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Results for “Peptide Synthesis”

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 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Nanoparticle-Mediated Assembly of Peptoid Nanosheets Functionalized with Solid-Binding Proteins: Designing Heterostructures for Hierarchy

The fabrication of ordered architectures that intimately integrate polymer, protein and inorganic components remains difficult. Two promising building blocks to tackle this challenge are peptoids, peptide mimics capable of self-assembly into well-defined structures, and solid-binding peptides, which offer a biological path to controlled inorganic assembly. Here, we report on the synthesis of 3.3 nm-thick, thiol-reactive peptoid nanosheets from equimolar mixtures of unmodified and maleimide-derivatized versions of the Nbpe 6 Nce 6 oligomer, optimize the location of engineered cysteine residues in silica-binding derivatives of superfolder green fluorescent protein for maleimide conjugation, and react the two components to form proteinpeptoid hybrids exhibiting partial or uniform protein coverage on both of their sides. Using 10 nm silica nanoparticles, we trigger the stacking of these 2D structures into a multi-layer material comprised of alternating peptoid, protein and organic layers. This simple and modular approach to hierarchical hybrid synthesis should prove useful in bioimaging and photocatalysis applications.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2025

Cell-Free-Based Thermophilic Biocatalyst for the Synthesis of Amino Acids from One-Carbon Feedstocks

Bioproduction from one-carbon compounds, such as formate, is an attractive prospect due to reduced energy requirements and the possibility for using CO 2 as a sustainable feedstock. Formate-fixing pathways engineered using Escherichia coli lysate-based cell-free expression (CFE) biocatalysts have the potential to route 100% of feedstock carbon toward chemical synthesis but are undermined by siphoning of in-pathway metabolites and cofactors by the CFE background metabolism. To address this limitation, we engineer a CFE-based thermophilic multienzyme biocatalyst for the synthesis of serine and glycine from formate, bicarbonate, and ammonia. After expression of the thermophilic formate-to-serine pathway in a one-pot reaction, the mesophilic E. coli CFE background machinery is removed by simple heat denaturation, eliminating the siphoning of cofactors, inpathway metabolites, and products. After bioprocess optimization, including pathway gene expression duration and chemical synthesis temperature, we achieve near stoichiometric conversion of formate and bicarbonate to serine and glycine, reaching 97% of stoichiometric yield. The use of a moderately thermophilic biocatalyst allowed chemical synthesis to take place at mesophilic temperatures, enabling the balance of optimal enzyme activity with minimal metabolite/cofactor thermal degradation. In a fed-batch experiment, the biocatalyst shows sustained chemical synthesis rates for 8 h, paving the way toward a continuous bioprocess. Finally, a sensitivity analysis of cofactor usage revealed that the most expensive cofactors, THF and NADPH, can be reduced by 5-fold without significantly lowering product yields. To the best of our knowledge, this is the first instance of expressing a thermophilic pathway in an E. coli lysate-based CFE system to generate a thermophilic biocatalyst for use at mesophilic temperatures. The CFEbased thermophilic formate-to-serine biocatalyst triples the combined serine and glycine yield previously obtained by a CFE-based mesophilic formate-to-serine biocatalyst (30%), and quadruple the yield obtained by a purified enzyme system (22%). Ultimately, this work opens the door to using E. coli lysate-based CFE for thermophilic biocatalyst generation to achieve high chemical synthesis yields.

bacteria↗
NASA NTRS1990

Prebiotic synthesis of histidyl-histidine

Histidyl-histidine (His-His) has been synthesized in a yield of up to 14.4% under plausible prebiotic conditions using histidine (His), cyanamide, and 4-amino-5-imidazole carboxamide. A trace amount of His trimer was also detected. Because the imidazole group of His is involved in a number of important enzymatic reactions, and His-His has been shown to catalyze the prebiotic synthesis of glycyl-glycine, we expect this work will stimulate further studies on the catalytic activities of simple His-containing peptides in prebiotic reactions.

