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Catalytic physiological amyloids. 催化生理淀粉样蛋白。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-27 DOI: 10.1016/bs.mie.2024.01.014
Elad Arad, Raz Jelinek
{"title":"Catalytic physiological amyloids.","authors":"Elad Arad, Raz Jelinek","doi":"10.1016/bs.mie.2024.01.014","DOIUrl":"10.1016/bs.mie.2024.01.014","url":null,"abstract":"<p><p>Amyloid fibrils have been identified in many protein systems, mostly linked to progression and cytotoxicity in neurodegenerative diseases and other pathologies, but have also been observed in normal physiological systems. A growing body of work has shown that amyloid fibrils can catalyze chemical reactions. Most studies have focused on catalysis by de-novo synthetic amyloid-like peptides; however, recent studies reveal that physiological, native amyloids are catalytic as well. Here, we discuss methodologies and major experimental aspects pertaining to physiological catalytic amyloids. We highlight analyzes of kinetic parameters related to the catalytic activities of amyloid fibrils, structure-function considerations, characterization of the catalytic active sites, and deciphering of catalytic mechanisms.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"697 ","pages":"77-112"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180196","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
Identification and functional/structural analyses of large terpene synthases. 大型萜烯合成酶的鉴定和功能/结构分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-09 DOI: 10.1016/bs.mie.2024.03.017
Daijiro Ueda, Tohru Abe, Masahiro Fujihashi, Tsutomu Sato
{"title":"Identification and functional/structural analyses of large terpene synthases.","authors":"Daijiro Ueda, Tohru Abe, Masahiro Fujihashi, Tsutomu Sato","doi":"10.1016/bs.mie.2024.03.017","DOIUrl":"10.1016/bs.mie.2024.03.017","url":null,"abstract":"<p><p>Large terpene synthases (large-TSs) are a new family of TSs. The first large-TS discovered was from Bacillus subtilis (BsuTS), which is involved in the biosynthesis of a C<sub>35</sub> sesquarterpene. Large-TSs are the only enzymes that enable the biosynthesis of sesquarterpenes and do not share any sequence homology with canonical Class I and II TSs. Thus, the investigation of large-TSs is promising for expanding the chemical space in the terpene field. In this chapter, we describe the experimental methods used for identifying large-TSs, as well as their functional and structural analyses. Additionally, several enzymes related to the biosynthesis of large-TS substrates have been described.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"477-512"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469490","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 tethering methods for membrane proteins. 膜蛋白的单分子拴系方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-15 DOI: 10.1016/bs.mie.2023.12.013
Daehyo Lee, Duyoung Min
{"title":"Single-molecule tethering methods for membrane proteins.","authors":"Daehyo Lee, Duyoung Min","doi":"10.1016/bs.mie.2023.12.013","DOIUrl":"10.1016/bs.mie.2023.12.013","url":null,"abstract":"<p><p>Molecular tethering of a single membrane protein between the glass surface and a magnetic bead is essential for studying the structural dynamics of membrane proteins using magnetic tweezers. However, the force-induced bond breakage of the widely-used digoxigenin-antidigoxigenin tether complex has imposed limitations on its stable observation. In this chapter, we describe the procedures of constructing highly stable single-molecule tethering methods for membrane proteins. These methods are established using dibenzocyclooctyne click chemistry, traptavidin-biotin binding, SpyCatcher-SpyTag conjugation, and SnoopCatcher-SnoopTag conjugation. The molecular tethering approaches allow for more stable observation of structural transitions in membrane proteins under force.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"694 ","pages":"263-284"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140476","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
Immunofluorescence microscopy of G-quadruplexes and R-loops. G 型四联体和 R 型环的免疫荧光显微镜。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-23 DOI: 10.1016/bs.mie.2024.01.008
Giulia Miglietta, Jessica Marinello, Giovanni Capranico
{"title":"Immunofluorescence microscopy of G-quadruplexes and R-loops.","authors":"Giulia Miglietta, Jessica Marinello, Giovanni Capranico","doi":"10.1016/bs.mie.2024.01.008","DOIUrl":"10.1016/bs.mie.2024.01.008","url":null,"abstract":"<p><p>A large variety of non-B secondary structures can be formed between DNA and RNA. In this chapter, we focus on G-quadruplexes (G4) and R-loops, which can have a close structural interplay. In recent years, increasing evidence pointed to the fact that they can strongly influence each other in vivo, both having physiological and pathological roles in normal and cancer cells. Here, we detail specific and accurate methods for purification of BG4 and S9.6 antibodies, and their subsequent use in immunofluorescence microscopy, enabling single-cell analysis of extent and localization of G4s and R-loops.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"695 ","pages":"103-118"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194172","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
