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At least 1,297 records · Page 72

A Novel Bacterial Polymeric Silk-Like Protein from a Petroleum Origin Bacillus sp. strain NE: Isolation and Characterization

This paper explores a novel silk-like protein (named Bacillus NE silk (BNES)) isolated from a petroleum origin bacterial strain belongs to the genus Bacillus. The successful characterizations for its chemical structure determination and protein composition have been performed. The results demonstrate that BNES protein is a new type of silk-like protein and has a prospective for applying as a new bacterial silk-like biomaterial. The amino acids constituents of silk-like protein showed that (Ala-Pro-Gly) rich-block protein, are the main major amino acids, while the protein M{sub wt}., was recorded around > ~ 400 kDa, which excelled those similar of natural silk. The degummed or purified silk-like protein was successfully electro-spinned for wet-electrospun silk-like nanofibers using electrospinning technique. Silk-like nanofibers gave an adequate and uniform ribbon-shape structure with average diameter of nanofiber around 110 nm. Meanwhile, the block silk-like protein exhibited high thermal degradation monitored at 140–373 °C, which close those of natural silkworm silk. The purified silk-like protein showed no toxic effect against Vero kidney normal cell-line even after 48 h of incubation, using MTT-assay test. The obtained results demonstrate that, silk-like protein from wild type bacterial origin has potential for use as novel fibrous biomaterials for biomedical and environmental applications.

60 APPLIED LIFE SCIENCES↗

The synthesis of glutamic acid in the absence of enzymes: Implications for biogenesis

This paper reports on the non-enzymatic aqueous phase synthesis of amino acids from keto acids, ammonia and reducing agents. The facile synthesis of key metabolic intermediates, particularly in the glycolytic pathway, the citric acid cycle, and the first step of amino acid synthesis, lead to new ways of looking at the problem of biogenesis.

Morowitz, Harold↗

CO 2 absorption from simulated flue gas in a bubble column

Carbon dioxide (CO 2 ) absorption from power plant generated flue gas is studied for CO 2 emissions reduction. Amino acid alkaline solvents have the potential to reduce the energy required for solvent regeneration. Here, we focus on CO 2 absorption by amino acid alkaline solvents in a bubble column. Important parameters characterizing the reactor, e.g., bubble size, liquid-phase dispersion coefficient, and gas holdup, are experimentally determined. A theoretical model based upon transient mass and energy balances for the chemical species involved is presented. Simulation results are compared to experimental data. The model can be used to predict, optimize, and control CO 2 absorption in industrial-scale applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Further Investigations of Racemic and Chiral Molecular Sieves of the STW Topology

Large single crystals of pure-silica STW-type molecular sieves are prepared using 1,2,3,4,5-pentamethylimidazolium (12345PMI) and 2-ethyl-1,3,4-trimethylimidazolium (2E134TMI) as organic structure-directing agents (OSDAs) and α-amino acid additives. The effects of the amount and type of amino acid on the crystallization of pure-silica STW materials are investigated. Room-temperature single-crystal X-ray diffractometry shows that 12345PMI better fits the STW cage than 2E134TMI and is fully consistent with computed simulations. The synthesis of an enantioenriched R-STW-type molecular sieve having a Si/Ge ratio of 2.2 is achieved using a chiral, dicationic OSDA that contains two stereocenters. Furthermore, the conformation of the chiral OSDA in the chiral framework has been obtained from a Rietveld refinement using synchrotron powder diffraction data and is the first structure refinement of an enantioenriched, inorganic molecular sieve prepared from a pure, asymmetric OSDA.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response

Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNA His . While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain ofChaetomium thermophilumGcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNA His . Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2 HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD–CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2 HisRS of two highly conserved structural domains for binding both ends of tRNA—each crucial for Gcn2 function—supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.

Science & Technology - Other Topics↗

The Adaptive Potential of the Middle Domain of Yeast Hsp90

Abstract The distribution of fitness effects (DFEs) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in others. So far, results regarding the cost of adaptation across environments have been mixed, and most studies have sampled random mutations across different genes. Here, we quantify systematically how costs of adaptation vary along a large stretch of protein sequence by studying the distribution of fitness effects of the same ≈2,300 amino-acid changing mutations obtained from deep mutational scanning of 119 amino acids in the middle domain of the heat shock protein Hsp90 in five environments. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-terminal-Middle and Middle-C-terminal interdomains, and regulation of ATPase–chaperone activity. Interestingly, we find that fitness correlates well across diverse stressful environments, with the exception of one environment, diamide. Consistent with this result, we find little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client-binding interfaces, or residues that are involved in ATPase–chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information.