NASA Discipline Exobiology↗
DOE OSTI2022

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Does Chirality Influence the Stability of Amino Acid –Cu Complexes in the Salt-Induced Peptide Formation Reaction? Insights from Density Functional Theory Calculations

The polymerization of amino acids into peptides and ultimately proteins is critical to evolution of life. Ribosomally-synthesized proteins are homochiral, composed entirely of L-amino acids. Abiotic syntheses of amino acids, however, produce racemic mixtures(equal amounts of L and D enantiomers), meaning that the pool of monomers available for abiotic protein synthesis would be racemic or nearly so. There are no prebiotically plausible mechanisms known to yield homochiral peptides from racemic pools of amino acids. While some mechanisms capable of producing or amplifying enantiomeric excesses have been observed, these excesses are small or not applicable to all biologically relevant amino acids. As a result, a prebiotically-feasible mechanism to create enantiopure pools of amino acids has not been identified. However, it is not clear that these enantio-enriched or enantiopure pools are necessary to produce homochiral peptides. Steric and electronic effects, or a combination of both, may influence how different enantiomers interact with each other, potentially leading to homochiral peptides from racemic mixtures. Here, we use density functional theory (DFT) calculations to assess the stability of homochiral and heterochiral reactive complexes produced during polymerization via the salt-induced peptide formation (SIPF) reaction. Experimental studies of the SIPF reaction have shown that it enables the formation of peptides under diverse environmental conditions, making it a plausible pathway to peptide formation on early Earth. In the SIPF reaction, NaCl acts as a condensation reagent while a divalent metal cation, primarily Cu2+, facilitates polymerization by complexing and activating amino acids forming a monochlorocuprate complex. To assess if hetero-or homochiral monochlorocuprate complexes were energetically favored, we compared the stability of LL, DD, and LD enantiomers of Cu2+–(alanine)2and Cu2+–(valine) 2 complexes. Gaussian 09 density DFT energy-minimization calculations were made for each Cu –amino acid complex in the cis and trans configuration. Models were energy minimized using the BH and HLYP/6-31++G(d,p) level of theory and free energies were compared to determine the most stable configuration.

A C Fox↗
NASA NTRS1998

Prebiotic Amino Acid Thioester Synthesis: Thiol-Dependent Amino Acid Synthesis from Formose substrates (Formaldehyde and Glycolaldehyde) and Ammonia

Formaldehyde and glycolaldehyde (substrates of the formose autocatalytic cycle) were shown to react with ammonia yielding alanine and homoserine under mild aqueous conditions in the presence of thiol catalysts. Since similar reactions carried out without ammonia yielded alpha-hydroxy acid thioesters, the thiol-dependent synthesis of alanine and homoserine is presumed to occur via amino acid thioesters-intermediates capable of forming peptides. A pH 5.2 solution of 20 mM formaldehyde, 20 mM glycolaldehyde, 20 mM ammonium chloride, 23 mM 3-mercaptopropionic acid, and 23 mM acetic acid that reacted for 35 days at 40 C yielded (based on initial formaldehyde) 1.8% alanine and 0.08% homoserine. In the absence of thiol catalyst, the synthesis of alanine and homoserine was negligible. Alanine synthesis required both formaldehyde and glycolaldehyde, but homoserine synthesis required only glycolaldehyde. At 25 days the efficiency of alanine synthesis calculated from the ratio of alanine synthesized to formaldehyde reacted was 2.1%, and the yield (based on initial formaldehyde) of triose and tetrose intermediates involved in alanine and homoserine synthesis was 0.3 and 2.1%, respectively. Alanine synthesis was also seen in similar reactions containing only 10 mM each of aldehyde substrates, ammonia, and thiol. The prebiotic significance of these reactions that use the formose reaction to generate sugar intermediates that are converted to reactive amino acid thioesters is discussed.

