Methods in enzymology最新文献_第3页

In vitro import of mitochondrial precursor proteins into yeast mitochondria. 体外将线粒体前体蛋白导入酵母线粒体。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-21 DOI: 10.1016/bs.mie.2024.07.016

Soraya Badrie, Julian Alexander Draken, Dejana Mokranjac

引用次数: 0

Analysis of protein trafficking between mitochondria and the endoplasmic reticulum by fluorescence microscopy. 利用荧光显微镜分析线粒体和内质网之间的蛋白质运输。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-16 DOI: 10.1016/bs.mie.2024.07.041

Shunsuke Matsumoto, Suzuka Ono, Toshiya Endo

引用次数: 0

Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins. 基于质谱的蛋白质组学研究线粒体转运蛋白的突变体和相互作用组。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-23 DOI: 10.1016/bs.mie.2024.07.059

Silke Oeljeklaus, Lakshita Sharma, Julian Bender, Bettina Warscheid

{"title":"Mass spectrometry-based proteomics to study mutants and interactomes of mitochondrial translocation proteins.","authors":"Silke Oeljeklaus, Lakshita Sharma, Julian Bender, Bettina Warscheid","doi":"10.1016/bs.mie.2024.07.059","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.059","url":null,"abstract":"The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are \"born\" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. In the last unifying part, we describe methods for LC-MS, data processing, statistical analysis and visualization of quantitative MS data, and provide a Python code for effective, customizable analysis.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"101-152"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564480","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 activation of one key enzyme in anaerobic CO₂ fixation: Carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans. 厌氧二氧化碳固定过程中一种关键酶的表达、纯化和活化：Carboxydothermus hydrogenoformans 的一氧化碳脱氢酶 II。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-29 DOI: 10.1016/bs.mie.2024.10.016

Kareem Aboulhosn, Stephen Wiley Ragsdale

{"title":"Expression, purification, and activation of one key enzyme in anaerobic CO2 fixation: Carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans.","authors":"Kareem Aboulhosn, Stephen Wiley Ragsdale","doi":"10.1016/bs.mie.2024.10.016","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.10.016","url":null,"abstract":"Climate change due to anthropomorphic emissions will increase global temperature by at least 1.5 °C by the year 2030. One strategy to reduce the severity of the effects of climate change is to sequester carbon dioxide via natural biochemical cycles. Carbon monoxide dehydrogenase (CODH) has the remarkable ability to catalyze the reversible reduction of CO2 to CO without an overpotential and without reducing protons. It also is a key enzyme in the Wood-Ljungdahl pathway (WLP), which is the only known anaerobic carbon fixation pathway and fixes 10 % of carbon on earth every year. Characterization of this pathway is crucial because it may enable tools to mitigate climate change by using CO2 to produce biofuels, chemical feedstocks, and polymers. In the WLP, CODH associates with Acetyl-Coenzyme A synthase (ACS), which catalyzes the condensation of CO from CODH, a methyl group from a B12-dependent methyltransferase, and CoA to form acetyl-CoA. In this complex, CO is shuttled through a 138 Å gas tunnel between the two enzymes. One valuable model for studying the CODH component of CODH/ACS is CODH-II from Carboxydothermus hydrogenoformans because it is stand-alone and is conducive to recombinant expression. Here we describe a detailed protocol for producing high-activity CODH-II in E. coli.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"708 ","pages":"237-256"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142687420","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

Characterization of guanidine carboxylases. 胍基羧化酶的特征。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-11-07 DOI: 10.1016/bs.mie.2024.10.013