Cote-Hammarlof, Pamela A.↗

Structural Insights into Adeno-Associated Virus Serotype 5

The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between βH and βI of the core β-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. Here, this structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity.

59 BASIC BIOLOGICAL SCIENCES↗

Wet chemistry instrument prototype

A wet chemistry instrument prototype for detecting amino acids in planetary soil samples was developed. The importance of amino acids and their condensation products to the development of life forms is explained. The characteristics of the instrument and the tests which were conducted to determine the materials compatibility are described. Diagrams are provided to show the construction of the instrument. Data obtained from the performance tests are reported.

Source record↗

Enantiomeric and Isotopic Analysis of Organic Compounds in Carbonaceous Meteorites

Carbonaceous meteorites are relatively enriched in soluble organic compounds. The Murchison and Murray meteorites contain numerous compounds of interest in the study of early solar system organic chemistry and organic compounds of potential importance for the origin of life. These include: amino acids, amides, carboxylic acids, and polyols. This talk will focus on the enantiomeric and isotopic analysis of individual meteoritic compounds - primarily polyol acids. The analyses will determine if, in addition to certain amino acids from Murchison, another potentially important class of prebiotic compounds also contains enantiomeric excesses, i.e., excesses that could have contributed to the current homochirality of life. Preliminary enantiomeric and isotopic (C- 13) measurements of Murchison glyceric acid show that it is indeed extraterrestrial. C-13 and D isotope analysis of meteoritic sugar alcohols (glycerol, threitol, ribitol, etc.) has shown that they are also indigenous to the meteorite.

Cooper, George↗

Versatility of Synthetic tRNAs in Genetic Code Expansion

Transfer RNA (tRNA) is a dynamic molecule used by all forms of life as a key component of the translation apparatus. Each tRNA is highly processed, structured, and modified, to accurately deliver amino acids to the ribosome for protein synthesis. The tRNA molecule is a critical component in synthetic biology methods for the synthesis of proteins designed to contain non-canonical amino acids (ncAAs). The multiple interactions and maturation requirements of a tRNA pose engineering challenges, but also offer tunable features. Major advances in the field of genetic code expansion have repeatedly demonstrated the central importance of suppressor tRNAs for efficient incorporation of ncAAs. Here we review the current status of two fundamentally different translation systems (TSs), selenocysteine (Sec)- and pyrrolysine (Pyl)-TSs. Idiosyncratic requirements of each of these TSs mandate how their tRNAs are adapted and dictate the techniques used to select or identify the best synthetic variants.

59 BASIC BIOLOGICAL SCIENCES↗

Evolutionary conservation of transferrin genomic organization and expression characterization in seven freshwater turtles

Highlights: • Transferrin have evolutionary conservation in genomic organization in freshwater turtles. • Transferrin have evolutionary conservation in expression characterization in freshwater turtles. • 12 positive selected sites were detected in freshwater turtles which were mainly located on the surface of Tf proteins. Serum transferrin (tf), encoding an iron-binding glycoprotein, has been revealed to play important roles in iron transportation and immune response, and it also has been demonstrated to be valuable for phylogenetic analysis in vertebrates. However, the evolutionary conservation, expression profiles and positive selection of transferrin genes among freshwater turtle species remain largely unclear. Here, the genomic DNA and coding sequences of transferrin genes were cloned and characterized in seven freshwater turtles including Mauremys mutica, Mauremys sinensis, Cyclemys dentate, Mauremyssi reevesi, Heosemys grandis, Trachemys scripta and Chrysemys picta. The isolated coding sequences of turtles' tf genes were 2118 bp or 2121 bp, encoding 706 or 707 amino acids. The predicted Tf proteins of turtles share high identities with M. mutica Tf, up to 91%–98% and the M. mutica Tf has the highest identity (91%) in amino acid with the Chelomia mydas Tf, the moderate with other reptiles' Tfs (65%–59%), chicken (58%), and Human Tf (∼55%), and the lowest with zebrafish Tf (41%). Additionally, tf genes were consistently composed of 17 exons and 16 introns with the same splicing sites in introns in all the turtles examined. Moreover, 12 positive selected sites were detected in these turtles’ Tf and mainly distributed on the surface of transferrin protein. Importantly, it was found that transferrin genes in all turtles examined were predominantly expressed in adult liver via real-time quantitative PCR. The molecular characterizations and expression profiles of transferrin would shed new insights into understanding the conversations and divergences of transferrin genes in turtles, even in vertebrates.

