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Metal co-factors to enhance catalytic activity of short prion-derived peptide sequences. 增强朊病毒衍生短肽序列催化活性的金属辅助因子。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-28 DOI: 10.1016/bs.mie.2024.02.003
Nimisha A Mavlankar, Antarlina Maulik, Asish Pal
{"title":"Metal co-factors to enhance catalytic activity of short prion-derived peptide sequences.","authors":"Nimisha A Mavlankar, Antarlina Maulik, Asish Pal","doi":"10.1016/bs.mie.2024.02.003","DOIUrl":"10.1016/bs.mie.2024.02.003","url":null,"abstract":"<p><p>Development of biomolecular enzyme mimics to efficiently catalyse biochemical reactions are of prime relevance for the bulk scale production of industrially relevant biocatalyst. In this regard, amyloidogenic peptides act as suitable self-assembling scaffolds, providing stable nanostructures with high surface area facilitating biocatalysis. Herein, we rationally design two positional amyloidogenic peptide isomers, \"Fmoc-VYYAHH (1)\" and \"Fmoc-VHHAYY (2)\" considering catalytic and metal binding affinity of histidine and tyrosine when placed in periphery vs. inner core of the peptide sequence. With an ultimate objective of designing metalloenzyme mimic, we choose Co<sup>2+</sup> and Cu<sup>2+</sup> as divalent transition metal cations for peptide complexation to aid in catalysis. After optimizing self-assembly of innate peptides, we investigate metal-peptide binding ratio and co-ordination, finally selecting 1:1 peptide metal complex suitable for biocatalysis. Metallopeptides act as better catalysts than the innate peptides as acyl esterase when tyrosines were present at the periphery. Kinetic parameters for assessing hydrolysis rate were calculated by fitting data into Michaelis-Menten and Lineweaver Burk plots. Catalytic activity is altered depending on the stability of peptide metal complexes. 2-Cu acting as the best biocatalyst with a kcat/K<sub>M</sub> = 0.08 M/s. The protocols mentioned in this chapter meticulously cover the design, synthesis, self-assembly and enzyme kinetics.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"697 ","pages":"473-498"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180222","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
Designing and synthesizing peptide-based quorum sensing modulators. 设计和合成基于肽的法定人数感应调制器。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-27 DOI: 10.1016/bs.mie.2024.04.017
Xiaotian Gong, Carter J Brand, Michael A Bertucci
{"title":"Designing and synthesizing peptide-based quorum sensing modulators.","authors":"Xiaotian Gong, Carter J Brand, Michael A Bertucci","doi":"10.1016/bs.mie.2024.04.017","DOIUrl":"10.1016/bs.mie.2024.04.017","url":null,"abstract":"<p><p>Quorum sensing (QS) is a density-dependent bacterial communication system that uses small molecules as regulatory modulators. Synthetic changes to these molecules can up-or-down-regulate this system, leading to control of phenotypes, like competence and virulence factor production, that have implications in human health. In this chapter, a methodology for library design and screening of synthetic autoinducing peptides (AIPs) to uncover QS SARs is delineated. Additionally, procedures for the synthesis, purification and analysis of linear and cyclic AIPs are detailed. This includes solutions for potential synthetic challenges including diketopiperazine formation when using N-methyl amino acids and cyclization of peptides containing N-terminal cysteine residues. These procedures have and are currently being applied to develop potent QS modulators in Streptococcus pneumoniae, Bacillus cereus, Streptococcus gordonii and Lactiplantibacillus plantarum.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"698 ","pages":"263-299"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141419862","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
Catalytic amyloids for nucleotide hydrolysis. 用于核苷酸水解的催化淀粉。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-08 DOI: 10.1016/bs.mie.2024.01.017
Daniel Carrillo, Eva Duran-Meza, Claudio Castillo-Caceres, Diego Eduardo Alarcon, Hardy Guzman, Rodrigo Diaz-Espinoza
{"title":"Catalytic amyloids for nucleotide hydrolysis.","authors":"Daniel Carrillo, Eva Duran-Meza, Claudio Castillo-Caceres, Diego Eduardo Alarcon, Hardy Guzman, Rodrigo Diaz-Espinoza","doi":"10.1016/bs.mie.2024.01.017","DOIUrl":"10.1016/bs.mie.2024.01.017","url":null,"abstract":"<p><p>The design of small peptides that assemble into catalytically active intermolecular structures has proven to be a successful strategy towards developing minimalistic catalysts that exhibit some of the unique functional features of enzymes. Among these, catalytic amyloids have emerged as a fruitful source to unravel many different activities. These assemblies can potentially have broad applications that range from biotechnology to prebiotic chemistry. Although many peptides that assemble into catalytic amyloids have been developed in recent years, the elucidation of convergent mechanistic aspects of the catalysis and the structure/function relationship is still a challenge. Novel catalytic activities are necessary to better address these issues and expand the current repertoire of applicability. In this chapter, we described a methodology to produce catalytic amyloids that are specifically active towards the hydrolysis of phosphoanhydride bonds of nucleotides. The design of potentially active amyloid-prone peptide sequences is explored using as template the active site of enzymes with nucleotidyltransferase activity. The procedures include an approach for sequence design, in vitro aggregation assays, morphological characterization of the amyloid state and a comprehensive methodology to measure activity in vitro using nucleoside and deoxynucleosides triphosphates as model substrates. The proposed strategy can also be implemented to explore different types of activities for the design of future catalytic amyloids.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"697 ","pages":"269-291"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180191","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
