Methods in enzymology最新文献_第8页

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

Characterization of amyloid-like metal-amino acid assemblies with remarkable catalytic activity. 具有显著催化活性的淀粉样金属-氨基酸组合体的特征。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-07 DOI: 10.1016/bs.mie.2024.01.018

Om Shanker Tiwari, Ehud Gazit

{"title":"Characterization of amyloid-like metal-amino acid assemblies with remarkable catalytic activity.","authors":"Om Shanker Tiwari, Ehud Gazit","doi":"10.1016/bs.mie.2024.01.018","DOIUrl":"10.1016/bs.mie.2024.01.018","url":null,"abstract":"While enzymes are potentially useful in various applications, their limited operational stability and production costs have led to an extensive search for stable catalytic agents that will retain the efficiency, specificity, and environmental-friendliness of natural enzymes. Despite extensive efforts, there is still an unmet need for improved enzyme mimics and novel concepts to discover and optimize such agents. Inspired by the catalytic activity of amyloids and the formation of amyloid-like assemblies by metabolites, our group pioneered the development of novel metabolite-metal co-assemblies (bio-nanozymes) that produce nanomaterials mimicking the catalytic function of common metalloenzymes that are being used for various technological applications. In addition to their notable activity, bio-nanozymes are remarkably safe as they are purely composed of amino acids and minerals that are harmless to the environment. The bio-nanozymes exhibit high efficiency and exceptional robustness, even under extreme conditions of temperature, pH, and salinity that are impractical for enzymes. Our group has recently also demonstrated the formation of ordered amino acid co-assemblies showing selective and preferential interactions comparable to the organization of residues in folded proteins. The identified bio-nanozymes can be used in various applications including environmental remediation, synthesis of new materials, and green energy.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"697 ","pages":"181-209"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180197","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

Structural studies of catalytic peptides using molecular dynamics simulations. 利用分子动力学模拟对催化肽进行结构研究。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-06 DOI: 10.1016/bs.mie.2024.01.019

Parth Rathee, Sreerag N Moorkkannur, Rajeev Prabhakar

{"title":"Structural studies of catalytic peptides using molecular dynamics simulations.","authors":"Parth Rathee, Sreerag N Moorkkannur, Rajeev Prabhakar","doi":"10.1016/bs.mie.2024.01.019","DOIUrl":"10.1016/bs.mie.2024.01.019","url":null,"abstract":"Many self-assembling peptides can form amyloid like structures with different sizes and morphologies. Driven by non-covalent interactions, their aggregation can occur through distinct pathways. Additionally, they can bind metal ions to create enzyme like active sites that allow them to catalyze diverse reactions. Due to the non-crystalline nature of amyloids, it is quite challenging to elucidate their structures using experimental spectroscopic techniques. In this aspect, molecular dynamics (MD) simulations provide a useful tool to derive structures of these macromolecules in solution. They can be further validated by comparing with experimentally measured structural parameters. However, these simulations require a multi-step process starting from the selection of the initial structure to the analysis of MD trajectories. There are multiple force fields, parametrization protocols, equilibration processes, software and analysis tools available for this process. Therefore, it is complicated for non-experts to select the most relevant tools and perform these simulations effectively. In this chapter, a systematic methodology that covers all major aspects of modeling of catalytic peptides is provided in a user-friendly manner. It will be helpful for researchers in this critical area of research.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"697 ","pages":"151-180"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180261","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

Bioinformatic analysis of microbial type terpene synthase genes in plants. 植物中微生物型萜烯合成酶基因的生物信息学分析。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-12 DOI: 10.1016/bs.mie.2024.02.014

Xinlu Chen, Jin Han, Feng Chen

引用次数: 0

Structural biology of terpene synthases. 萜烯合成酶的结构生物学。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-31 DOI: 10.1016/bs.mie.2024.03.012

Baiying Xing, Zhenyu Lei, Zhaoye Bai, Guowei Zang, Yuxian Wang, Chenyu Zhang, Minren Chen, Yucheng Zhou, Jiahao Ding, Donghui Yang, Ming Ma

引用次数: 0

Vanadium haloperoxidases as noncanonical terpene synthases. 作为非典型萜烯合成酶的钒卤过氧化物酶

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-17 DOI: 10.1016/bs.mie.2024.03.024

Jackson T Baumgartner, Lia I Lozano Salazar, Lukas A Varga, Gabriel H Lefebre, Shaun M K McKinnie

引用次数: 0