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In vitro assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays. 茄属植物和玉米中的 9-顺式环氧类胡萝卜素二加氧酶（NCED）的体外测定和抑制作用。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-06 DOI: 10.1016/bs.mie.2024.05.012
Peter J Harrison, Jake Chandler, Andrew J Thompson, Timothy D H Bugg
{"title":"In vitro assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays.","authors":"Peter J Harrison, Jake Chandler, Andrew J Thompson, Timothy D H Bugg","doi":"10.1016/bs.mie.2024.05.012","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.012","url":null,"abstract":"<p><p>The article reports methods for the expression and assay of 9-cis-epoxycarotenoid cleavage dioxygenase (NCED), an enzyme involved in the biosynthesis of phytohormone abscisic acid in plants. A method for the preparation of the unstable substrate 9'-cis-neoxanthin from fresh spinach is described. The inhibition of Solanum lycopersicum NCED by a series of aryl hydroxamic acid inhibitors is illustrated, and inhibitors D2 and D4 are assayed against NCED isozymes from Zea mays.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"704 ","pages":"291-312"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291298","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
Whole-cell Rieske non-heme iron biocatalysts. 全细胞 Rieske 非血红素铁生物催化剂。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-22 DOI: 10.1016/bs.mie.2024.05.008
Meredith B Mock, Shuyuan Zhang, Ryan M Summers
{"title":"Whole-cell Rieske non-heme iron biocatalysts.","authors":"Meredith B Mock, Shuyuan Zhang, Ryan M Summers","doi":"10.1016/bs.mie.2024.05.008","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.008","url":null,"abstract":"<p><p>Rieske non-heme iron oxygenases (ROs) possess the ability to catalyze a wide range of reactions. Their ability to degrade aromatic compounds is a unique characteristic and makes ROs interesting for a variety of potential applications. However, purified ROs can be challenging to work with due to low stability and long, complex electron transport chains. Whole cell biocatalysis represents a quick and reliable method for characterizing the activity of ROs and harnessing their metabolic potential. In this protocol, we outline a step-by-step protocol for the overexpression of ROs for whole cell biocatalysis and characterization. We have utilized a caffeine-degrading, N-demethylation system, expressing the RO genes ndmA and ndmD, as an example of this method.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"243-262"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291212","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
Analysis of the mitochondria-associated degradation pathway (MAD) in yeast cells. 分析酵母细胞中线粒体相关降解途径（MAD）。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-20 DOI: 10.1016/bs.mie.2024.09.001
Pin-Chao Liao, Liza A Pon
{"title":"Analysis of the mitochondria-associated degradation pathway (MAD) in yeast cells.","authors":"Pin-Chao Liao, Liza A Pon","doi":"10.1016/bs.mie.2024.09.001","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.09.001","url":null,"abstract":"<p><p>Mitochondria are critical for cellular function in health, disease and aging. Mitochondria-associated degradation (MAD), a pathway for quality control of the organelle, recognizes and ubiquitinates unfolded mitochondrial proteins, removes them from the organelle using a conserved segregase complex, which contains an AAA-ATPase Cdc48 and its cofactors, and degrades them using the ubiquitin-proteasome system (UPS). Here, we describe an approach to (1) study the turnover and ubiquitination of candidate MAD substrates, (2) assay retrotranslocation and export of MAD substrates from the mitochondrial matrix in vitro, and (3) study interactions between MAD substrates and Cdc48 using the budding yeast, Saccharomyces cerevisiae, as a model organism.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"585-610"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564301","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
A modified procedure for separating yeast peroxisomes from mitochondria. 从线粒体中分离酵母过氧物酶体的改进程序。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-14 DOI: 10.1016/bs.mie.2024.07.046
Nitya Aravindan, Doron Rapaport
{"title":"A modified procedure for separating yeast peroxisomes from mitochondria.","authors":"Nitya Aravindan, Doron Rapaport","doi":"10.1016/bs.mie.2024.07.046","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.046","url":null,"abstract":"<p><p>Mitochondria and peroxisomes are mutually dependent organelles that share several membrane proteins that carry out the same function in both organelles. To study the unique features of these dually localized proteins in each of the two organelles, it is essential to separate mitochondria from peroxisomes. Isolating organelles from cells of Baker's yeast, Saccharomyces cerevisiae, is crucial for our understanding of the biogenesis and functions of proteins. Traditionally, subcellular fractionation and isolation of individual organelles by differential centrifugation benefit from the specific and unique density of each organelle. However, when yeast cells are grown under normal conditions, certain organelles like mitochondria and peroxisomes share strikingly similar densities. This similarity challenges the separation of these organelles from one another. In this chapter, we describe an optimized procedure to address this task. We depict growth conditions that would favor stimulation of peroxisomes to increase their number and density, and portray organellar isolation followed by gradient centrifugation, enabling an improved separation of both organelles. Additionally, we illustrate the advantage of the procedure to study the dual localization of the membrane protein Fis1.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"37-57"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504037","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
Super-resolution microscopy methods to study membrane pores in situ. 原位研究膜孔的超分辨率显微镜方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-31 DOI: 10.1016/bs.mie.2024.03.020
Timo Dellmann, Raed Shalaby, Ana J Garcia-Saez
