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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
{"title":"In-gel staining methods of G4 DNA and RNA structures.","authors":"Philipp Schult, Katrin Paeschke","doi":"10.1016/bs.mie.2023.12.002","DOIUrl":"10.1016/bs.mie.2023.12.002","url":null,"abstract":"<p><p>G-quadruplexes (G4) are functionally important nucleic acid structures, involved in many cellular pathways. They are often dynamically regulated in cells, which makes detecting them in vivo challenging and dependent on sophisticated technical equipment. Therefore, in vitro studies are commonly performed as a first step to confirm a candidate sequence folds into a G4. Several methods have been developed, each with its individual pros and cons. A highly accessible and quick approach, without the need for specialized equipment, is the detection of G4s in native gels using light-up probes. These molecules become fluorescent after specifically binding to G4s. Several different classes have been discovered, emitting light in various colors, and some possess specificity for certain G4 topologies, which makes them highly versatile tools for G4 visualization. Here, we will explore the general procedure using the light-up probe NMM on RNA G4s and discuss advantages and limitations of this method.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"695 ","pages":"29-43"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194174","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 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":"<p><p>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.</p>","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":"<p><p>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.</p>","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
{"title":"Bioinformatic analysis of microbial type terpene synthase genes in plants.","authors":"Xinlu Chen, Jin Han, Feng Chen","doi":"10.1016/bs.mie.2024.02.014","DOIUrl":"10.1016/bs.mie.2024.02.014","url":null,"abstract":"<p><p>Plants are prolific producers of terpenoids. Terpenoid biosynthesis is initiated by terpene synthases (TPS). In plants, two types of terpenes synthase genes are recognized: typical plant TPS genes and microbial-terpene synthase like-genes (MTPSL). While TPS genes are ubiquitous in land plants, MTPSL genes appear to be restricted to non-seed land plants. Evolutionarily, TPS genes are specific to land plants, whereas MTPSL genes have related counterparts in other organisms, especially fungi and bacteria. The presence of microbial type TPS in plants, fungi and bacteria, with the latter two often being associated with plants, poses a challenge in accurately identifying bona fide MTPSL genes in plants. In this chapter, we present bioinformatic procedures designed to identify MTPSL genes in sequenced plant genomes and/or transcriptomes. Additionally, we outline validation methods for confirming the identified microbial-type TPS genes as genuine plant genes. The method described in this chapter can also be adopted to analyze microbial type TPS in organisms other than plants.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"293-310"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469484","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 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
{"title":"Structural biology of terpene synthases.","authors":"Baiying Xing, Zhenyu Lei, Zhaoye Bai, Guowei Zang, Yuxian Wang, Chenyu Zhang, Minren Chen, Yucheng Zhou, Jiahao Ding, Donghui Yang, Ming Ma","doi":"10.1016/bs.mie.2024.03.012","DOIUrl":"10.1016/bs.mie.2024.03.012","url":null,"abstract":"<p><p>Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"59-87"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469497","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
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
{"title":"Vanadium haloperoxidases as noncanonical terpene synthases.","authors":"Jackson T Baumgartner, Lia I Lozano Salazar, Lukas A Varga, Gabriel H Lefebre, Shaun M K McKinnie","doi":"10.1016/bs.mie.2024.03.024","DOIUrl":"10.1016/bs.mie.2024.03.024","url":null,"abstract":"<p><p>Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize vanadate, an aqueous halide ion, and hydrogen peroxide to produce an electrophilic halogen species that can be incorporated into electron rich organic substrates. This halogen species can react with terpene substrates and trigger halonium-induced cyclization in a manner reminiscent of class II terpene synthases. While not all VHPOs act in this capacity, several notable examples from algal and actinobacterial species have been characterized to catalyze regio- and enantioselective reactions on terpene and meroterpenoid substrates, resulting in complex halogenated cyclic terpenes through the action of single enzyme. In this article, we describe the expression, purification, and chemical assays of NapH4, a difficult to express characterized VHPO that catalyzes the chloronium-induced cyclization of its meroterpenoid substrate.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"447-475"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469500","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. 动态蛋白质晶体学中的多路复用方法。
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
{"title":"Multiplexing methods in dynamic protein crystallography.","authors":"Margaret A Klureza, Yelyzaveta Pulnova, David von Stetten, Robin L Owen, Godfrey S Beddard, Arwen R Pearson, Briony A Yorke","doi":"10.1016/bs.mie.2024.10.009","DOIUrl":"10.1016/bs.mie.2024.10.009","url":null,"abstract":"<p><p>Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"709 ","pages":"177-206"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142751362","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
Processing serial synchrotron crystallography diffraction data with DIALS. 用DIALS处理串行同步加速器晶体衍射数据。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-29 DOI: 10.1016/bs.mie.2024.10.004
James Beilsten-Edmands, James M Parkhurst, Graeme Winter, Gwyndaf Evans
{"title":"Processing serial synchrotron crystallography diffraction data with DIALS.","authors":"James Beilsten-Edmands, James M Parkhurst, Graeme Winter, Gwyndaf Evans","doi":"10.1016/bs.mie.2024.10.004","DOIUrl":"10.1016/bs.mie.2024.10.004","url":null,"abstract":"<p><p>This chapter describes additions to the DIALS software package for processing serial still-shot crystallographic data, and the implementation of a pipeline, xia2.ssx, for processing and merging serial crystallography data using DIALS programs. To integrate partial still-shot diffraction data, a 3D gaussian profile model was developed that can describe anisotropic spot shapes. This model is optimised by maximum likelihood methods using the pixel-intensity distributions of strong diffraction spots, enabling simultaneous refinement of the profile model and Ewald-sphere offsets. We demonstrate the processing of an example SSX dataset where the improved partiality estimates lead to better model statistics compared with post-refined isotropic models. We also demonstrate some of the workflows available for merging SSX data, including processing time/dose resolved data series, where data can be separated at the point of merging after scaling and discuss the program outputs used to investigate the data throughout the pipeline.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"709 ","pages":"207-244"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142751367","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
Time-resolved IR spectroscopy for monitoring protein dynamics in microcrystals. 时间分辨红外光谱用于监测微晶体中的蛋白质动力学。
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":"<p><p>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.</p>","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
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
{"title":"Single-molecule observation of G-quadruplex and R-loop formation induced by transcription.","authors":"Jihee Hwang, Bradleigh Palmer, Sua Myong","doi":"10.1016/bs.mie.2024.01.001","DOIUrl":"10.1016/bs.mie.2024.01.001","url":null,"abstract":"<p><p>Potential G-quadruplex forming sequences (PQS) are enriched in cancer-related genes and immunoglobulin class-switch recombination. They are prevalent in the 5'UTR of transcriptionally active genes, thereby contributing to the regulation of gene expression. We and others previously demonstrated that the PQS located in the non-template strand leads to an R-loop formation followed by a G-quadruplex (G4) formation during transcription. These structural changes increase mRNA production. Here, we present how single-molecule technique was used to observe cotranscriptional G4 and R-loop formation and to examine the impact on transcription, particularly for the initiation and elongation stages.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"695 ","pages":"71-88"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11756578/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194179","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
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