Methods in enzymology最新文献

Direct, ensemble FRET approaches to monitor transient state kinetics of human DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis. 直接、集合 FRET 方法监测人类 DNA 聚合酶 δ 全酶组装和 DNA 合成启动的瞬态动力学。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-28 DOI: 10.1016/bs.mie.2024.08.002

Jessica L Norris, Mark Hedglin

{"title":"Direct, ensemble FRET approaches to monitor transient state kinetics of human DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.","authors":"Jessica L Norris, Mark Hedglin","doi":"10.1016/bs.mie.2024.08.002","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.08.002","url":null,"abstract":"In humans, DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand replication, the initiation of leading strand DNA replication as well as most of the major DNA damage repair pathways. In each of these contexts, pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process that involves the PCNA clamp loader, replication factor C and, depending on the DNA synthesis pathway, the major single strand DNA-binding protein complex, replication protein A (RPA). In a recent report from our laboratory, we designed and utilized direct, ensemble Förster Resonance Energy Transfer approaches to monitor the transient state kinetics of pol δ holoenzyme assembly and initiation of DNA synthesis on P/T junctions engaged by RPA. In this chapter, we detail the original approaches and discuss adaptations that can be utilized to monitor fast kinetic reactions in the millisecond (ms) timescale. All approaches described in this chapter utilize a commercially-available fluorescence spectrophotometer, can be readily evolved for alternative DNA polymerases and P/T DNA substrates, and permit incorporation of protein posttranslational modifications, accessory factors, DNA covalent modifications, accessory factors, enzymes, etc. Hence, these approaches are widely accessible and broadly applicable for characterizing DNA polymerase holoenzyme assembly and initiation of DNA synthesis during any PCNA-dependent DNA synthesis pathway.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"271-309"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400652","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

PAR-dCLIP: Enabling detection of RNA binding protein target transcripts bound at 5' termini through the incorporation of a decapping step. PAR-dCLIP：通过加入解帽步骤，检测结合在 5'末端的 RNA 结合蛋白目标转录本。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-07 DOI: 10.1016/bs.mie.2024.08.003

Samantha Lisy, Katherine Rothamel, Yelena Perevalova-Pinzul, Manuel Ascano

{"title":"PAR-dCLIP: Enabling detection of RNA binding protein target transcripts bound at 5' termini through the incorporation of a decapping step.","authors":"Samantha Lisy, Katherine Rothamel, Yelena Perevalova-Pinzul, Manuel Ascano","doi":"10.1016/bs.mie.2024.08.003","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.08.003","url":null,"abstract":"RNA binding proteins (RBPs) are responsible for facilitating a wealth of post-transcriptional gene regulatory functions. The role of an RBP on regulated transcripts can be investigated through a pull-down of the RBP and high-throughput sequencing (HTS) of the associated transcripts. Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), is one such pull-down method that isolates, detects, and sequences the cDNA of RBP-associated transcripts. PAR-CLIP relies on a photoactivatable ribonucleoside analogue, 4-thiouridine, to facilitate covalent RNA-protein crosslinks at 365 nm. These crosslinks permit stringent wash conditions and result in T to C mismatch incorporations during reverse transcription, a unique parameter for the computational analysis of high-confidence binding sites. However, until now, RBPs that bind at the 5'-termini of RNAs have been uniquely restricted from the full potential bandwidth of autoradiographic detection and HTS library preparation. The 5'-termini of RNAs are highly modified, including the most common Pol-II derived modification: the 7-methylguanosine (m7G) cap. In the conventional PAR-CLIP protocol, cap-binding proteins protect the m7G cap from the RNase treatment that generates the necessary substrate for autoradiographic detection and 5' adapter ligation-thus occluding entire populations of RNA from visualization and HTS. Here, we introduce decapping-PAR-CLIP or PAR-dCLIP. We incorporate a decapping step into the PAR-CLIP protocol to generate the necessary substrate to sequence m7G capped transcripts. While PAR-dCLIP was originally targeted towards known m7G-cap binding proteins, we argue that all RBP inquiries, and particularly those suspected to regulate translation, should incorporate this decapping step to ensure that all possible populations of bound transcripts are identified.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"159-222"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400658","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 vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01. 荧光假单胞菌 IP01 的三组分 Rieske 加氧酶--积烯二加氧酶的体外分析。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-12 DOI: 10.1016/bs.mie.2024.05.013

Niels A W de Kok, Hui Miao, Sandy Schmidt

{"title":"In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01.","authors":"Niels A W de Kok, Hui Miao, Sandy Schmidt","doi":"10.1016/bs.mie.2024.05.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.013","url":null,"abstract":"Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"167-192"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291291","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

Purification of functional recombinant human mitochondrial Hsp60. 纯化功能性重组人线粒体 Hsp60。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-31 DOI: 10.1016/bs.mie.2024.07.049

Celeste Weiss, Alberto G Berruezo, Shaikhah Seraidy, Avital Parnas, Igor Tascón, Iban Ubarretxena-Belandia, Abdussalam Azem

{"title":"Purification of functional recombinant human mitochondrial Hsp60.","authors":"Celeste Weiss, Alberto G Berruezo, Shaikhah Seraidy, Avital Parnas, Igor Tascón, Iban Ubarretxena-Belandia, Abdussalam Azem","doi":"10.1016/bs.mie.2024.07.049","DOIUrl":"10.1016/bs.mie.2024.07.049","url":null,"abstract":"The mitochondrial 60 kDa heat shock protein (mHsp60) is an oligomeric, barrel-like structure that mediates protein folding in cooperation with its cochaperonin Hsp10, in an ATP-dependent manner. In contrast to the extremely stable oligomeric structure of the bacterial chaperonin, GroEL, the human mHsp60 exists in equilibrium between single and double heptameric units, which dissociate easily to inactive monomers under laboratory conditions. Consequently, purification and manipulation of active mHsp60 oligomers is not straightforward. In this manuscript, we present an improved protocol for the purification of functional mHsp60, following its expression in bacteria. This method is based upon a previously published strategy that exploits the notorious instability of mHsp60 to purify the monomeric form, which is subsequently reconstituted to functional oligomers under controlled conditions. In our protocol, we use affinity chromatography on a Ni NTA-agarose resin as the initial step, facilitating purification of substantial amounts of highly pure active protein. The resulting Hsp60 is suitable for both functional and structural analyses, including crystallography and electron cryo-microscopy (cryo-EM) studies, to obtain high resolution structures of the mHsp60 oligomers alone and in various complexes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"423-440"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564646","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

Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach. 揭示半胱胺二氧化酶的机制：HPLC-MS测定与金属置换相结合的方法。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-22 DOI: 10.1016/bs.mie.2024.05.018

Ran Duan, Jiasong Li, Aimin Liu

{"title":"Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach.","authors":"Ran Duan, Jiasong Li, Aimin Liu","doi":"10.1016/bs.mie.2024.05.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.018","url":null,"abstract":"Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"147-166"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291211","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

Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria. 监测哺乳动物线粒体前体蛋白的体外导入和组装。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-28 DOI: 10.1016/bs.mie.2024.07.034

Jordan J Crameri, Diana Stojanovski

引用次数: 0

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