scholarly journals Structured proteins are abundant in unevolved sequence space

2021 ◽  
Author(s):  
Vyacheslav Tretyachenko ◽  
Jiří Vymětal ◽  
Tereza Neuwirthová ◽  
Jiří Vondrášek ◽  
Kosuke Fujishima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sequence Space ◽  
Structural Characteristics ◽  
Random Sequence ◽  
Biological Evolution ◽  
Compact Structure ◽  
Hsp70 Chaperone ◽  
Protein Libraries ◽  
A Minor ◽  
Structured Proteins

AbstractNatural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact structure occurrence (i) is abundant (up to 40%) in random sequence space, (ii) is independent of general Hsp70 chaperone system activity, and (iii) is not granted solely by “late” and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and stability of the canonical alphabet but has only a minor impact on the “early” library. The early alphabet proteins are inherently more stable and soluble, possibly assisted by salts and cofactors in the cell-like environment in which these assays were performed.Our work indicates that natural protein space may have been selected to some extent by chance rather than unique structural characteristics.

scholarly journals Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

2010 ◽  
Vol 84 (21) ◽  
pp. 10999-11009 ◽  
Author(s):  
Pablo Gastaminza ◽  
Kelly A. Dryden ◽  
Bryan Boyd ◽  
Malcolm R. Wood ◽  
Mansun Law ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Characteristics ◽  
Buoyant Density ◽  
Hcv Rna ◽  
Membrane Bilayer ◽  
Biophysical Characterization ◽  
Negative Stain ◽  
A Minor

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

scholarly journals Characterization of porcine osteonectin extracted from foetal calvariae

1988 ◽  
Vol 253 (1) ◽  
pp. 139-151 ◽  
Author(s):  
C Domenicucci ◽  
H A Goldberg ◽  
T Hofmann ◽  
D Isenman ◽  
S Wasi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Secondary Structure ◽  
Type I Collagen ◽  
Culture Shock ◽  
Gel Filtration ◽  
Alpha Helix ◽  
Type I ◽  
Homologous Proteins ◽  
Compact Structure ◽  
Significant Difference

Osteonectin, extracted from foetal porcine calvariae with 0.5 M-EDTA, was purified to homogeneity by using gel filtration and polyanion anion-exchange fast protein liquid chromatography under dissociative conditions without the need of reducing agents. The purified protein migrated with an Mr of 40,300 on SDS/polyacrylamide gels and was similar to bovine osteonectin in both amino acid composition and in its ability to bind to hydroxyapatite in the presence of 4 M-guanidinium hydrochloride (GdmCl). However, unlike the bovine protein, porcine osteonectin did not bind selectively to hydroxyapatite when EDTA tissue extracts were used. In addition, purified porcine osteonectin did not show any apparent affinity for either native or denatured type I collagen, but did bind to serum albumin. Primary sequence analysis revealed an N-terminal alanine residue, with approximately one-half of the subsequent 35 residues identified as small hydrophobic amino acids and one-quarter as acidic amino acids. The only significant difference between the N-terminal sequences of the bovine and porcine proteins was the deletion of the tripeptide Val-Ala-Glu in porcine osteonectin. In contrast with bovine osteonectin, far-u.v.c.d. of porcine osteonectin revealed considerable secondary structure, of which 27% was alpha-helix and 39% was beta-sheet. Cleavage of the molecule with CNBr under non-reducing conditions generated five fragments, of which two major fragments (Mr 27,900 and 12,400) stained blue with Stains All, a reagent that stains sialic-acid-rich proteins/phosphate-containing proteins and/or Ca2+-binding proteins blue while staining other proteins pink. The 12,400-Mr fragment bound 45Ca2+ selectively, indicating a Ca2+-binding site in this part of the molecule. The 27,900-Mr fragment did not bind Ca2+, and since biosynthetic studies with 32PO4(3-) did not show phosphorylation of porcine osteonectin, this fragment is likely to be highly acidic. The incomplete cleavage of the molecule with CNBr and the ability of the molecule to regain its secondary structure after exposure to 7 M-urea are features consistent with the molecule having a compact structure that is stabilized by numerous disulphide bridges. The chemical and binding properties of porcine osteonectin are closely similar to the recently described ‘culture shock’, SPARC and BM-40 proteins, indicating that these are homologous proteins.

scholarly journals Secondary Structure Libraries for Artificial Evolution Experiments

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1671
Author(s):  
Ráchel Sgallová ◽  
Edward A. Curtis
Keyword(s):  
Secondary Structure ◽  
Sequence Space ◽  
In Vitro Selection ◽  
Random Sequence ◽  
Random Mutagenesis ◽  
Nucleotide Composition ◽  
Artificial Evolution ◽  
Wide Range ◽  
Range Of Functions

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.

scholarly journals Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering

2017 ◽  
Author(s):  
Sean A. Higgins ◽  
Sorel Ouonkap ◽  
David F. Savage
Keyword(s):  
Structure Function ◽  
Sequence Space ◽  
Protein Function ◽  
Fluorescent Protein ◽  
One Pot ◽  
Protein Mutagenesis ◽  
Protein Mutants ◽  
Protein Libraries

ABSTRACTComprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and unbiased, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple one-pot protocol.

