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A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts. 一种检测低丰度转录本中A-to- i编辑的探针捕获富集方法。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-02 DOI: 10.1016/bs.mie.2024.11.033
Emma Lamb, Dyuti Pant, Boyoon Yang, Heather A Hundley
{"title":"A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts.","authors":"Emma Lamb, Dyuti Pant, Boyoon Yang, Heather A Hundley","doi":"10.1016/bs.mie.2024.11.033","DOIUrl":"10.1016/bs.mie.2024.11.033","url":null,"abstract":"<p><p>Exactly two decades ago, the ability to use high-throughput RNA sequencing technology to identify sites of editing by ADARs was employed for the first time. Since that time, RNA sequencing has become a standard tool for researchers studying RNA biology and led to the discovery of RNA editing sites present in a multitude of organisms, across tissue types, and in disease. However, transcriptome-wide sequencing is not without limitations. Most notably, RNA sequencing depth of a given transcript is correlated with expression, and sequencing depth impacts the ability to robustly detect RNA editing events. This chapter focuses on a method for enrichment of low-abundance transcripts that can facilitate more efficient sequencing and detection of RNA editing events. An important note is that while we describe aspects of the protocol important for capturing intron-containing transcripts, this probe-based enrichment method could be easily modified to assess editing within any low-abundance transcript. We also provide some perspectives on the current limitations as well as important future directions for expanding this technology to gain more insights into how RNA editing can impact transcript diversity.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"55-75"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Assay methods and colorimetric screens for lignin-degrading microbes and lignin-oxidising enzymes. 木质素降解微生物和木质素氧化酶的测定方法和比色筛。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-16 DOI: 10.1016/bs.mie.2025.01.055
Timothy D H Bugg, Mark Ahmad, Charles R Taylor, Marina Konstantopoulou, Goran M M Rashid
{"title":"Assay methods and colorimetric screens for lignin-degrading microbes and lignin-oxidising enzymes.","authors":"Timothy D H Bugg, Mark Ahmad, Charles R Taylor, Marina Konstantopoulou, Goran M M Rashid","doi":"10.1016/bs.mie.2025.01.055","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.055","url":null,"abstract":"<p><p>Assaying enzymes and microbes for activity for degradation of polymeric lignin is inherently challenging to do. This article describes several methods that our research group has developed for assay of lignin-oxidising enzymes and lignin-degrading microbes. The assay methods involve (1) colorimetric assays involving chemically nitrated lignin; (2) changes in molecular weight using gel filtration chromatography; (3) delignification of lignocellulose using Klason assay; (4) colorimetric assays for release of low molecular weight phenols and carbonyl compounds.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"716 ","pages":"105-123"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144294053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Design, construction and characterization of laccase-xylanase chimeras by insertional fusion. 插入融合漆酶-木聚糖酶嵌合体的设计、构建与表征。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-18 DOI: 10.1016/bs.mie.2025.01.044
Lucas F Ribeiro, Gilvan P Furtado, Marcos R Lourenzoni, Richard J Ward
{"title":"Design, construction and characterization of laccase-xylanase chimeras by insertional fusion.","authors":"Lucas F Ribeiro, Gilvan P Furtado, Marcos R Lourenzoni, Richard J Ward","doi":"10.1016/bs.mie.2025.01.044","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.044","url":null,"abstract":"<p><p>The broad substrate specificity of laccases makes these enzymes suitable for a wide range of applications. The use of protein engineering strategies to modulate the catalytic properties of these enzymes is a promising strategy to expand their use in the sustainable economy. Here we describe the construction of laccase-xylanase bifunctional enzyme by insertional fusion using a procedure based on the rational design starting with the analysis of the 3D-structure of laccase to select positions for the insertion of the xylanase domain, followed by the creation of the fusion construct by ligation of overlapping fragments generated by PCR. Finally, the heterologous expression and biochemical characterization of the laccase and xylanase activities of the fusion protein is described and demonstrate significant increase in the laccase activity. These protocols can be applied to the fusion of any pair of proteins.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"716 ","pages":"217-238"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144294056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Preface. 前言。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00251-4