M Sinn, J Techel, A Joachimi, J S Hartig

{"title":"Characterization of guanidine carboxylases.","authors":"M Sinn, J Techel, A Joachimi, J S Hartig","doi":"10.1016/bs.mie.2024.10.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.10.013","url":null,"abstract":"Guanidine metabolism has been an overlooked aspect of the global nitrogen cycle until RNA sensors (riboswitches) were discovered in bacteria that bind the nitrogen-rich compound. The associated genes were initially proposed to detoxify guanidine from the cells. We were intrigued by a genetic organization where the guanidine riboswitch is located upstream of an operon comprising a carboxylase, two putative hydrolases, and an assigned allophanate hydrolase. An ABC transporter is located on the same operon with a periplasmic binding domain that is indicative of an importer. Therefore, we hypothesized that certain bacteria actively import guanidine and assimilate the nitrogen. To test this hypothesis, we searched for bacteria that were able to assimilate guanidine. We isolated three enterobacteria (Raoultella terrigena str. JH01, Erwinia rhapontici str. JH02 and Klebsiella michiganensis str. JH07) that utilize guanidine efficiently as a nitrogen source. Proteome analyses demonstrate that the expression of the guanidine riboswitch-associated carboxylase, in conjunction with associated hydrolases and transport genes, is markedly elevated in the presence of guanidine. Subsequent analysis of the carboxylases that are homologous to urea carboxylase confirmed the substrate preference of guanidine over urea. This chapter outlines a procedure for the isolation of guanidine-assimilating bacteria and the analysis of their proteome to identify enzymes responsible for guanidine degradation. Finally, an assay for the characterization of the endogenous guanidine carboxylases in comparison with the endogenous urea carboxylase from E. rhapontici is described.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"708 ","pages":"105-123"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142687413","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

Evaluation of functional transbilayer coupling in live cells by controlled lipid exchange and imaging fluorescence correlation spectroscopy. 通过受控脂质交换和成像荧光相关光谱评估活细胞中的跨膜功能耦合。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-25 DOI: 10.1016/bs.mie.2024.04.001

Arpita Tripathy, Sudipti Priyadarsinee, Nirmalya Bag

{"title":"Evaluation of functional transbilayer coupling in live cells by controlled lipid exchange and imaging fluorescence correlation spectroscopy.","authors":"Arpita Tripathy, Sudipti Priyadarsinee, Nirmalya Bag","doi":"10.1016/bs.mie.2024.04.001","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.04.001","url":null,"abstract":"Biophysical coupling between the inner and outer leaflets, known as inter-leaflet or transbilayer coupling, is a fundamental organizational principle in the plasma membranes of live mammalian cells. Lipid-based interactions between the two leaflets are proposed to be a primary mechanism underlying transbilayer coupling. However, there are only a few experimental evidence supporting the existence of such interactions in live cells. This is seemingly due to the lack of experimental strategies to perturb the lipid composition in one leaflet and quantitative techniques to evaluate the biophysical properties of the opposite leaflet. The existing strategies often dependent on immobilization and clustering a component in one of the leaflets and technically demanding biophysical tools to evaluate the effects on the opposing leaflet. In the recent years, the London group developed a simple but elegant method, namely methyl-alpha-cyclodextrin catalyzed lipid exchange (LEX), to efficiently exchange outer leaflet lipids with an exogenous lipid of choice. Here, we adopted this method to perturb outer leaflet lipid composition. The corresponding changes in the inner leaflet is evaluated by comparing the diffusion of lipid probes localized in this leaflet in unperturbed and perturbed conditions. We employed highly multiplexed imaging fluorescence correlation spectroscopy (ImFCS), realized in a commercially available or home-built total internal reflection fluorescence microsocope equipped with a fast and sensitive camera, to determine diffusion coefficient of the lipid probes. Using the combination of LEX and ImFCS, we directly demonstrate lipid-based transbilayer coupling that does not require immobilization of membrane components in live mast cells in resting conditions. Overall, we present a relatively straightforward experimental strategy to evaluate transbilayer coupling quantitively in live cells.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"1-32"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545032","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

Force-fluorescence setup for observing protein-DNA interactions under load. 用于观察负载下蛋白质-DNA 相互作用的力荧光装置。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-16 DOI: 10.1016/bs.mie.2024.01.003