Synthesis and Structural Characteristics of Geminally Disubstituted β-Amino Acids

Synlett ◽  
2004 ◽  
pp. 1409-1413 ◽  
Author(s):  
Anna K. Mapp ◽  
Amelia A. Fuller ◽  
Bin Chen ◽  
Aaron R. Minter
Keyword(s):  
Amino Acids ◽  
Structural Characteristics

The effect of cycloheximide on amide formation in maize roots

1973 ◽  
Vol 51 (1) ◽  
pp. 91-95 ◽  
Author(s):  
Ann Oaks ◽  
F. J. Johnson
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Root Tip ◽  
Minor Effect ◽  
Root Tips ◽  
Corn Root ◽  
Basic Amino Acids ◽  
Maize Roots ◽  
A Minor ◽  
Mature Root

Cycloheximide inhibits the incorporation of acetate-2-14C into protein and into asparagine in corn root tips. It also causes an accumulation of glutamine and, over a concentration range of 0.4 to 5.0 μg/ml, a transient accumulation of the neutral and basic amino acids. In mature sections, cycloheximide inhibits protein synthesis but causes an increase in the incorporation of radioactivity into both glutamine and asparagine. Azaserine, a glutamine analogue, also inhibits the formation of asparagine in root-tip sections but has only a minor effect on protein synthesis. In mature root sections, there is an accumulation of glutamine but no effect on asparagine formation when azaserine is used. Glutamine additions to root tips or mature root sections affect neither asparagine formation nor protein synthesis. We conclude that cycloheximide is behaving as a glutamine analogue in its effect on asparagine biosynthesis, and that its effect as a glutamine analogue is lost as cells mature.

scholarly journals Exploring Antibody Recognition of Sequence Space through Random-Sequence Peptide Microarrays

2010 ◽  
Vol 10 (3) ◽  
pp. M110.000786 ◽  
Author(s):  
Rebecca F. Halperin ◽  
Phillip Stafford ◽  
Stephen Albert Johnston
Keyword(s):  
Sequence Space ◽  
Random Sequence ◽  
Antibody Recognition ◽  
Peptide Microarrays

EXPLORING PROTEIN'S OPTIMAL HP CONFIGURATIONS BY SELF-ORGANIZING MAPPING

2005 ◽  
Vol 03 (02) ◽  
pp. 385-400 ◽  
Author(s):  
XIANG-SUN ZHANG ◽  
YONG WANG ◽  
ZHONG-WEI ZHAN ◽  
LING-YUN WU ◽  
LUONAN CHEN
Keyword(s):  
Amino Acids ◽  
Search Method ◽  
Training Phase ◽  
Self Organizing Map ◽  
Compact Structure ◽  
Hp Model ◽  
Self Organized ◽  
Input And Output ◽  
Competition Rules ◽  
Self Organizing

Self-organizing map (SOM) has been used in protein folding prediction when the HP model is employed. The existing work uses a square-like shape lattice with l = m×n points to represent the optimal compact structure of a sequence of l amino acids. In this paper, a general l′-size sequence of amino acids is self-organized in a two dimensional lattice with l (> l′) points. The obtained minimum configuration then has a flexible shape, in contrast to the compact structure limited in the lattice. To fulfil this extension, a new self-organizing map (SOM) technique is proposed to deal with the difficulty of the unsymmetric input and output spaces. New competition rules in the training phase are introduced and a local search method is applied to overcome the multi-mapping phenomena. Several HP benchmark examples with up to 36 amino acids are tested to verify the effectiveness of the proposed approach in this paper.

scholarly journals The fate of absorbed and exogenous ammonia as influenced by forage or forage–concentrate diets in growing sheep

1996 ◽  
Vol 76 (2) ◽  
pp. 231-248 ◽  
Author(s):  
G. E. Lobley ◽  
P. J. M. Weijs ◽  
A. Connell ◽  
A. G. Calder ◽  
D. S. Brown ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ammonium Salt ◽  
Free Amino ◽  
Essential Amino Acids ◽  
Whole Body ◽  
Minor Role ◽  
N Removal ◽  
A Minor ◽  
Branched Chains