Weber, Arthur L.↗
DOE OSTI2021

Designing Sequence-Defined Peptoids for Biomimetic Control over Inorganic Crystallization

Crystallization defines what the world is all about, ranging from biomineralization in living organisms to various materials in our lives. During these processes, biomineralization set a superexcellent example of the additive-controlled crystallization. However, the high complexity and low stability of peptides and proteins preclude their wide applications in controlling inorganic crystallization beyond the biological systems. Peptoids are one type of sequence-defined biomimetic polymers with a simple structure, high stability, protein-like molecular recognition, and distinctive self-assembly properties. They provide a promising alternative to mimic peptides and proteins for controlling inorganic crystallization and extend its application to predictive materials synthesis. In this review article, we extract the main concepts of peptoid engineering and highlight the recent advances in peptoid-controlled inorganic crystallization. Our focus is to understand the principles of sequence engineering that lead to the predictable physiochemical properties of these substances, which will give insights into other inorganic systems.

36 MATERIALS SCIENCE↗
NASA NTRS1997

Redox Energy and Sulfur Chemistry in Prebiotic Polymer Synthesis and Replication

In the past year we have made significant progress in three research areas: (1) Most importantly, we discovered a new pathway of prebiotic amino acid synthesis in which formaldehyde and glycolaldehyde (substrates of the formose reaction) react with ammonia yielding alanine and homoserine in the presence of thiol catalysts. This thiol-dependent synthesis of amino acids undoubtedly occurs via amino acid thioester intermediates capable of forming peptides. This 'one-pot' reaction system operates under mild aqueous conditions, and like modern amino acid biosynthesis, uses sugar intermediates which are converted to amino acids by energy-yielding redox disproportionation. (2) Finally, in preparation for the analysis of Martian meteorite samples, we upgraded our HPLC system and developed an improved method capable of detecting a I femtomole of amino acid enantiomers. (3) We completed our analysis of the energetics of metabolism that revealed that life depends on biosynthetic processes driven by chemical energy made available by the redox disproportionation of carbon groups of sugars. We established that the favorable energy of redox disproportionation is based on the universal reduction potentials of carbon groups. We concluded that it is hard to imagine any other organic molecule besides sugars (formaldehyde oligomers) having the energy and reactivity needed to drive either modem biosynthesis or the chemical processes behind its origin.

Weber, Arthur L.↗
NASA NTRS1998

Prebiotic Polymer Synthesis and the Origin of Glycolytic Metabolism

Our research resulted in several discoveries which contributed to understanding the origin and operation of life. (1) Most importantly, we discovered a new pathway of prebiotic amino acid synthesis in which formaldehyde and glycolaldehyde (formose reaction substrates) react with ammonia to give alanine and homoserine in the presence of thiol catalysts. The thiol-dependent synthesis of amino acids undoubtedly occurs via amino acid thioester intermediates capable of forming peptides. This 'one-pot' reaction system operates under mild aqueous conditions, and like modern amino acid biosynthesis, uses sugar intermediates which are converted to amino acids by energy-yielding redox disproportionation. Preliminary evidence suggests that this type of process can be "evolved" by a serial transfer methods that lead to enrichment of autocatalytic molecules. (2) We established that prebiotic peptide polymers can be made by condensation of amino acid thioesters (homocysteine thiolactone and S-(N-beta-orotidyl- diaminopropionic acid) ethanethiol), and that prebiotic polydisulfide polymers can be generated by oxidation of dithiols with iron(III) in minerals. (3) In our analysis of metabolism we discovered the primary energy source of biosynthesis -- chemical energy made available by the redox disproportionation of substrate carbon groups. We concluded that the energy and reactivity of sugars make them the optimal substrate for the origin and operation of terrestrial (or extraterrestrial) life. (4) Since it is likely that the use of optimal sugar substrates in biosynthesis sets the average oxidation number of functional biocarbon throughout the Universe near 0.0 (the reduction level of formaldehyde), we proposed that a line(s) in the microwave spectrum of formaldehyde could be rationally selected as a frequency for interstellar communication that symbolizes life. (5) Finally, in preparation for the analysis of Martian meteorite samples, we upgraded our HPLC system to one femtomole sensitivity, and developed a new electrophoretic method of sample preparation for HPLC analysis of the meteoritic amino acids. In a sample of the KT boundary layer from Sussex Wyoming, we found about 300 picomoles per gram of meteoritic alpha-aminoisobutyric acid per gram of KT layer.

