scholarly journals InDel assembly: A novel framework for engineering protein loops through length and compositional variation

2017 ◽  
Author(s):  
Pedro A. G. Tizei ◽  
Emma Harris ◽  
Marleen Renders ◽  
Vitor B. Pinheiro
Keyword(s):  
Substrate Specificity ◽  
Building Blocks ◽  
Sequence Length ◽  
Compositional Variation ◽  
Length Variation ◽  
Dna Assembly ◽  
Sequence Composition ◽  
Efficient Route ◽  
Generation Sequencing ◽  
Protein Loops

AbstractInsertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with no available platforms capable of systematically generating or analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 β-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum β-lactamases with loops of modified length and composition areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.

scholarly journals A novel framework for engineering protein loops exploring length and compositional variation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pedro A. G. Tizei ◽  
Emma Harris ◽  
Shamal Withanage ◽  
Marleen Renders ◽  
Vitor B. Pinheiro
Keyword(s):  
Substrate Specificity ◽  
Building Blocks ◽  
Sequence Length ◽  
Compositional Variation ◽  
Length Variation ◽  
Dna Assembly ◽  
Sequence Composition ◽  
Efficient Route ◽  
Generation Sequencing ◽  
Protein Loops

AbstractInsertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 β-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum β-lactamases with loops of modified length and composition—areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.

scholarly journals Variations in the Community Structure of Fungal Microbiota Associated with Apple Fruit Shaped by Fruit Bagging-Based Practice

2021 ◽  
Vol 7 (9) ◽  
pp. 764
Author(s):  
Punda Khwantongyim ◽  
Somying Wansee ◽  
Xi Lu ◽  
Wei Zhang ◽  
Guangyu Sun
Keyword(s):  
Management Practice ◽  
Agricultural Practices ◽  
Apple Fruit ◽  
Fungal Communities ◽  
Compositional Variation ◽  
Fungal Microbiota ◽  
Abundance And Diversity ◽  
Generation Sequencing ◽  
Fruit Part ◽  
Fruit Bagging

The various fungal communities that adhere to apple fruit are influenced by agricultural practices. However, the effects of fruit bagging-based management practice on the fungal microbiota are still unknown, and little is known about the fungal communities of bagged apple fruit. We conducted a study using apple fruit grown in a conventionally managed orchard where pesticide use is an indispensable practice. Fungal communities were collected from the calyx-end and peel tissues of bagged and unbagged fruit and characterized using barcode-type next-generation sequencing. Fruit bagging had a stronger effect on fungal richness, abundance, and diversity of the fungal microbiota in comparison to non-bagging. In addition, bagging also impacted the compositional variation of the fungal communities inhabiting each fruit part. We observed that fruit bagging had a tendency to maintain ecological equilibrium since Ascomycota and Basidiomycota were more distributed in bagged fruit than in unbagged fruit. These fungal communities consist of beneficial fungi rather than potentially harmful fungi. Approximately 50 dominant taxa were detected in bagged fruit, for example, beneficial genera such as Articulospora, Bullera, Cryptococcus, Dioszegia, Erythrobasidium, and Sporobolomyces, as well as pathogenic genera such as Aureobasidium and Taphrina. These results suggested that fruit bagging could significantly increase fungal richness and promote healthy fungal communities, especially the harmless fungal communities, which might be helpful for protecting fruit from the effects of pathogens. This study provides a foundation for understanding the impacts of bagging-based practice on the associated fungal microbiota.

scholarly journals First steps towards mitochondrial pan-genomics: detailed analysis of Fusarium graminearum mitogenomes

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5963 ◽  
Author(s):  
Balázs Brankovics ◽  
Tomasz Kulik ◽  
Jakub Sawicki ◽  
Katarzyna Bilska ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Fusarium Graminearum ◽  
Population Analysis ◽  
Gene Copy ◽  
Sequence Length ◽  
Length Variation ◽  
Group I Introns ◽  
Group I ◽  
Pooled Sequencing ◽  
Mitochondrial Introns

