Methods in enzymology最新文献_第9页

Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies. 通过动力学结构研究可视化CRISPR-Cas9的构象景观。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.004

Grace N Hibshman, David W Taylor

{"title":"Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies.","authors":"Grace N Hibshman, David W Taylor","doi":"10.1016/bs.mie.2025.01.004","DOIUrl":"10.1016/bs.mie.2025.01.004","url":null,"abstract":"CRISPR-Cas9 has transformed genome editing through its programmability and versatility. Its DNA cleavage activity involves dynamic conformational changes during gRNA binding, DNA recognition, R-loop formation, and endonuclease activation. Understanding these molecular transitions is critical for improving the specificity and efficiency of Cas9, but this remains challenging precisely due to these rapid structural rearrangements. Early structural studies provided foundational insights but were limited to static states under catalytically inactive conditions. Cryo-EM has since enabled visualization of the dynamic nature of active Cas9, by enriching for specific conformations. This chapter introduces a kinetics-informed cryo-EM approach to capture the stepwise activation of Cas9 in real time. With thorough kinetic analyses, such as stopped-flow measurements of R-loop formation, we describe how to identify optimal timepoints to visualize key conformational states with cryo-EM. Integration of kinetic and structural data enables precise mapping of the conformational landscape of Cas9 and other dynamic enzymes, advancing our understanding of their molecular mechanisms and providing a framework for engineering enhanced variants.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"41-53"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692837","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

Cloning and validating systems for high throughput molecular recording. 克隆和验证系统的高通量分子记录。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-09 DOI: 10.1016/bs.mie.2025.01.015

Anqi Zhao, Michelle M Chan

引用次数: 0

Design of fusion proteins for biocatalysis. 生物催化融合蛋白的设计。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-12 DOI: 10.1016/bs.mie.2025.01.013

Beyzanur Celebi, Janina Lawniczek, David Angelo V Guanzon, Anna Christina R Ngo

{"title":"Design of fusion proteins for biocatalysis.","authors":"Beyzanur Celebi, Janina Lawniczek, David Angelo V Guanzon, Anna Christina R Ngo","doi":"10.1016/bs.mie.2025.01.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.013","url":null,"abstract":"The use of enzymes to convert substrates into valuable products has been an integral part of biocatalysis. However, some reactions are energy-demanding that requires the use of NAD(P)H to proceed. This NAD(P)H can be costly impeding the progress of enzyme usage at a bigger scale. The rise of sophisticated cloning methods has allowed the possibility of constructing multi-enzyme complexes such as coupling NAD(P)H-requiring enzymes with NADH-regeneration systems such as formate dehydrogenases. This allows a more-efficient way to recycle co-factors or co-substrates with cheaper sacrificial substrate such as formate for formate dehydrogenases or glucose for glucose dehydrogenases. However, the design of fusion proteins requires careful attention especially on the peptide linker that will be used to connect two protein domains. The length and the property of the linker and even the orientation of the genes encoding for the proteins in the open reading frame can significantly affect the outcome of the fusion protein. In this chapter, we present a step-by-step procedure for the design of a fusion protein construct via Gibson assembly and how to design linker libraries from one construct using site-directed mutagenesis.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"393-406"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143972168","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

Tungsten containing aldehyde oxidoreductase (AOR)-family enzymes; past, present and future production strategies. 含钨醛氧化还原酶家族酶；过去、现在和未来的生产策略。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2025.01.027

Deborah M Boes, Rob A Schmitz, Peter-Leon Hagedoorn

{"title":"Tungsten containing aldehyde oxidoreductase (AOR)-family enzymes; past, present and future production strategies.","authors":"Deborah M Boes, Rob A Schmitz, Peter-Leon Hagedoorn","doi":"10.1016/bs.mie.2025.01.027","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.027","url":null,"abstract":"The transition metals tungsten and molybdenum are the heaviest metals found in biological systems and are embedded in the cofactor of several metalloenzymes. As a result of their redox activity, they provide great catalytic power in these enzymes and facilitate chemical reactions that would not occur using only the functionalities of natural amino acids. For their functionality these enzymes depend on a metal cofactor, which consists of at least one metal binding pterin (MPT) and a tungsten or molybdenum ion, but the complete make-up of the cofactor differs per enzyme group. One of these enzyme groups comprises the AOR-family enzymes. These enzymes have the ability to oxidize a range of aldehyde substrates into their corresponding carboxylic acid products. Next to this, they are also the only known catalysts able to perform the thermodynamically challenging reduction reaction of carboxylic acids to aldehydes. These enzymes are currently obtained by purification from the hyperthermophilic archaeon Pyrococcus furiosus. This process, however, does not yield a large amount of enzyme, since it is naturally expressed at moderate levels. For that reason, other production methods need to be considered if the enzyme is to be used on a large scale. These alternatives include the use of a recombinant expression system. The recombinant expression of W-dependent enzymes in different host organisms, such as Escherichia coli, has already been attempted for different enzymes, but with varying success. This shows that more research on the production, and especially incorporation of the metal cofactor, is necessary to achieve a successful production and use of recombinant AOR-family enzymes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"313-336"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143972172","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

Programmable C-to-U editing to track endogenous proteins. 可编程C-to-U编辑跟踪内源性蛋白质。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-02 DOI: 10.1016/bs.mie.2024.11.039

Min Hao, Tao Liu

引用次数: 0

Engineering unspecific peroxygenases by structure-guided in vivo recombination of homologous protein blocks. 通过结构引导同源蛋白块的体内重组工程非特异性过氧酶。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-06 DOI: 10.1016/bs.mie.2025.01.008