60 APPLIED LIFE SCIENCES↗

Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV

Highlights: • NMR analysis on BTV VP6 reveals two large loop regions. • The loss of a loop (aa 34–130) does not affect the overall fold of the protein. • A region of VP6 (aa 34–92) is not required for BTV replication. • A region of VP6 (aa 93–130) plays an essential role in the virus replication. - Abstract: Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.

60 APPLIED LIFE SCIENCES↗

How to correct relative voxel scale factors for calculations of vector-difference Fourier maps in cryo-EM

The atomic coordinates derived from cryo-electron microscopy (cryo-EM) maps can be inaccurate when the voxel scaling factors are not properly calibrated. Here, we describe a method for correcting relative voxel scaling factors between pairs of cryo-EM maps for the same or similar structures that are expanded or contracted relative to each other. We find that the correction of scaling factors reduces the amplitude differences of Fourier-inverted structure factors from voxel-rescaled maps by up to 20–30%, as shown by two cryo-EM maps of the SARS-CoV-2 spike protein measured at pH 4.0 and pH 8.0. This allows for the calculation of the difference map after properly scaling, revealing differences between the two structures for individual amino acid residues. Unexpectedly, the analysis uncovers two previously overlooked differences of amino acid residues in structures and their local structural changes. Furthermore, we demonstrate the method as applied to two cryo-EM maps of monomeric apo-photosystem II from the cyanobacteria Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus. The resulting difference maps reveal many changes in the peripheral transmembrane PsbX subunit between the two species.

59 BASIC BIOLOGICAL SCIENCES↗

Atomic Force Microscopy to Characterize Antimicrobial Peptide-Induced Defects in Model Supported Lipid Bilayers

Antimicrobial peptides (AMPs) interact with bacterial cell membranes through a variety of mechanisms, causing changes extending from nanopore formation to microscale membrane lysis, eventually leading to cell death. Several AMPs also disrupt mammalian cell membranes, despite their significantly different lipid composition and such collateral hemolytic damage hinders the potential therapeutic applicability of the AMP as an anti-microbial. Elucidating the mechanisms underlying the AMP–membrane interactions is challenging due to the variations in the chemical and structural features of the AMPs, the complex compositional variations of cell membranes and the inadequacy of any single experimental technique to comprehensively probe them. (1) Background: Atomic Force Microscopy (AFM) imaging can be used in combination with other techniques to help understand how AMPs alter the orientation and structural organization of the molecules within cell membranes exposed to AMPs. The structure, size, net charge, hydrophobicity and amphipathicity of the AMPs affect how they interact with cell membranes of differing lipid compositions. (2) Methods: Our study examined two different types of AMPs, a 20-amino acid, neutral, α-helical (amphipathic) peptide, alamethicin, and a 13-amino acid, non-α-helical cationic peptide, indolicidin (which intramolecularly folds, creating a hydrophobic core), for their interactions with supported lipid bilayers (SLBs). Robust SLB model membranes on quartz supports, incorporating predominantly anionic lipids representative of bacterial cells, are currently not available and remain to be developed. Therefore, the SLBs of zwitterionic egg phosphatidylcholine (PC), which represents the composition of a mammalian cell membrane, was utilized as the model membrane. This also allows for a comparison with the results obtained from the Quartz Crystal Microbalance with Dissipation (QCM-D) experiments conducted for these peptides interacting with the same zwitterionic SLBs. Further, in the case of alamethicin, because of its neutrality, the lipid charge may be less relevant for understanding its membrane interactions. (3) Results: Using AFM imaging and roughness analysis, we found that alamethicin produced large, unstable defects in the membrane at 5 µM concentrations, and completely removed the bilayer at 10 µM. Indolicidin produced smaller holes in the bilayer at 5 and 10 µM, although they were able to fill in over time. The root-mean-square (RMS) roughness values for the images showed that the surface roughness caused by visible defects peaked after peptide injection and gradually decreased over time. (4) Conclusions: AFM is useful for helping to uncover the dynamic interactions between different AMPs and cell membranes, which can facilitate the selection and design of more efficient AMPs for use in therapeutics and antimicrobial applications.

