Methods in enzymology最新文献_第6页

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":"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.","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

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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

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

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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":"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.","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

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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

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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

引用次数: 0

Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity. 将跨孔膜作为研究横向脂膜异质性的工具。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-05 DOI: 10.1016/bs.mie.2024.02.009

Larissa Socrier, Claudia Steinem

{"title":"Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity.","authors":"Larissa Socrier, Claudia Steinem","doi":"10.1016/bs.mie.2024.02.009","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.02.009","url":null,"abstract":"Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (ld) and liquid-ordered (lo) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a lo phase enriched in the high-melting lipids and cholesterol and a ld phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated lo/ld phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with lo/ld phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"455-483"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545036","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 use of hemifusion to create asymmetric giant unilamellar vesicles: Insights on induced order domains. 利用半融合技术制造非对称巨型单拉米尔囊泡：关于诱导阶域的见解。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-06 DOI: 10.1016/bs.mie.2024.03.025

Thais A Enoki

{"title":"The use of hemifusion to create asymmetric giant unilamellar vesicles: Insights on induced order domains.","authors":"Thais A Enoki","doi":"10.1016/bs.mie.2024.03.025","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.025","url":null,"abstract":"The natural asymmetry of the lipid bilayer in biological membranes is, in part, a testament to the complexity of the structure and function of this barrier limiting and protecting cells (or organelles). These lipid bilayers consist of two lipid leaflets with different lipid compositions, resulting in unique interactions within each leaflet. These interactions, combined with interactions between the two leaflets, determine the overall behavior of the membrane. Model membranes provide the most suitable option for investigating the fundamental interactions of lipids. This report describes a comprehensive method to make asymmetric giant unilamellar vesicles (aGUVs) using the technique of hemifusion. In this method, calcium ions induce the hemifusion of giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB), both having different lipid compositions. During hemifusion, a stalk, or a more commonly seen hemifusion diaphragm, connects the outer leaflets of GUVs and the SLB. The lateral diffusion of lipids naturally promotes the lipid exchange between the connected outer leaflets. After calcium chelation to prevent further fusion, a mechanical shear detaches aGUVs from the SLB. A fluorescence quench assay is employed to test the extent of bilayer asymmetry. A fluorescence quenching assay tests bilayer asymmetry and verifies dye and lipid migration to a GUV's outer leaflet.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"127-159"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545060","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