Weber, Arthur L.↗
DOE OSTI2023

Dendritic gold nanoparticles and methods of synthesis

A method of producing dendritic gold nanoparticles by combining a gold precursor solution, a reducing agent, and a bifunctional peptide having an amine-rich amino acid sequence into a buffered aqueous solution in a single container, and agitating the mixture causing the formation of the dendritic gold nanoparticles having a surface with a positive charge and a second end portion of the bifunctional peptide exposed on the surface of the dendritic gold nanoparticles. The dendritic gold nanoparticles may be used to deliver therapeutic, diagnostic, and/or immunogenic amino acid sequences as portions of the bifunctional peptide.

Mao, Chuanbin↗
DOE OSTI2021

Solid-Binding Proteins: Bridging Synthesis, Assembly, and Function in Hybrid and Hierarchical Materials Fabrication

There is considerable interest in the development of hybrid organic–inorganic materials because of the potential for harvesting the unique capabilities that each system has to offer. Proteins are an especially attractive organic component owing to the high amount of chemical information encoded in their amino acid sequence, their amenability to molecular and computational (re)design, and the many structures and functions they specify. Genetic installation of solid-binding peptides (SBPs) within protein frameworks affords control over the position and orientation of adhesive and morphogenetic segments, and a path toward predictive synthesis and assembly of functional materials and devices, all while harnessing the built-in properties of the host scaffold. Furthermore, we review the current understanding of the mechanisms through which SBPs bind to technologically relevant interfaces, with an emphasis on the variables that influence the process, and highlight the last decade of progress in the use of solid-binding proteins for hybrid and hierarchical materials synthesis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1973

Investigation of the effect of stress on the chemistry, metabolism, and biophysics of collagen

The research is reported concerning the effect of stress on the chemistry in the connective tissue of the rat. It was found that within a day after administration of cortisol (stress harmone), a significant amount of the insoluble collagen disappeared from the skin. It is concluded that the abrupt catabolism of cutaneous collagen releases peptides. These peptides are rapidly degraded to free amino acids which are active in effecting liver glycogen synthesis, and provide a major energy source to assist the animal in the fight or flight reaction. It is proposed that cutaneous collagen represents a reserve energy pool, which can be mobilized via stress harmones.

Houck, J. C.↗
NASA NTRS1994

Fusion proteins as alternate crystallization paths to difficult structure problems

The three-dimensional structure of a peptide fusion product with glutathione transferase from Schistosoma japonicum (SjGST) has been solved by crystallographic methods to 2.5 A resolution. Peptides or proteins can be fused to SjGST and expressed in a plasmid for rapid synthesis in Escherichia coli. Fusion proteins created by this commercial method can be purified rapidly by chromatography on immobilized glutathione. The potential utility of using SjGST fusion proteins as alternate paths to the crystallization and structure determination of proteins is demonstrated.

Carter, Daniel C.↗
DOE OSTI2022

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Improved Protein Semi-Synthesis Enables Biophysical Studies of Thioamide Destabilization of β-Sheet Interactions

Abstract Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide-containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the β-sheet region of the B1 domain of protein G (GB1) as well as β-strand interactions in amyloid fibrils of the Parkinson’s disease protein α-synuclein. In both contexts, we observe dramatic destabilization of the β-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of α-synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen-bonding networks and prompt future study of both systems.

Yanagawa, Evan S. K. [University of Pennsylvania ,↗
DOE OSTI2020

Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu∙GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu∙GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

59 BASIC BIOLOGICAL SCIENCES↗