Swana, Kathleen W.↗

Geochemistry and the origin of life

The origin of life on earth is examined from a viewpoint stressing the validity of the concept of chemical evolution. The different geological formations supporting the mechanisms of the theory are described; the stage of chemical evolution (preceding that of biological evolution) would have taken place from the time of the origin of the earth and meteorites, 4.6 billion years ago, to the early Precambrian period, about 3.2 billion years ago. Specific aspects of the problem discussed include amino acids from spark discharges and their comparison with the Murchison meteorite amino acids, the properties and theory of genesis of the carbonaceous complex within the cold Bokevelt meteorite, ammonion ion concentration in the primitive ocean, the oxygen isotope chemistry of ancient charts, the origin and rise of oxygen concentration in the earth's atmosphere, Precambrian microorganisms and evolutionary events prior to the origin of vascular plants, and biogenicity and significance of the oldest known stromatolites.

Kvenvolden, K. A.↗

The polymer-like organic material in the Orgueil meteorite

Results are reported for analysis of polymeric organic material contained in powder from the Orgueil chondrite, using a stepwise high-vacuum pyrolysis-gas chromatography-mass spectrometry technique. Pyrolysis products obtained include a series of alkanes and alkenes to C8, an extensive series of alkylbenzene isomers, thiophene, alkylthiophenes, benzothiophene, acetonitrile, acrylonitrile, benzonitrile, acetone, and phenol. Most of these products are shown to be similar both qualitatively and quantitatively to those previously obtained from solvent-extracted Allende powder, indicating a basically aromatic and heteroaromatic polymer matrix with short aliphatic bridges or side chains. The production of acrylonitrile, acetonitrile, and benzonitrile (common breakdown products of amino acids) from the insoluble organic material is taken to suggest that amino acids exist in an insoluble form, perhaps as peptides, in the meteorite's polymeric component. Similarities between the structure of the Orgueil polymeric material and terrestrial kerogen are discussed which raise the possibility that both might have been produced in part by similar reactions.

Bandurski, E. L.↗

Protein-based forensic identification using genetically variant peptides in human bone

Bone tissue contains organic material that is useful for forensic investigations and may contain preserved endogenous protein that can persist in the environment for extended periods of time over a range of conditions. Single amino acid polymorphisms in these proteins reflect genetic information since they result from non-synonymous single nucleotide polymorphisms (SNPs) in DNA. Detection of genetically variant peptides (GVPs) — those peptides that contain amino acid polymorphisms — in digests of bone proteins allows for the corresponding SNP alleles to be inferred. Resulting genetic profiles can be used to calculate statistical measures of association between a bone sample and an individual. In this study proteomic analysis on rib cortical bone samples from 10 recently deceased individuals demonstrates this concept. A straight-forward acidic demineralization protocol yielded proteins that were digested with trypsin. Tryptic digests were analyzed by liquid chromatography mass spectrometry. A total of 1736 different proteins were identified across all resulting datasets. On average, individual samples contained 454 ± 121 ($\bar{x}$ ± σ) proteins. Thirty-five genetically variant peptides were identified from 15 observed proteins. Overall, 134 SNP inferences were made based on proteomically detected GVPs, which were confirmed by sequencing of subject DNA. Inferred individual SNP genetic profiles ranged in random match probability (RMP) from 1/6 to 1/42,472 when calculated with European population frequencies in the 1000 Genomes Project, Phase 3. Similarly, RMPs based on African population frequencies were calculated for each SNP genetic profile and likelihood ratios (LR) were obtained by dividing each European RMP by the corresponding African RMP. In conclusion, resulting LR values ranged from 1.4 to 825 with a median value of 16. GVP markers offer a basis for the identification of compromised skeletal remains independent of the presence of DNA template.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome

The Escherichia coli (E. coli) ribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains in vitro but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives–3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)–the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high-resolution cryo-EM structures of the ribosome with each of these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A-site). The structures reveal how the aromatic ring of each monomer sterically blocks the positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the bound water network that is believed to facilitate formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a mechanistic rationale for differences in reactivity of aminobenzoic acid derivatives relative to l-α-amino acids and each other and identify stereochemical constraints on the size and geometry of non-monomers that can be accepted efficiently by wild-type ribosomes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