Potentiometric titrations to study ligand interactions with DNA i-motifs. 电位滴定法研究配体与 DNA i-motifs 的相互作用。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2023-12-04 DOI: 10.1016/bs.mie.2023.11.005
Joseph Boissieras, Anton Granzhan
{"title":"Potentiometric titrations to study ligand interactions with DNA i-motifs.","authors":"Joseph Boissieras, Anton Granzhan","doi":"10.1016/bs.mie.2023.11.005","DOIUrl":"10.1016/bs.mie.2023.11.005","url":null,"abstract":"<p><p>i-Motifs are non-canonical secondary structures of DNA formed by mutual intercalation of hemi-protonated cytosine-cytosine base pairs, most typically in slightly acidic conditions (pH<7.0). These structures are well-studied in vitro and have recently been suggested to exist in cells. Despite nearly a decade of active research, the quest for small-molecule ligands that could selectively bind to and stabilize i-motifs continues, and no reference, bona fide i-motif ligand is currently available. This is, at least in part, due to the lack of robust methods to assess the interaction of ligands with i-motifs, since many techniques well-established for studies of other secondary structures (such as CD-, UV-, and FRET-melting) may generate artifacts when applied to i-motifs. Here, we describe an implementation of automated, potentiometric (pH) titrations as a robust isothermal method to assess the impact of ligands or cosolutes on thermodynamic stability of i-motifs. This approach is validated through the use of a cosolute previously known to stabilize i-motifs (PEG2000) and three small-molecule ligands that are able to stabilize, destabilize, or have no effect on the stability of i-motifs, respectively.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"695 ","pages":"233-254"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194176","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
NMR methods to detect fluoride binding and transport by membrane proteins. 用核磁共振方法检测膜蛋白对氟化物的结合和转运。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-03 DOI: 10.1016/bs.mie.2023.12.009
Jin Zhang, Juan Li, Yusong Wang, Chaowei Shi
{"title":"NMR methods to detect fluoride binding and transport by membrane proteins.","authors":"Jin Zhang, Juan Li, Yusong Wang, Chaowei Shi","doi":"10.1016/bs.mie.2023.12.009","DOIUrl":"10.1016/bs.mie.2023.12.009","url":null,"abstract":"<p><p>Solid-state nuclear magnetic resonance (NMR) methods can probe the motions of membrane proteins in liposomes at the atomic level, and propel the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. High-resolution crystallography snapshots have provided a structural basis for fluoride channels. NMR is a powerful tool to build upon these snapshots and depict a dynamic picture of fluoride channels in native-like lipid bilayers. In this contribution, we discuss solid-state and solution NMR experiments to detect fluoride binding and transport by fluoride channels. Ongoing developments in membrane protein sample preparation and ssNMR methodology, particularly in using <sup>1</sup>H, <sup>19</sup>F and <sup>13</sup>C-detection schemes, offer additional opportunities to study structure and functional aspects of fluoride channels.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"696 ","pages":"25-42"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140849240","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
Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans. 酿酒酵母和白色念珠菌中氟化物通道的电生理学。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-20 DOI: 10.1016/bs.mie.2024.01.005
Alberto Rivetta, Clifford Slayman
{"title":"Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans.","authors":"Alberto Rivetta, Clifford Slayman","doi":"10.1016/bs.mie.2024.01.005","DOIUrl":"10.1016/bs.mie.2024.01.005","url":null,"abstract":"<p><p>Tight regulation of molecules moving through the cell membrane is particularly important for free-living microorganisms because of their small cell volumes and frequent changes in the chemical composition of the extracellular environment. This is true for nutrients, but even more so for toxic molecules. Traditionally, the transport of these diverse molecules in microorganisms has been studied on cell populations rather than on single cells, mainly because of technical difficulties. The goal of this chapter is to make available a detailed method to prepare yeast spheroplasts to study the movement of fluoride ions across the plasma membrane of single cells by the patch-clamp technique. In this procedure, three steps are critical to achieve high resistance (GΩ) seals between the membrane and the glass electrode: (1) appropriate removal of the cell wall by enzymatic treatment; (2) balance between the osmotic strength of sealing solutions and cell membrane turgor; and (3) meticulous morphological inspection of spheroplasts suitable for gigaseal formation. We show now that this method, originally developed for Saccharomyces cerevisiae, can also be applied to Candida albicans, an opportunistic human pathogen.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"696 ","pages":"3-24"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140866344","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
