1 data. Among formula-assigned features, hidden quinone candidates had median (O+N)/C of 0.391 and normalized oxidation state of carbon of -0.281, consistent with relatively low polarity and low oxidation state. These results reveal a previously inaccessible pool of hidden redox-active compounds in PyOM and provide a framework for prioritizing quinone-like electrophiles for confirmation and incorporation into models of fire-driven biogeochemical cycling.

LC-MS/MS↗
DOE OSTI2025

SPE-CZE-MS Quantifies Zeptomole Amounts of Phosphorylated Peptides

Capillary zone electrophoresis (CZE) is gaining attention in the field of single-cell proteomics for its ultralow-flow and high-resolution separation abilities. Even more sample-limited yet rich in biological information are phosphoproteomics experiments, as the phosphoproteome composes only a fraction of the whole cellular proteome. Rapid analysis, high sensitivity, and maximization of sample utilization are paramount for single-cell analysis. Some challenges of coupling CZE analysis with mass spectrometry analysis (MS) of complex mixtures include 1. sensitivity due to volume loading limitations of CZE and 2. incompatibility of MS duty cycles with electropherographic time scales. Here, we address these two challenges as applied to singlecell- equivalent phosphoproteomics experiments by interfacing a microchip-based CZE device integrated with a solid-phaseextraction (SPE) bed with the Orbitrap Astral mass spectrometer. Using 225 phosphorylated peptide standards and phosphorylated peptide-enriched mouse brain tissue, we investigate microchip-based SPE-CZE functionality, quantitative performance, and complementarity to nano-LC-MS (nLC-MS) analysis. We highlight unique SPE-CZE separation mechanisms that can empower fitfor- purpose applications in single-cell-equivalent phosphoproteomics.

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

Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1’ site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Engineering Anti-Microbial Peptide Production in Fungi and Bioprocess Development (CRADA 502)

Crop Protection, specifically controlling agricultural diseases and pests, is essential for crop production at every agricultural scale. Biologics hold enormous potential in this equation to decrease use of more toxic pesticides. Currently, the widespread use of biologics in crop protection is severely hampered by the lack of available tools for their mass production. To ensure biologics are regulated under EPA Biopesticide regulations, the following must be true: The biopesticide is a naturally occurring substance or structurally similar and functionally identical to a naturally occurring substance with a history of exposure to humans and the environment demonstrating minimal toxicity. Since this pertains to the marketed product it is also essential that fermentation hosts have a history of exposure and safe use by humans This research focuses on developing a novel platform for the production and delivery of the cghSAMP peptide using the filamentous fungus Aspergillus niger. A. niger is a well-established industrial microorganism, widely recognized for its strong protein secretion capabilities, high-yield production of enzymes, and classification as a generally recognized as safe (GRAS) organism. By genetically modifying A. niger to express the cghSAMP peptide transgene, we aim to leverage the fungus's robust fermentation system to produce a high volume of the antimicrobial agent. This engineered system represents a potential breakthrough in sustainable disease management, offering an environmentally friendly method for localized application or large-scale production of a biopesticide designed to mitigate the effects of citrus greening disease. The main objectives of this project were: 1. Developing a high-yield and cost-efficient bioprocess to produce an AMP in a current ABF host or readily onboarded hosts. 2. Scale up the process in stirred tank reactors and produce at least 100 grams of AMP at 1- 5g/L

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

SPE-CZE-MS Quantifies Zeptomole Amounts of Phosphorylated Peptides

Using 225 phosphorylated peptide standards and phosphorylated peptide-enriched mouse brain tissue, we investigate microchip-based SPE-CZE functionality, quantitative performance, and complementarity to nano-LC-MS analysis.

Coon, Joshua J. [GLBRC - University of Wisconsin]↗
NASA NTRS1978

Boosting production yield of biomedical peptides

Nuclear magnetic resonance (NMR) technique is employed to monitor synthesis of biomedical peptides. Application of NMR technique may improve production yields of insulin, ACTH, and growth hormones, as well as other synthesized biomedical peptides.

Manatt, S. L.↗
NASA NTRS1980

Studies of the chemical basis of the origin of protein synthesis Initiation and direction of peptide growth

The data presented in this paper show that the ease of nonenzymatic activation of carboxylic acids by ATP at pH 5 varies directly with the pKa of the carboxyl group, and is consistent with the idea that it is the protonated form of the carboxyl group which participates in the activation reaction. Consequently, since most N-blocked amino acids have higher pKas than do their unblocked forms, they are activated more readily, and it has been demonstrated that this principle applies to peptides as well, which are activated more rapidly than single amino acids. It is proposed that this fact may be partly responsible for the origin of two important features still observed in contemporary protein synthesis: (1) initiation in prokaryotes is accomplished with an N-blocked amino acid, and (2) elongation in all living systems occurs at the carboxyl end of the growing peptide.

Mullins, D. W., Jr.↗
NASA NTRS1982

Oxidative peptide /and amide/ formation from Schiff base complexes

One hypothesis of the origin of pre-modern forms of life is that the original replicating molecules were specific polypeptides which acted as templates for the assembly of poly-Schiff bases complementary to the template, and that these polymers were then oxidized to peptide linkages, probably by photo-produced oxidants. A double cycle of such anti-parallel complementary replication would yield the original peptide polymer. If this model were valid, the Schiff base between an N-acyl alpha mino aldehyde and an amino acid should yield a dipeptide in aqueous solution in the presence of an appropriate oxidant. In the present study it is shown that the substituted dipeptide, N-acetyl-tyrosyl-tyrosine, is produced in high yield in aqueous solution at pH 9 through the action of H2O2 on the Schiff-base complex between N-acetyl-tyrosinal and tyrosine and that a great variety of N-acyl amino acids are formed from amino acids and aliphatic aldehydes under similar conditions.

Strehler, B. L.↗