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In organello silencing of mitochondrial gene expression. 在器官内沉默线粒体基因的表达。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-21 DOI: 10.1016/bs.mie.2024.07.035
Mats Koschel, Luis Daniel Cruz-Zaragoza
{"title":"In organello silencing of mitochondrial gene expression.","authors":"Mats Koschel, Luis Daniel Cruz-Zaragoza","doi":"10.1016/bs.mie.2024.07.035","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.035","url":null,"abstract":"<p><p>Mitochondria contain proteins from two genetic origins. Most mitochondrial proteins are encoded in the nuclear genome, translated in the cytosol, and subsequently imported into the different mitochondrial sub-compartments. A small number is encoded in the mitochondrial DNA (mtDNA). The manipulation of the mtDNA gene expression represents a challenge. Here, we present an in vitro approach using morpholinos chemically linked to a precursor protein to silence gene expression in purified human mitochondria. The protocol is demonstrated with a Jac1-morpholino chimera specifically targeting COX1 mRNA. The chimera import and mitochondrial translation requirements are described in a step-by-step procedure, where the dose-dependent effect of reducing COX1 translation is observed. The affinity and specificity of chimera-mRNA binding also show great applicability to purify transcript-associated proteins by using the imported chimera construct as bait for immunoprecipitation. This new strategy opens up the possibility to address mechanistic questions about gene expression and physiology in mitochondria.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"501-518"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504045","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
Methods to analyze mitochondrial protein translocation in plant mitochondria. 分析植物线粒体蛋白质转位的方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-24 DOI: 10.1016/bs.mie.2024.07.021
Saurabh Saha, Yanqiao Zhu, James Whelan, Monika W Murcha
{"title":"Methods to analyze mitochondrial protein translocation in plant mitochondria.","authors":"Saurabh Saha, Yanqiao Zhu, James Whelan, Monika W Murcha","doi":"10.1016/bs.mie.2024.07.021","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.021","url":null,"abstract":"<p><p>Complex processes have evolved in plants to import proteins into mitochondria. Investigating these processes in plants provides insights into the specialised machinery and pathways that have evolved to cope with; (1) the immobile nature of plants that results in exposure to environmental stresses, and (2) the more complex cell environment due to the presence of plastids, the most prevalent being chloropalst in leaves. In this chapter, we present detailed protocols for the isolation of respiratory competent, coupled mitochondria from Arabidopsis thaliana, conducting protein import assays, and analyzing protein assembly into large multi-subunit complexes. Additionally, we present straightforward protocols for examining the localization of fluorescently tagged proteins to organelles such as mitochondria through protoplast transfections.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"475-497"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504050","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
Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases. 非血红素铁（II）依赖性氧化酶/脱羧酶的异腈生物合成。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-29 DOI: 10.1016/bs.mie.2024.06.002
Antonio Del Rio Flores, Rui Zhai, Wenjun Zhang
{"title":"Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases.","authors":"Antonio Del Rio Flores, Rui Zhai, Wenjun Zhang","doi":"10.1016/bs.mie.2024.06.002","DOIUrl":"10.1016/bs.mie.2024.06.002","url":null,"abstract":"<p><p>The isonitrile group is a compact, electron-rich moiety coveted for its commonplace as a building block and bioorthogonal functionality in synthetic chemistry and chemical biology. Hundreds of natural products containing an isonitrile group with intriguing bioactive properties have been isolated from diverse organisms. Our recent discovery of a conserved biosynthetic gene cluster in some Actinobacteria species highlighted a novel enzymatic pathway to isonitrile formation involving a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase. Here, we focus this chapter on recent advances in understanding and probing the biosynthetic machinery for isonitrile synthesis by non-heme iron(II) and α-ketoglutarate-dependent dioxygenases. We will begin by describing how to harness isonitrile enzymatic machinery through heterologous expression, purification, synthetic strategies, and in vitro biochemical/kinetic characterization. We will then describe a generalizable strategy to probe the mechanism for isonitrile formation by combining various spectroscopic methods. The chapter will also cover strategies to study other enzyme homologs by implementing coupled assays using biosynthetic pathway enzymes. We will conclude this chapter by addressing current challenges and future directions in understanding and engineering isonitrile synthesis.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"143-172"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11424024/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291299","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, purification, kinetics, and crystallization of non-heme mononuclear iron enzymes: Biphenyl, Phthalate, and Terephthalate dioxygenases. 非血红素单核铁酶的表达、纯化、动力学和结晶：联苯、邻苯二甲酸盐和对苯二甲酸盐二氧酶。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-08 DOI: 10.1016/bs.mie.2024.05.014