Changes in splanchnic energy and N metabolism were studied in sheep, prepared with vascular catheters across the portal-drained viscera (PDV) and the Liver, and maintained on supramaintenance intakes of either grass or grass + barley pellets. The animals were challenged, on both diets, with 4 d intra- mesenteric vein infusions of NH4CI (25 µmol/min) plus NH4HCO3(at either 0 or 125 µmol/min). On the final day of each treatment the natural abundance NH4Cl was replaced with15NH4Cl over a 10 h infusion while over the same period [l-13C]leucine was infused via a jugular vein. Measurements were made of blood flow plus mass transfers of NH3, urea, free amino acids and O2, across the PDV and liver. Enrichments of [14N15N]urea and [15N15N]urea plus [15N]glutamine, aspartate and glutamate were also monitored. Whole-body urea flux was determined by infusion of [14C]urea. At the end of the study the animals were infused for 3 h with15NH4CI, killed and liver samples assayed for intracellular free amino acid enrichments and concentrations. Blood flows across the splanchnic region were unaffected by either diet or level of ammonium salt infusion. At the lower ammonium salt infusion there was a trend for greater absorption of NH3across the PDV (P<0·10) with grass + barley than with the grass diet, while removal of urea was unaltered. At the higher ammonium salt infusions there was a significantly greater appearance of NH, across the PDV and this exceeded the extra infused. Urea-N removal, however, was also elevated and by more than that required to account for the additional NH3. The PDV contributed 19–28% to whole-body O2consumption and the liver 23–32%. Hepatic extraction of absorbed NH3was complete on all treatments and systemic pH remained constant. The fractions of urea-N apparently derived from NH3, were similar on the grass (0·59–0·64) and grass + barley (0·64–0·67) diets. Hepatic production of urea agreed well with urea flux measurements. Between the two levels of ammonium salt infusion and within diets the additional NH3removed across the PDV was accounted for by the increased urea-N production. The [14N15N]: [15N15N] ratio of the urea produced was 97:3, while the enrichment of hepatic intracellular free aspartate was lower than that of [14N15N]urea. Glutamine enrichments were 0·23–0·37 those of [14N15N]urea, indicating a minor role for those hepatocytes (probably perivenous) which contain glutamine synthetase (EC6.3.1.2). Leucine kinetics, either for the whole body or splanchnic tissues, were not different between diets or level of ammonium salt infusion, except for oxidation which was less on the grassfbarley ration. Amino acid concentrations were lower on the grass + barley diet but net PDV absorptions were similar. The pattern of essential amino acids absorbed into the PDV showed good agreement with the published composition of mixed rumen microbial protein. Fractional disappearances of absorbed free essential amino acids across the liver varied from 0·4 (branched chains) to near unity (histidine, phenylalanine)

scholarly journals Engineering Translation Components Improve Incorporation of Exotic Amino Acids

2019 ◽  
Vol 20 (3) ◽  
pp. 522 ◽  
Author(s):  
Takayuki Katoh ◽  
Hiroaki Suga
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Active Site ◽  
Structural Characteristics ◽  
Elongation Factor ◽  
Lower Affinity ◽  
Nonsense Codon ◽  
Nascent Chain ◽  
Translation Systems ◽  
Incorporation Efficiency

Methods of genetic code manipulation, such as nonsense codon suppression and genetic code reprogramming, have enabled the incorporation of various nonproteinogenic amino acids into the peptide nascent chain. However, the incorporation efficiency of such amino acids largely varies depending on their structural characteristics. For instance, l-α-amino acids with artificial, bulky side chains are poorer substrates for ribosomal incorporation into the nascent peptide chain, mainly owing to the lower affinity of their aminoacyl-tRNA toward elongation factor-thermo unstable (EF-Tu). Phosphorylated Ser and Tyr are also poorer substrates for the same reason; engineering EF-Tu has turned out to be effective in improving their incorporation efficiencies. On the other hand, exotic amino acids such as d-amino acids and β-amino acids are even poorer substrates owing to their low affinity to EF-Tu and poor compatibility to the ribosome active site. Moreover, their consecutive incorporation is extremely difficult. To solve these problems, the engineering of ribosomes and tRNAs has been executed, leading to successful but limited improvement of their incorporation efficiency. In this review, we comprehensively summarize recent attempts to engineer the translation systems, resulting in a significant improvement of the incorporation of exotic amino acids.

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