There is a gradual shift from representing a species’ genome by a single reference genome sequence to a pan-genome representation. Pan-genomes are the abstract representations of the genomes of all the strains that are present in the population or species. In this study, we employed a pan-genomic approach to analyze the intraspecific mitochondrial genome diversity of Fusarium graminearum. We present an improved reference mitochondrial genome for F. graminearum with an intron-exon annotation that was verified using RNA-seq data. Each of the 24 studied isolates had a distinct mitochondrial sequence. Length variation in the F. graminearum mitogenome was found to be largely due to variation of intron regions (99.98%). The “intronless” mitogenome length was found to be quite stable and could be informative when comparing species. The coding regions showed high conservation, while the variability of intergenic regions was highest. However, the most important variable parts are the intron regions, because they contain approximately half of the variable sites, make up more than half of the mitogenome, and show presence/absence variation. Furthermore, our analyses show that the mitogenome of F. graminearum is recombining, as was previously shown in F. oxysporum, indicating that mitogenome recombination is a common phenomenon in Fusarium. The majority of mitochondrial introns in F. graminearum belongs to group I introns, which are associated with homing endonuclease genes (HEGs). Mitochondrial introns containing HE genes may spread within populations through homing, where the endonuclease recognizes and cleaves the recognition site in the target gene. After cleavage of the “host” gene, it is replaced by the gene copy containing the intron with HEG. We propose to use introns unique to a population for tracking the spread of the given population, because introns can spread through vertical inheritance, recombination as well as via horizontal transfer. We demonstrate how pooled sequencing of strains can be used for mining mitogenome data. The usage of pooled sequencing offers a scalable solution for population analysis and for species level comparisons studies. This study may serve as a basis for future mitochondrial genome variability studies and representations.

scholarly journals Sequence-length variation of mtDNA HVS-I C-stretch in Chinese ethnic groups

2009 ◽  
Vol 10 (10) ◽  
pp. 711-720 ◽  
Author(s):  
Feng Chen ◽  
Yong-hui Dang ◽  
Chun-xia Yan ◽  
Yan-ling Liu ◽  
Ya-jun Deng ◽  
...  
Keyword(s):  
Ethnic Groups ◽  
Sequence Length ◽  
Length Variation

Synthesis of both isomers of aziridine derived from the mixture of cis- and trans-limonene oxide

2009 ◽  
Vol 87 (8) ◽  
pp. 1117-1121 ◽  
Author(s):  
Hossein Mehrabi
Keyword(s):  
Scale Up ◽  
Building Blocks ◽  
Easy Access ◽  
Efficient Route ◽  
Limonene Oxide ◽  
Cis And Trans

A short and efficient route is described for both isomers of aziridine derived from the commercially available 1:1 mixture of limonene oxides. The process is amenable to scale-up and allows easy access to multigram quantities of these useful chiral building blocks.

Kinetic resolution of oxime esters with lipases — synthesis of enantiomerically enriched building blocks with quaternary carbon centers and formal total synthesis of perhydro histrionicotoxin

2002 ◽  
Vol 80 (6) ◽  
pp. 686-691 ◽  
Author(s):  
Nicole Diedrichs ◽  
Ralf Krelaus ◽  
Ina Gedrath ◽  
Bernhard Westermann
Keyword(s):  
Substrate Specificity ◽  
Total Synthesis ◽  
Solvent Effects ◽  
Kinetic Resolution ◽  
Building Blocks ◽  
Beckmann Rearrangement ◽  
Optically Active ◽  
Quaternary Carbon ◽  
Carbon Centers

Enantiomerically enriched oximes bearing stereogenic quaternary carbon centers can be obtained by lipase-catalyzed kinetic resolution of oxime esters. Substrate specificity, solvent effects, and the use of different lipases are discussed. Kinetic resolution of butyrylated oximes by lipase PS in the presence of n-butanol gave the best ee-values of both the saponified oxime and the residual oxime ester. Subsequent stereospecific Beckmann rearrangement of an enantiomerically enriched oxime provided lactams, which could be employed for the synthesis of optically active perhydro histrionicotoxin.Key words: oxime, lipase, kinetic resolution, Beckmann rearrangement, perhydro histrionicotoxin.

scholarly journals How Next-Generation Sequencing Has Aided Our Understanding of the Sequence Composition and Origin of B Chromosomes

Genes ◽  
2017 ◽  
Vol 8 (11) ◽  
pp. 294 ◽  
Author(s):  
Alevtina Ruban ◽  
Thomas Schmutzer ◽  
Uwe Scholz ◽  
Andreas Houben
Keyword(s):  
Next Generation Sequencing ◽  
B Chromosomes ◽  
Next Generation ◽  
Sequence Composition ◽  
Generation Sequencing

scholarly journals Sequence composition diversity in Alaskan glacier and other metagenomes