Jai Krishna Mahto, Arpan Kayastha, Pravindra Kumar
{"title":"Expression, purification, kinetics, and crystallization of non-heme mononuclear iron enzymes: Biphenyl, Phthalate, and Terephthalate dioxygenases.","authors":"Jai Krishna Mahto, Arpan Kayastha, Pravindra Kumar","doi":"10.1016/bs.mie.2024.05.014","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.014","url":null,"abstract":"<p><p>Non-heme iron oxygenases constitute a versatile enzyme family that is crucial for incorporating molecular oxygen into diverse biomolecules. Despite their importance, only a limited number of these enzymes have been structurally and functionally characterized. Surprisingly, there remains a significant gap in understanding how these enzymes utilize a typical architecture and reaction mechanism to catalyze a wide range of reactions. Improving our understanding of these catalysts holds promise for advancing both fundamental enzymology and practical applications. This chapter aims to outline methods for heterologous expression, enzyme preparation, in vitro enzyme assays, and crystallization of biphenyl dioxygenase, phthalate dioxygenase and terephthalate dioxygenase. These enzymes catalyze the dihydroxylation of biphenyl, phthalate and terephthalate molecules, serving as a model for functional and structural analysis of other non-heme iron oxygenases.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"39-58"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291218","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
Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis. 草嗪霉素生物合成过程中的非血红素铁依赖性单氧化酶 OzmD 的表达、纯化和特征。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-08 DOI: 10.1016/bs.mie.2024.05.006
Daan Ren, Yu-Hsuan Lee, Hung-Wen Liu
{"title":"Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis.","authors":"Daan Ren, Yu-Hsuan Lee, Hung-Wen Liu","doi":"10.1016/bs.mie.2024.05.006","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.006","url":null,"abstract":"<p><p>Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"113-142"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291216","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
Isotopic labelings for mechanistic studies. 用于机理研究的同位素标记。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-07 DOI: 10.1016/bs.mie.2024.01.011
Houchao Xu, Jeroen S Dickschat
{"title":"Isotopic labelings for mechanistic studies.","authors":"Houchao Xu, Jeroen S Dickschat","doi":"10.1016/bs.mie.2024.01.011","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.01.011","url":null,"abstract":"<p><p>The intricate mechanisms in the biosynthesis of terpenes belong to the most challenging problems in natural product chemistry. Methods to address these problems include the structure-based site-directed mutagenesis of terpene synthases, computational approaches, and isotopic labeling experiments. The latter approach has a long tradition in biosynthesis studies and has recently experienced a revival, after genome sequencing enabled rapid access to biosynthetic genes and enzymes. Today, this allows for a combined approach in which isotopically labeled substrates can be incubated with recombinant terpene synthases. These clearly defined reaction setups can give detailed mechanistic insights into the reactions catalyzed by terpene synthases, and recent developments have substantially deepened our understanding of terpene biosynthesis. This chapter will discuss the state of the art and introduce some of the most important methods that make use of isotopic labelings in mechanistic studies on terpene synthases.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"163-186"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469491","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
The density-threshold affinity: Calculating lipid binding affinities from unbiased coarse-grained molecular dynamics simulations. 密度-阈值亲和力：通过无偏粗粒度分子动力学模拟计算脂质结合亲和力。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-04 DOI: 10.1016/bs.mie.2024.03.008
Jesse W Sandberg, Ezry Santiago-McRae, Jahmal Ennis, Grace Brannigan