Timothy Bugg, Juan Carro
{"title":"Preface.","authors":"Timothy Bugg, Juan Carro","doi":"10.1016/S0076-6879(25)00251-4","DOIUrl":"https://doi.org/10.1016/S0076-6879(25)00251-4","url":null,"abstract":"","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"716 ","pages":"xix-xx"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144294091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Targeted genome mining for natural product discovery. 针对天然产物发现的基因组挖掘。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-15 DOI: 10.1016/bs.mie.2025.03.005
José D D Cediel-Becerra, Marc G Chevrette
{"title":"Targeted genome mining for natural product discovery.","authors":"José D D Cediel-Becerra, Marc G Chevrette","doi":"10.1016/bs.mie.2025.03.005","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.03.005","url":null,"abstract":"<p><p>Natural products are a rich source of bioactive compounds, which are encoded by the biosynthetic gene clusters (BGCs). Genome mining is an essential strategy for identifying and characterizing BGCs. Targeted genome mining excels in the identification of similar BGCs based on key biosynthetic enzymes across multiple genomes. This chapter details the use of both manual and automated targeted genome mining to identify members of the FK-family BGCs (rapamycin, FK520/506). We describe the process of selecting query proteins, evaluating genomic context, and determining the presence of putative BGCs. Additionally, to streamline the manual process, we used GATOR-GC, a computational tool that identifies similar BGCs using required and optional proteins, performs comparative genomic analysis, deduplicates redundant BGCs, and generates visualizations of gene conservation and BGC diversity. Applying this approach, we showed how to identify FK-family members, both by looking into the cluster conservation diagrams, and the clustered heatmap summarizing all-vs-all BGC comparisons. The methods outlined here can be adapted for mining other natural product families, offering a scalable framework for uncovering novel biosynthetic pathways. Beyond natural product discovery, GATOR-GC provides broader applications for analyzing gene cluster conservation, organization, and evolutionary patterns.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"717 ","pages":"267-298"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144619077","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Defining APOBEC-induced mutation signatures and modifying activities in yeast. 在酵母中定义apobecc诱导的突变特征和修饰活性。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-02 DOI: 10.1016/bs.mie.2024.11.041
Tony M Mertz, Zachary W Kockler, Margo Coxon, Cameron Cordero, Atri K Raval, Alexander J Brown, Victoria Harcy, Dmitry A Gordenin, Steven A Roberts
{"title":"Defining APOBEC-induced mutation signatures and modifying activities in yeast.","authors":"Tony M Mertz, Zachary W Kockler, Margo Coxon, Cameron Cordero, Atri K Raval, Alexander J Brown, Victoria Harcy, Dmitry A Gordenin, Steven A Roberts","doi":"10.1016/bs.mie.2024.11.041","DOIUrl":"10.1016/bs.mie.2024.11.041","url":null,"abstract":"<p><p>APOBEC cytidine deaminases guard cells in a variety of organisms from invading viruses and foreign nucleic acids. Recently, several human APOBECs have been implicated in mutating evolving cancer genomes. Expression of APOBEC3A and APOBEC3B in yeast allowed experimental derivation of the substitution patterns they cause in dividing cells, which provided critical links to these enzymes in the etiology of the COSMIC single base substitution (SBS) signatures 2 and 13 in human tumors. Additionally, the ability to scale yeast experiments to high-throughput screens allows use of this system to also investigate cellular pathways impacting the frequency of APOBEC-induced mutation. Here, we present validated methods utilizing yeast to determine APOBEC mutation signatures, genetic interactors, and chromosomal substrate preferences. These methods can be employed to assess the potential of other human APOBECs and APOBEC orthologs in different species to contribute to cancer genome evolution as well as define the pathways that protect the nuclear genome from inadvertent APOBEC activity during viral restriction.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"115-161"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12324072/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144035720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Expression and purification of methionine aminopeptidases and N-terminal acetyltransferases. 蛋氨酸氨基肽酶和n端乙酰转移酶的表达和纯化。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-05 DOI: 10.1016/bs.mie.2025.06.022
Marius Alexander Klein, Irmgard Sinning