2014 ◽  
Author(s):  
Sulbha Choudhari ◽  
Roman J Dial ◽  
Dibyendu Kumar ◽  
Daniel H Shain ◽  
Andrey Grigoriev
Keyword(s):  
Principal Component Analysis ◽  
Gc Content ◽  
Principal Component ◽  
Nucleotide Composition ◽  
Sequencing Technology ◽  
Sequence Composition ◽  
Comparative Metagenomics ◽  
Hypervariable Regions ◽  
Word Frequencies ◽  
Generation Sequencing

Metagenomics by next generation sequencing has become an important tool for interrogating complex microbial communities. In this study we analyzed several pairs of metagenomic samples obtained by different methods and observed biases, resulting in different nucleotide composition of the sequenced reads. The pairwise sample comparison was based on the principal component analysis of dinucleotide word frequencies in sequences obtained from different platforms. We found bias in the sequences obtained from the different platforms for the amplified hypervariable regions in 16S rRNA but not in shotgun metagenome reads aligned to such hypervariable regions. The differences and consistency of the distributions of the nucleotides suggest that the biases are likely due to a combination of biases introduced by PCR and different sequencing protocols, and they are related to the GC content of the reads produced. For this reason, caution should be exercised when interpreting the results of comparative metagenomics studies, as they may vary depending on the sequencing technology.

scholarly journals Dipeptides are minimalistic but sufficient for liquid-liquid phase separation

2021 ◽  
Author(s):  
Yiming Tang ◽  
Santu Bera ◽  
Yifei Yao ◽  
Jiyuan Zeng ◽  
Zenghui Lao ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Phase Behavior ◽  
General Property ◽  
Md Simulations ◽  
Building Blocks ◽  
Liquid Phase Separation ◽  
Sequence Length ◽  
Multivalent Interactions ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) of proteins mediates the assembly of biomolecular condensates involved in physiological and pathological processes. Identifying the minimalistic building blocks and the sequence determinant of protein phase separation is of urgent importance but remains challenging due to the enormous sequence space and difficulties of existing methodologies in characterizing the phase behavior of ultrashort peptides. Here we demonstrate computational tools to efficiently quantify the microscopic fluidity and density of liquid-condensates/solid-aggregates and the temperature-dependent phase diagram of peptides. Utilizing our approaches, we comprehensively predict the LLPS abilities of all 400 dipeptide combinations of coded amino acids based on 492 micro-second molecular dynamics simulations, and observe the occurrences of spontaneous LLPS. We identify 54 dipeptides that form solid-like aggregates and three categories of dipeptides with high LLPS propensity. Our predictions are validated by turbidity assays and differential interference contrast (DIC) microscopy on four representative dipeptides (WW, QW, GF, and VI). Phase coexistence diagrams are constructed to explore the temperature dependence of LLPS. Our results reveal that aromatic moieties are crucial for a dipeptide to undergo LLPS, and hydrophobic and polar components are indispensable. We demonstrate for the first time that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, suggesting that LLPS is a general property of peptides/proteins, independent of their sequence length. This study provides a computational and experimental approach to the prediction and characterization of the phase behavior of minimalistic peptides, and will be helpful for understanding the sequence-dependence and molecular mechanism of protein phase separation.SignificanceProtein liquid-liquid phase separation (LLPS) is associated with human health and diseases. Identifying the minimalistic building blocks and sequence determinants of LLPS is of urgent importance but remains computationally challenging partially due to the lack of methodologies characterizing the liquid condensates. Herein we provide approaches to evaluate LLPS ability of dipeptides, and screen all 400 dipeptides by MD simulations combined with multi-bead-per-residue models which capture key interactions driving LLPS that are missing in one-bead-per-residue models. Three categories of LLPS dipeptides are identified and the experimentally-verified QW dipeptide is by far the smallest LLPS system. Our results suggest that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, and LLPS is a general property of peptides/proteins, independent of their length.

scholarly journals Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1130
Author(s):  
Timo Meyer ◽  
Nadine Zumbrägel ◽  
Christina Geerds ◽  
Harald Gröger ◽  
Hartmut H. Niemann
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Catalytic Mechanism ◽  
Building Blocks ◽  
Secondary Amines ◽  
Hydrophobic Amino Acids ◽  
High Degree

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

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