{"title":"The density-threshold affinity: Calculating lipid binding affinities from unbiased coarse-grained molecular dynamics simulations.","authors":"Jesse W Sandberg, Ezry Santiago-McRae, Jahmal Ennis, Grace Brannigan","doi":"10.1016/bs.mie.2024.03.008","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.008","url":null,"abstract":"<p><p>Many membrane proteins are sensitive to their local lipid environment. As structural methods for membrane proteins have improved, there is growing evidence of direct, specific binding of lipids to protein surfaces. Unfortunately the workhorse of understanding protein-small molecule interactions, the binding affinity for a given site, is experimentally inaccessible for these systems. Coarse-grained molecular dynamics simulations can be used to bridge this gap, and are relatively straightforward to learn. Such simulations allow users to observe spontaneous binding of lipids to membrane proteins and quantify localized densities of individual lipids or lipid fragments. In this chapter we outline a protocol for extracting binding affinities from these localized distributions, known as the \"density threshold affinity.\" The density threshold affinity uses an adaptive and flexible definition of site occupancy that alleviates the need to distinguish between \"bound'' lipids and bulk lipids that are simply diffusing through the site. Furthermore, the method allows \"bead-level\" resolution that is suitable for the case where lipids share binding sites, and circumvents ambiguities about a relevant reference state. This approach provides a convenient and straightforward method for comparing affinities of a single lipid species for multiple sites, multiple lipids for a single site, and/or a single lipid species modeled using multiple forcefields.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"701 ","pages":"47-82"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723933","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
Modeling the mechanochemical feedback for membrane-protein interactions using a continuum mesh model. 利用连续网格模型建立膜蛋白相互作用的机械化学反馈模型。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-15 DOI: 10.1016/bs.mie.2024.03.016
Christopher T Lee, Padmini Rangamani
{"title":"Modeling the mechanochemical feedback for membrane-protein interactions using a continuum mesh model.","authors":"Christopher T Lee, Padmini Rangamani","doi":"10.1016/bs.mie.2024.03.016","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.016","url":null,"abstract":"<p><p>The Helfrich free energy is widely used to model the generation of membrane curvature due to different physical and chemical components. The governing equations resulting from the energy minimization procedure are a system of coupled higher order partial differential equations. Simulations of membrane deformation for obtaining quantitative comparisons against experimental observations require computational schemes that will allow us to solve these equations without restrictions to axisymmetric coordinates. Here, we describe one such tool that we developed in our group based on discrete differential geometry to solve these equations along with examples.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"701 ","pages":"387-424"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723926","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
Quantification of membrane geometry and protein sorting on cell membrane protrusions using fluorescence microscopy. 利用荧光显微镜对细胞膜突起的膜几何形状和蛋白质分类进行量化。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-16 DOI: 10.1016/bs.mie.2024.01.023
Shilong Yang, Zheng Shi
{"title":"Quantification of membrane geometry and protein sorting on cell membrane protrusions using fluorescence microscopy.","authors":"Shilong Yang, Zheng Shi","doi":"10.1016/bs.mie.2024.01.023","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.01.023","url":null,"abstract":"<p><p>Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences of membrane proteins.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"385-411"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545053","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
Thickness determination of hydroperoxidized lipid bilayers from medium-resolution cryo-TEM images. 从中等分辨率的低温 TEM 图像中确定氢过氧化脂质双分子层的厚度。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-23 DOI: 10.1016/bs.mie.2024.02.008
Eulalie Lafarge, Carlos M Marques, Marc Schmutz, Pierre Muller, André P Schroder
{"title":"Thickness determination of hydroperoxidized lipid bilayers from medium-resolution cryo-TEM images.","authors":"Eulalie Lafarge, Carlos M Marques, Marc Schmutz, Pierre Muller, André P Schroder","doi":"10.1016/bs.mie.2024.02.008","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.02.008","url":null,"abstract":"<p><p>As the primary products of lipid oxidation, lipid hydroperoxides constitute an important class of lipids generated by aerobic metabolism. However, despite several years of effort, the structure of the hydroperoxidized bilayer has not yet been observed under electron microscopy. Here we use a 200 kV Cryo-TEM to image small unilamellar vesicles (SUVs) made (i) of pure POPC or SOPC, (ii) of their pure hydroperoxidized form, and (iii) of their equimolar mixtures. We show that the challenges posed by the determination of the thickness of the hydroperoxidized bilayers under these observation conditions can be addressed by an image analysis method that we developed and describe here.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"329-348"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545061","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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