Jaehun Jung, Subin Kim, Sang-Hyun Rah, Jayil Lee, Min Ju Shon

引用次数: 0

Magnetic nano-tweezer for interrogating mechanosensitive signaling proteins in space and time. 用于在空间和时间上检测机械敏感信号蛋白的磁性纳米镊子。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-20 DOI: 10.1016/bs.mie.2024.01.009

Minsuk Kwak

{"title":"Magnetic nano-tweezer for interrogating mechanosensitive signaling proteins in space and time.","authors":"Minsuk Kwak","doi":"10.1016/bs.mie.2024.01.009","DOIUrl":"10.1016/bs.mie.2024.01.009","url":null,"abstract":"Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to understand how extracellular cues are converted into biochemical signals and differentially regulate cellular responses. Using single-cell perturbation tools such as optogenetics, specific biochemical cues can be delivered to selective molecules or cells at any desired location and time. By measuring cellular responses to provided perturbations, investigators have decoded and deconstructed the working mechanisms of a variety of neuroelectric and biochemical signaling processes. However, analogous methods for deciphering the working mechanisms of mechanosensitive signaling by regulating mechanical inputs to cell receptors have remained elusive. To address this unmet need, we have recently developed a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. This tool combines a magnetofluorescent nanoparticle (MFN) actuator, which provides precise spatial and mechanical signals to receptors via target-specific one-to-one interaction, with a micromagnetic tweezers that remotely controls the force exerted on a single nanoparticle. This chapter provides comprehensive experimental protocols of mechanogenetics consisting of four stages: (i) chemical synthesis of MFNs, (ii) bio-conjugation and purification of monovalent MFNs, (iii) establishment of cells with genetically encoded mechanosensitive proteins, and (iv) modular targeting and control of cell-surface receptors in live cells. The entire procedure takes up to 1 week. This mechanogenetic tool can be generalized to study many outstanding questions related to the dynamics of cell signaling and transcriptional control, including the mechanism of mechanically activated receptor.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"694 ","pages":"303-320"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140471","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

Magnetic tweezers in cell mechanics. 细胞力学中的磁镊。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-05 DOI: 10.1016/bs.mie.2023.12.007

Claudia Tanja Mierke

引用次数: 0

Construction and operation of high-resolution magnetic tape head tweezers for measuring single-protein dynamics under force. 用于测量单个蛋白质受力动态的高分辨率磁带头镊子的构造和操作。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-05 DOI: 10.1016/bs.mie.2023.12.010

Rafael Tapia-Rojo

{"title":"Construction and operation of high-resolution magnetic tape head tweezers for measuring single-protein dynamics under force.","authors":"Rafael Tapia-Rojo","doi":"10.1016/bs.mie.2023.12.010","DOIUrl":"10.1016/bs.mie.2023.12.010","url":null,"abstract":"Mechanical forces are critical to protein function across many biological contexts-from bacterial adhesion to muscle mechanics and mechanotransduction processes. Hence, understanding how mechanical forces govern protein activity has developed into a central scientific question. In this context, single-molecule magnetic tweezers has recently emerged as a valuable experimental tool, offering the capability to measure single proteins over physiologically relevant forces and timescales. In this chapter, we present a detailed protocol for the assembly and operation of our magnetic tape head tweezers instrument, specifically tailored to investigate protein dynamics. Our instrument boasts a simplified microscope design and incorporates a magnetic tape head as the force-generating apparatus, facilitating precise force control and enhancing its temporal stability, enabling the study of single protein mechanics over extended timescales spanning several hours or even days. Moreover, its straightforward and cost-effective design ensures its accessibility to the wider scientific community. We anticipate that this technique will attract widespread interest within the growing field of mechanobiology and expect that this chapter will provide facilitated accessibility to this technology.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"694 ","pages":"83-107"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140465","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