Alejandro Beltran-Nogal, Ivan Mateljak, David Gonzalez-Perez, Miguel Alcalde

引用次数: 0

EndoVIA for quantifying A-to-I editing and mapping the subcellular localization of edited transcripts. EndoVIA用于量化A-to-I编辑和绘制编辑转录本的亚细胞定位。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.029

Alexandria L Quillin, Benoît Arnould, Steve D Knutson, Tatiana F Flores, Jennifer M Heemstra

{"title":"EndoVIA for quantifying A-to-I editing and mapping the subcellular localization of edited transcripts.","authors":"Alexandria L Quillin, Benoît Arnould, Steve D Knutson, Tatiana F Flores, Jennifer M Heemstra","doi":"10.1016/bs.mie.2024.11.029","DOIUrl":"10.1016/bs.mie.2024.11.029","url":null,"abstract":"Adenosine-to-inosine (A-to-I) editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a prevalent post-transcriptional modification that is vital for numerous biological functions. Given that this modification impacts global gene expression, RNA localization, and innate cellular immunity, dysregulation of A-to-I editing has unsurprisingly been linked to a variety of cancers and other diseases. However, our current understanding of the underpinning mechanisms that connect dysregulated A-to-I editing and disease processes remains limited. Widely used methods require RNA extraction and pooling that ultimately erases subcellular localization and cell-to-cell variation, which may be critical to understanding misregulation. To overcome these challenges, we recently developed Endonuclease V Immunostaining Assay (EndoVIA) to selectively detect and visualize A-to-I edited RNA in situ. In this chapter, we describe in detail how to prepare cell samples, stain A-to-I edited transcripts with EndoVIA, quantify global inosine abundance, and visualize the subcellular localization of inosine-containing RNAs at the single molecule level.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"99-130"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908505/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052987","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

An in vitro cytidine deaminase assay to monitor APOBEC activity on DNA. 体外胞苷脱氨酶测定监测APOBEC对DNA的活性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-07 DOI: 10.1016/bs.mie.2024.11.037

Ambrocio Sanchez, Rémi Buisson

{"title":"An in vitro cytidine deaminase assay to monitor APOBEC activity on DNA.","authors":"Ambrocio Sanchez, Rémi Buisson","doi":"10.1016/bs.mie.2024.11.037","DOIUrl":"10.1016/bs.mie.2024.11.037","url":null,"abstract":"APOBEC enzymes promote the deamination of cytosine (C) to uracil (U) in DNA to defend cells against viruses but also serve as a predominant source of mutations in cancer genomes. This protocol describes an assay to monitor APOBEC deaminase activity in vitro on a synthetic DNA oligonucleotide. The method described here focuses specifically on APOBEC3B to illustrate the different steps of the assay. However, the protocol can be applied to monitor the DNA deaminase activity of any other member of the APOBEC family, such as APOBEC3A. This assay involves preparing APOBEC3B-expressing cell extract or purifying APOBEC3B by immunoprecipitation, followed by incubation with a single-stranded DNA containing a TpC motif. The deaminated cytosine is then removed by recombinant Uracil DNA Glycosylase present in the reaction to form an abasic site. The abasic site creates a weakness in the DNA's backbone, causing the DNA to be cleaved under high temperatures and alkaline conditions. Denaturing gel electrophoresis is used to separate cleaved DNA from full-length DNA, enabling the quantification of the percentage of deamination induced by APOBEC3B. This protocol can be used to determine the presence of APOBEC and the regulation of APOBEC activity in specific cell lines, to study substrate preference targeted by different members of the APOBEC family and different APOBEC mutants, or to determine the efficiency and specificity of inhibitor compounds against APOBEC enzymes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"201-219"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12083365/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144064144","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

Quantification of spermine oxidase (SMOX) activity in tissues by HPLC. 高效液相色谱法测定组织中精胺氧化酶（SMOX）活性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-17 DOI: 10.1016/bs.mie.2025.01.075

Jackson R Foley, Cassandra E Holbert

{"title":"Quantification of spermine oxidase (SMOX) activity in tissues by HPLC.","authors":"Jackson R Foley, Cassandra E Holbert","doi":"10.1016/bs.mie.2025.01.075","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.075","url":null,"abstract":"The polyamine oxidases, SMOX and PAOX, are enzymes involved in the normal metabolism of polyamines. Both enzymes are implicated in numerous human diseases including cancer, reperfusion injury, and neurodegenerative diseases. The ability to directly measure the activity of these enzymes is imperative in understanding their role in human health and disease. Most assays currently used to measure both SMOX and PAOX activity use a coupled reaction with horse radish peroxidase (HRP). These assays cannot be used when evaluating certain compounds for potential polyamine oxidase inhibition if these compouds also affect HRP activity. Additionally, since most assays use H2O2 production as an indicator of oxidase activity they cannot be used to evaluate polyamine oxidase activity in the presence of iron or other divalent metals. This prevents the use of these assays to evaluate polyamine oxidase activity in tissue samples. Here we describe the protocols for determining polyamine oxidase activity in an HRP-independent manner via an HPLC-based assay allowing for evaluation of both compounds that may interfere with HRP activity and polyamine oxidase activity in tissue samples.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"183-199"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094327","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

Cell-based determination of HDAC10-mediated polyamine deacetylase activity. 基于细胞的hdac10介导的多胺去乙酰酶活性测定。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-22 DOI: 10.1016/bs.mie.2025.01.047

Ishika Gupta, Ashley Nwafor, Robert A Casero, Tracy Murray Stewart

引用次数: 0