Functional analysis of transmembrane terpene cyclases involved in fungal meroterpenoid biosynthesis. 参与真菌经萜生物合成的跨膜萜环酶的功能分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-24 DOI: 10.1016/bs.mie.2024.02.007
Jia Tang, Yudai Matsuda
{"title":"Functional analysis of transmembrane terpene cyclases involved in fungal meroterpenoid biosynthesis.","authors":"Jia Tang, Yudai Matsuda","doi":"10.1016/bs.mie.2024.02.007","DOIUrl":"10.1016/bs.mie.2024.02.007","url":null,"abstract":"<p><p>Pyr4-family terpene cyclases are noncanonical transmembrane class II terpene cyclases that catalyze a variety of cyclization reactions in the biosynthesis of microbial terpenoids, such as meroterpenoids. However, although these cyclases are widely distributed in microorganisms, their three-dimensional structures have not been determined, possibly due to the transmembrane locations of these enzymes. In this chapter, we describe procedures for the functional analysis of transmembrane terpene cyclases based on their model structures generated using AlphaFold2. We used AdrI, the Pyr4-family terpene cyclase required for the biosynthesis of andrastin A and its homologs, as an example.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"419-445"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469489","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 UbiA terpene synthases with a precursor overproduction system in Escherichia coli. 大肠杆菌中带有前体过量生产系统的 UbiA 萜烯合成酶的特征。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-21 DOI: 10.1016/bs.mie.2024.02.001
Tyler A Alsup, Melvin Osei Opoku, Jeffrey D Rudolf
{"title":"Characterization of UbiA terpene synthases with a precursor overproduction system in Escherichia coli.","authors":"Tyler A Alsup, Melvin Osei Opoku, Jeffrey D Rudolf","doi":"10.1016/bs.mie.2024.02.001","DOIUrl":"10.1016/bs.mie.2024.02.001","url":null,"abstract":"<p><p>Expression and purification of membrane-bound proteins remains a challenge and limits enzymology efforts, contributing to a substantial knowledge gap in the biochemical functions of many proteins found in nature. Accordingly, the study of bacterial UbiA terpene synthases (TSs) has been limited due to the experimental hurdles required to purify active enzymes for characterization in vitro. Previous work employed the use of microsomes or crude membrane fractions to test enzyme activity; however, these methods can be labor intensive, require access to an ultracentrifuge, or may not be suitable for all membrane-bound TSs. We detail here an alternative strategy for the in vivo expression and biochemical characterization of the membrane associated UbiA TSs by employing a precursor overproduction system in Escherichia coli.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"395-417"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11216710/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469486","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
Methods for the discovery and characterization of octocoral terpene cyclases. 章鱼萜环化酶的发现和表征方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-05 DOI: 10.1016/bs.mie.2024.02.011
Immo Burkhardt, Lara Dürr, Natalie E Grayson, Bradley S Moore
{"title":"Methods for the discovery and characterization of octocoral terpene cyclases.","authors":"Immo Burkhardt, Lara Dürr, Natalie E Grayson, Bradley S Moore","doi":"10.1016/bs.mie.2024.02.011","DOIUrl":"10.1016/bs.mie.2024.02.011","url":null,"abstract":"<p><p>Octocorals are the most prolific source of terpenoids in the marine environment, with more than 4000 different compounds known from the phylum to date. However, the biochemical and genetic origin of their production remained elusive until recent studies showed that octocorals encode genes responsible for the biosynthesis of terpenoids in their own chromosomal DNA rather than from microbial symbionts as originally proposed. The identified coral genes include those encoding a new group of class I terpene cyclases (TCs) clustered among other candidate classes of tailoring enzymes. Phylogenetic analyses established octocoral TCs as a monophyletic clade, distinct from TCs of plants, bacteria, and other organisms. The newly discovered group of TCs appears to be ubiquitous in octocorals and is evolutionarily ancient. Given the recent discovery of octocoral terpenoid biochemistry and only limited genomic data presently available, there is substantial potential for discovering new biosynthetic pathways from octocorals for terpene production. The following chapter outlines practical experimental procedures for octocoral DNA and RNA extraction, genome and transcriptome assembly and mining, TC cloning and gene expression, protein purification, and in vitro analyses.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"699 ","pages":"343-371"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11590171/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141469493","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
Robust magnetic tweezers for membrane protein folding studies. 用于膜蛋白折叠研究的强力磁镊。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-16 DOI: 10.1016/bs.mie.2023.12.014
Seoyoon Kim, Duyoung Min