{"title":"Super-resolution microscopy methods to study membrane pores in situ.","authors":"Timo Dellmann, Raed Shalaby, Ana J Garcia-Saez","doi":"10.1016/bs.mie.2024.03.020","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.020","url":null,"abstract":"","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"161-187"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545057","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. 序言
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 DOI: 10.1016/S0076-6879(24)00271-4
Tobias Baumgart, Markus Deserno
{"title":"Preface.","authors":"Tobias Baumgart, Markus Deserno","doi":"10.1016/S0076-6879(24)00271-4","DOIUrl":"https://doi.org/10.1016/S0076-6879(24)00271-4","url":null,"abstract":"","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"xxiii-xxv"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545037","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 spectral phasor approach to resolving membrane order with environmentally sensitive dyes. 用环境敏感染料解析膜阶的光谱相量法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-05 DOI: 10.1016/bs.mie.2024.01.024
Agustín Mangiarotti, Rumiana Dimova
{"title":"The spectral phasor approach to resolving membrane order with environmentally sensitive dyes.","authors":"Agustín Mangiarotti, Rumiana Dimova","doi":"10.1016/bs.mie.2024.01.024","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.01.024","url":null,"abstract":"<p><p>Hyperspectral imaging is a technique that captures a three-dimensional array of spectral information at each spatial location within a sample, enabling precise characterization and discrimination of biological structures, materials, and chemicals, based on their unique spectral features. Nowadays most commercially available confocal microscopes allow hyperspectral imaging measurements, providing a valuable source of spatially resolved spectroscopic data. Spectral phasor analysis quantitatively and graphically transforms the fluorescence spectra at each pixel of a hyperspectral image into points in a polar plot, offering a visual representation of the spectral characteristics of fluorophores within the sample. Combining the use of environmentally sensitive dyes with phasor analysis of hyperspectral images provides a powerful tool for measuring small changes in lateral membrane heterogeneity. Here, we focus on applications of spectral phasor analysis for the probe LAURDAN on model membranes to resolve packing and hydration. The method is broadly applicable to other dyes and to complex systems such as cell membranes.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"105-126"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545059","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
Free energy calculations for membrane morphological transformations and insights to physical biology and oncology. 膜形态转化的自由能计算以及对物理生物学和肿瘤学的启示。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-14 DOI: 10.1016/bs.mie.2024.03.028
Kshitiz Parihar, Seung-Hyun Ko, Ryan Bradley, Phillip Taylor, N Ramakrishnan, Tobias Baumgart, Wei Guo, Valerie M Weaver, Paul A Janmey, Ravi Radhakrishnan
{"title":"Free energy calculations for membrane morphological transformations and insights to physical biology and oncology.","authors":"Kshitiz Parihar, Seung-Hyun Ko, Ryan Bradley, Phillip Taylor, N Ramakrishnan, Tobias Baumgart, Wei Guo, Valerie M Weaver, Paul A Janmey, Ravi Radhakrishnan","doi":"10.1016/bs.mie.2024.03.028","DOIUrl":"10.1016/bs.mie.2024.03.028","url":null,"abstract":"<p><p>In this chapter, we aim to bridge basic molecular and cellular principles surrounding membrane curvature generation with rewiring of cellular signals in cancer through multiscale models. We describe a general framework that integrates signaling with other cellular functions like trafficking, cell-cell and cell-matrix adhesion, and motility. The guiding question in our approach is: how does a physical change in cell membrane configuration caused by external stimuli (including those by the extracellular microenvironment) alter trafficking, signaling and subsequent cell fate? We answer this question by constructing a modeling framework based on stochastic spatial continuum models of cell membrane deformations. We apply this framework to explore the link between trafficking, signaling in the tumor microenvironment, and cell fate. At each stage, we aim to connect the results of our predictions with cellular experiments.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"701 ","pages":"359-386"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11258396/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723923","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
Building complex membranes with Martini 3. 用 Martini 3 构建复杂的膜。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-09 DOI: 10.1016/bs.mie.2024.03.010
Tugba Nur Ozturk, Melanie König, Timothy S Carpenter, Kasper B Pedersen, Tsjerk A Wassenaar, Helgi I Ingólfsson, Siewert J Marrink
{"title":"Building complex membranes with Martini 3.","authors":"Tugba Nur Ozturk, Melanie König, Timothy S Carpenter, Kasper B Pedersen, Tsjerk A Wassenaar, Helgi I Ingólfsson, Siewert J Marrink","doi":"10.1016/bs.mie.2024.03.010","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.010","url":null,"abstract":"<p><p>The Martini model is a popular force field for coarse-grained simulations. Membranes have always been at the center of its development, with the latest version, Martini 3, showing great promise in capturing more and more realistic behavior. In this chapter we provide a step-by-step tutorial on how to construct starting configurations, run initial simulations and perform dedicated analysis for membrane-based systems of increasing complexity, including leaflet asymmetry, curvature gradients and embedding of membrane proteins.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"701 ","pages":"237-285"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723919","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. 序言
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 DOI: 10.1016/S0076-6879(24)00284-2
Tobias Baumgart, Markus Deserno
{"title":"Preface.","authors":"Tobias Baumgart, Markus Deserno","doi":"10.1016/S0076-6879(24)00284-2","DOIUrl":"https://doi.org/10.1016/S0076-6879(24)00284-2","url":null,"abstract":"","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"701 ","pages":"xxiii-xxv"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723930","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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