{"title":"Expression and purification of methionine aminopeptidases and N-terminal acetyltransferases.","authors":"Marius Alexander Klein, Irmgard Sinning","doi":"10.1016/bs.mie.2025.06.022","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.06.022","url":null,"abstract":"<p><p>Methionine aminopeptidases (MetAPs) and N-terminal acetyltransferases (NATs) function co-translationally at the ribosome to enzymatically modify the emerging nascent chain. Eukaryotes express two types of MetAPs, namely MetAP1 and MetAP2, which can both carry out N-terminal methionine excision (NME) at the ribosome during translation. Following NME, the most abundant NAT, NatA, can acetylate the penultimate amino acid of the nascent chain, under regulation of the NatA inhibitor HypK. Alternatively, NatA can accommodate a second enzyme, called NAA50, to form the NatE complex capable of acetylating the initiator methionine. The abundant N-terminal modifications facilitated by MetAP1/2 and NatA/E-HypK impinge on protein function, interactions, lifetime and overall proteostasis. Robust and reliable methods for the expression and purification of MetAPs and NATs set the stage for targeted functional and structural studies of these enzymes. Established methods for the production of pure ribosome-associated enzymes and stochiometric complexes will be delineated in this chapter.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"718 ","pages":"1-18"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961148","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Proteome analysis of puromycin-labeled nascent polypeptides. 嘌呤霉素标记新生多肽的蛋白质组学分析。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-08 DOI: 10.1016/bs.mie.2025.06.018
Koshi Imami
{"title":"Proteome analysis of puromycin-labeled nascent polypeptides.","authors":"Koshi Imami","doi":"10.1016/bs.mie.2025.06.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.06.018","url":null,"abstract":"<p><p>Emerging nascent polypeptides from ribosomes and their protein N-termini have significant impact on protein stability, folding, interaction and subcellular targeting. To profile elongating nascent polypeptides on a proteome-wide scale, here we present a streamlined protocol that combined a biochemical enrichment of puromycin-labeled nascent polypeptides and mass spectrometry-based quantitative proteomics. This chapter includes the detailed protocol for metabolic pulse labeling with puromycin and SILAC amino acids, immunoprecipitation for nascent polypeptides, fractionation and enrichment of N-terminal acetylated peptides as well as MS and data analysis. These protocols are illustrated using HeLa cells treated with cycloheximide, a protein synthesis inhibitor, but can be broadly applied to any cell culture systems, including primary cultures, or any treatments (e.g., drugs).</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"719 ","pages":"1-23"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145138137","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Tuning the function of de novo designed artificial Cu proteins by modulating reorganization energies. 通过调节重组能调节从头设计的人工铜蛋白的功能。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-28 DOI: 10.1016/bs.mie.2025.06.033
Divyansh Prakash, Simran Sony, Saumen Chakraborty
{"title":"Tuning the function of de novo designed artificial Cu proteins by modulating reorganization energies.","authors":"Divyansh Prakash, Simran Sony, Saumen Chakraborty","doi":"10.1016/bs.mie.2025.06.033","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.06.033","url":null,"abstract":"<p><p>Artificial metalloenzyme (ArM) design is an attractive approach for deciphering the functional determinants of native enzymes or imparting new functions. Metalloproteins with redox cofactors catalyze critical reactions enabled by optimized primary, secondary, and outer-sphere interactions that facilitate efficient electron transfer. Controlling outer-sphere interactions to tune reactivity remains a challenge. Inspired by the common coordination motifs of copper (Cu) proteins, we have designed artificial Cu proteins (ArCuPs), extensively characterized them, and demonstrated their H<sub>2</sub>O<sub>2</sub>, O<sub>2</sub>, and C-H oxidation reactivity to abiotic substrates. By selectively modifying outer-sphere solvent reorganization energy, we have shown that we can control C-H peroxidation activity. This chapter describes methods to design ArCuPs and their detailed characterization, including electrochemical C-H oxidation, and the procedures for determining reorganization energies using electrochemistry as a readily available laboratory tool.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"720 ","pages":"349-375"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232842","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Preface. 前言。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00380-5
{"title":"Preface.","authors":"","doi":"10.1016/S0076-6879(25)00380-5","DOIUrl":"https://doi.org/10.1016/S0076-6879(25)00380-5","url":null,"abstract":"","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"720 ","pages":"xix-xx"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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