{"title":"Robust magnetic tweezers for membrane protein folding studies.","authors":"Seoyoon Kim, Duyoung Min","doi":"10.1016/bs.mie.2023.12.014","DOIUrl":"10.1016/bs.mie.2023.12.014","url":null,"abstract":"<p><p>Single-molecule magnetic tweezers have recently been adapted for monitoring the interactions between transmembrane helices of membrane proteins within lipid bilayers. In this chapter, we describe the procedures of conducting studies on membrane protein folding using a robust magnetic tweezer method. This tweezer method is capable of observing thousands of (un)folding transitions over extended periods of several to tens of hours. Using this approach, we can dissect the folding pathways of membrane proteins, determine their folding time scales, and map the folding energy landscapes, with a higher statistical reliability. Our robust magnetic tweezers also allow for estimating the folding speed limit of helical membrane proteins, which serves as a link between the kinetics and barrier energies.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"694 ","pages":"285-301"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140475","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 of bona fide RNA G-quadruplex binding proteins. 鉴定真正的 RNA G-四叠体结合蛋白。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2023-12-21 DOI: 10.1016/bs.mie.2023.12.001
Prakash Kharel, Pavel Ivanov
{"title":"Identification of bona fide RNA G-quadruplex binding proteins.","authors":"Prakash Kharel, Pavel Ivanov","doi":"10.1016/bs.mie.2023.12.001","DOIUrl":"10.1016/bs.mie.2023.12.001","url":null,"abstract":"<p><p>RNAs often accomplish their diverse functions through direct interactions with RNA-binding proteins (RBPs) in a sequence- and/or structure-dependent manner. RNA G-quadruplexes (rG4s) are unique secondary structures formed by guanine-rich RNA sequences which impact RNA function independently and in combination with RBPs. Efforts from several labs have identified dozens of rG4 specific RBPs (rG4BPs), although the research is still in the growing phase. Here we present methods for the systematic identification of rG4BPs using a pull-down approach that takes advantage of the chemical modification of guanine bases. This allows abolishing the rG4 structures while still maintaining the base composition intact, and hence helps in recognizing true rG4BPS (in contrast to G-rich motif binders). In combination with other biochemical assays, such an approach can be efficiently used for the identification and characterization of bona fide rG4BPs.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"695 ","pages":"255-274"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140194171","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
Lanthanum-fluoride electrode-based methods to monitor fluoride transport in cells and reconstituted lipid vesicles. 基于氟化镧电极的方法，用于监测细胞和重组脂质囊泡中的氟迁移。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-09 DOI: 10.1016/bs.mie.2024.01.012
Chia-Yu Kang, Minjun An, Randy B Stockbridge
{"title":"Lanthanum-fluoride electrode-based methods to monitor fluoride transport in cells and reconstituted lipid vesicles.","authors":"Chia-Yu Kang, Minjun An, Randy B Stockbridge","doi":"10.1016/bs.mie.2024.01.012","DOIUrl":"10.1016/bs.mie.2024.01.012","url":null,"abstract":"<p><p>Fluoride (F<sup>-</sup>) export proteins, including F<sup>-</sup> channels and F<sup>-</sup> transporters, are widespread in biology. They contribute to cellular resistance against fluoride ion, which has relevance as an ancient xenobiotic, and in more modern contexts like organofluorine biosynthesis and degradation or dental medicine. This chapter summarizes quantitative methods to measure fluoride transport across membranes using fluoride-specific lanthanum-fluoride electrodes. Electrode-based measurements can be used to measure unitary fluoride transport rates by membrane proteins that have been purified and reconstituted into lipid vesicles, or to monitor fluoride efflux into living microbial cells. Thus, fluoride electrode-based measurements yield quantitative mechanistic insight into one of the major determinants of fluoride resistance in microorganisms, fungi, yeasts, and plants.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"696 ","pages":"43-63"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140851424","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
Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans. 模式真菌 Cunninghamella elegans 对含氟药物和异种生物的生物转化。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-03-16 DOI: 10.1016/bs.mie.2023.12.016
Mohd Faheem Khan, Carina Hof, Patricie Niemcova, Cormac D Murphy
{"title":"Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans.","authors":"Mohd Faheem Khan, Carina Hof, Patricie Niemcova, Cormac D Murphy","doi":"10.1016/bs.mie.2023.12.016","DOIUrl":"10.1016/bs.mie.2023.12.016","url":null,"abstract":"<p><p>Some species of the genus Cunninghamella (C. elegans, C. echinulata and C. blaskesleeana) produce the same phase I and phase II metabolites when incubated with xenobiotics as mammals, and thus are considered microbial models of mammalian metabolism. This had made these fungi attractive for metabolism studies with drugs, pesticides and environmental pollutants. As a substantial proportion of pharmaceuticals and agrochemicals are fluorinated, their biotransformation has been studied in Cunninghamella fungi and C. elegans in particular. This article details the methods employed for cultivating the fungi in planktonic and biofilm cultures, and extraction and analysis of fluorinated metabolites. Furthermore, protocols for the heterologous expression of Cunninghamella cytochromes P450 (CYPs), which are the enzymes associated with phase I metabolism, are described.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"696 ","pages":"251-285"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140859737","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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