Methods in enzymology最新文献_第2页

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

In-gel staining methods of G4 DNA and RNA structures. G4 DNA 和 RNA 结构的凝胶内染色法。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2023-12-22 DOI: 10.1016/bs.mie.2023.12.002

Philipp Schult, Katrin Paeschke

引用次数: 0

Single-molecule observation of G-quadruplex and R-loop formation induced by transcription. 单分子观测转录诱导的 G 型四联体和 R 型环的形成。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-16 DOI: 10.1016/bs.mie.2024.01.001

Jihee Hwang, Bradleigh Palmer, Sua Myong

引用次数: 0

Time-resolved IR spectroscopy for monitoring protein dynamics in microcrystals.

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-22 DOI: 10.1016/bs.mie.2024.10.006

Wataru Sato, Daichi Yamada, Minoru Kubo

{"title":"Time-resolved IR spectroscopy for monitoring protein dynamics in microcrystals.","authors":"Wataru Sato, Daichi Yamada, Minoru Kubo","doi":"10.1016/bs.mie.2024.10.006","DOIUrl":"10.1016/bs.mie.2024.10.006","url":null,"abstract":"Analysis of protein dynamics is crucial for understanding the molecular mechanisms underlying protein function. To gain insights into the structural changes in proteins, time-resolved X-ray crystallography has been greatly advanced by the development of X-ray free-electron lasers. This tool has the potential to trace structural changes at atomic resolution; however, data interpretation and extrapolation to the solution state is often not straightforward as the in crystallo environment is not the same as it is in solution. On the other hand, time-resolved spectroscopy techniques, which have long been used for tracking protein dynamics, offer the advantage of being applicable irrespective of whether the target proteins are in crystalline or solution phase. Time-resolved IR spectroscopy is a particularly powerful technique, as it can be used on various proteins, including those that are colorless, and provides information on the chemical structures of functional sites of proteins and ligands which complements X-ray crystallography. This chapter presents the protocol for time-resolved IR microspectroscopic measurements of protein microcrystals. It includes an overview of the measurement system assembly, sample preparation, setting of experimental conditions, and time-resolved data analysis. It also describes, with examples, the usefulness of time-resolved IR measurements for comparing the dynamics between crystalline and solution conditions.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"709 ","pages":"161-176"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142750263","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

Multiplexing methods in dynamic protein crystallography.

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-24 DOI: 10.1016/bs.mie.2024.10.009

Margaret A Klureza, Yelyzaveta Pulnova, David von Stetten, Robin L Owen, Godfrey S Beddard, Arwen R Pearson, Briony A Yorke

引用次数: 0