Measuring carbonic anhydrase activity in alpha-carboxysomes using stopped-flow. 利用停流法测量α-羧酶体中碳酸酐酶的活性
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-28 DOI: 10.1016/bs.mie.2024.10.012
Nikoleta Vogiatzi, Cecilia Blikstad
{"title":"Measuring carbonic anhydrase activity in alpha-carboxysomes using stopped-flow.","authors":"Nikoleta Vogiatzi, Cecilia Blikstad","doi":"10.1016/bs.mie.2024.10.012","DOIUrl":"10.1016/bs.mie.2024.10.012","url":null,"abstract":"<p><p>Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO<sub>2</sub> concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO<sub>2</sub> fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO<sub>2</sub>. This creates a high CO<sub>2</sub> concentration around Rubisco, ensuring efficient carboxylation. In this chapter, we present a general method for purifying α-carboxysomes and measuring carbonic anhydrase activity within these purified compartments. We exemplify this with α-carboxysomes purified from the chemoautotroph Halothiobacillus neapolitanus c2, a model organism for the α-carboxysome based CCM. However, this purification protocol can be adapted for other species, such as carboxysomes from α-cyanobacteria or carboxysomes expressed in heterologous hosts. Further, we describe the Khalifah/pH indicator assay for measuring steady-state kinetics of carbonic anhydrase catalyzed CO<sub>2</sub> hydration. This method allows us to determine the kinetic parameters k<sub>cat</sub>, K<sub>M</sub> and k<sub>cat</sub>/K<sub>M</sub> for the purified α-carboxysomes. It uses a stopped-flow spectrometer for rapid mixing and detection, crucial for capturing the fast equilibrium between CO<sub>2</sub> and bicarbonate. The reaction progress is monitored by absorbance via a pH indicator that changes color due to the proton release. While the method specifically focuses on measuring carbonic anhydrase activity on carboxysomes, it can be used to measure activity on carbonic anhydrases from other contexts as well.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"708 ","pages":"297-322"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142687427","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 growth of microcrystals for time resolved serial crystallography.
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-10-29 DOI: 10.1016/bs.mie.2024.10.003
Alexander McPherson
{"title":"The growth of microcrystals for time resolved serial crystallography.","authors":"Alexander McPherson","doi":"10.1016/bs.mie.2024.10.003","DOIUrl":"10.1016/bs.mie.2024.10.003","url":null,"abstract":"<p><p>The production of enzyme microcrystals for time resolved serial crystallography employing free electron laser or synchrotron radiation is a relatively new variation on traditional macromolecular crystallization for conventional single crystal X-ray analysis. While the fundamentals of macromolecular crystal growth are the same, some modifications and special considerations are in order if the objective is to produce uniform size, microcrystals in very large numbers for serial data collection. Presented here are the basic principles of protein crystal growth with particular attention to the approaches best employed to achieve the goal of microcrystals and some novel techniques, as well as old, that may be useful. Also discussed are the advantages of particular precipitants and certain methods of growing protein crystals that might be advantageous for serial data recording.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"709 ","pages":"1-27"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142751308","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
Radical-relay C(sp3)-H azidation catalyzed by an engineered nonheme iron enzyme. 工程非血红素铁酶催化的自由基接力 C(sp3)-H 叠氮化反应。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-07-23 DOI: 10.1016/bs.mie.2024.07.003
Qun Zhao, Jinyan Rui, Xiongyi Huang
{"title":"Radical-relay C(sp<sup>3</sup>)-H azidation catalyzed by an engineered nonheme iron enzyme.","authors":"Qun Zhao, Jinyan Rui, Xiongyi Huang","doi":"10.1016/bs.mie.2024.07.003","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.003","url":null,"abstract":"<p><p>Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp<sup>3</sup>)-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"195-213"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291296","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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