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A method for in situ self-assembly of the catalytic peptide in enzymatic compartments of glucan particles. 在葡聚糖颗粒的酶区中原位自组装催化肽的方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-10 DOI: 10.1016/bs.mie.2024.01.021
Tiezheng Pan, Yaling Wang, Chunqiu Zhang
{"title":"A method for in situ self-assembly of the catalytic peptide in enzymatic compartments of glucan particles.","authors":"Tiezheng Pan, Yaling Wang, Chunqiu Zhang","doi":"10.1016/bs.mie.2024.01.021","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.01.021","url":null,"abstract":"<p><p>Drawing inspiration from cellular compartmentalization, enzymatic compartments play a pivotal role in bringing enzymes and substrates into confined environments, offering heightened catalytic efficiency and prolonged enzyme lifespan. Previously, we engineered bioinspired enzymatic compartments, denoted as TPE-Q18H@GPs, achieved through the spatiotemporally controllable self-assembly of the catalytic peptide TPE-Q18H within hollow porous glucan particles (GPs). This design strategy allows substrates and products to freely traverse, while retaining enzymatic aggregations. The confined environment led to the formation of catalytic nanofibers, resulting in enhanced substrate binding affinity and a more than two-fold increase in the second-order kinetic constant (k<sub>cat</sub>/K<sub>m</sub>) compared to TPE-Q18H nanofibers in a dispersed system. In this work, we will introduce how to synthesize the above-mentioned enzymatic compartments using salt-responsive catalytic peptides and GPs.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180157","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 self-templating catalytic amyloids. 自模板催化淀粉的特性。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-16 DOI: 10.1016/bs.mie.2024.04.004
Saroj K Rout, David Rhyner, Jason Greenwald, Roland Riek
{"title":"Characterization of self-templating catalytic amyloids.","authors":"Saroj K Rout, David Rhyner, Jason Greenwald, Roland Riek","doi":"10.1016/bs.mie.2024.04.004","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.04.004","url":null,"abstract":"<p><p>Amyloid aggregates with unique periodic structures have garnered significant attention due to their association with numerous diseases, including systemic amyloidoses and the neurodegenerative diseases Parkinson's, Alzheimer's, and Creutzfeld-Jakob. However, more recent investigations have expanded our understanding of amyloids, revealing their diverse functional biological roles. Amyloids have also been proposed to have played a significant role in prebiotic molecular evolution because of their exceptional stability, spontaneous formation in a prebiotic environment, catalytic and templating abilities, and cooperative interaction with fatty acids, polysaccharides, and nucleic acids. This chapter summarizes methods and techniques associated with studying short amyloidogenic peptides, including detailed procedures for investigating cross-templating and autocatalytic templating reactions. Since the work with amyloidogenic peptides and their aggregates present unique challenges, we have attempted to address these with essential details throughout the procedures. The lessons herein may be used in any amyloid-related research to ensure more reproducible results and reduce entrance barriers for researchers new to the field.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180201","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
Computational approaches to investigate fluoride binding, selectivity and transport across the membrane. 研究氟化物结合、选择性和跨膜运输的计算方法。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-01-22 DOI: 10.1016/bs.mie.2024.01.006
Kira R Mills, Hedieh Torabifard
{"title":"Computational approaches to investigate fluoride binding, selectivity and transport across the membrane.","authors":"Kira R Mills, Hedieh Torabifard","doi":"10.1016/bs.mie.2024.01.006","DOIUrl":"10.1016/bs.mie.2024.01.006","url":null,"abstract":"<p><p>The use of molecular dynamics (MD) simulations to study biomolecular systems has proven reliable in elucidating atomic-level details of structure and function. In this chapter, MD simulations were used to uncover new insights into two phylogenetically unrelated bacterial fluoride (F<sup>-</sup>) exporters: the CLC<sup>F</sup> F<sup>-</sup>/H<sup>+</sup> antiporter and the Fluc F<sup>-</sup> channel. The CLC<sup>F</sup> antiporter, a member of the broader CLC family, has previously revealed unique stoichiometry, anion-coordinating residues, and the absence of an internal glutamate crucial for proton import in the CLCs. Through MD simulations enhanced with umbrella sampling, we provide insights into the energetics and mechanism of the CLC<sup>F</sup> transport process, including its selectivity for F<sup>-</sup> over HF. In contrast, the Fluc F<sup>-</sup> channel presents a novel architecture as a dual topology dimer, featuring two pores for F<sup>-</sup> export and a central non-transported sodium ion. Using computational electrophysiology, we simulate the electrochemical gradient necessary for F<sup>-</sup> export in Fluc and reveal details about the coordination and hydration of both F<sup>-</sup> and the central sodium ion. The procedures described here delineate the specifics of these advanced techniques and can also be adapted to investigate other membrane protein systems.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140870790","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
Mining and engineering activity in catalytic amyloids. 催化淀粉的采矿和工程活动。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-20 DOI: 10.1016/bs.mie.2024.03.002
Samuel Peña-Díaz, Pedro Ferreira, Maria João Ramos, Daniel E Otzen
{"title":"Mining and engineering activity in catalytic amyloids.","authors":"Samuel Peña-Díaz, Pedro Ferreira, Maria João Ramos, Daniel E Otzen","doi":"10.1016/bs.mie.2024.03.002","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.002","url":null,"abstract":"<p><p>This chapter describes how to test different amyloid preparations for catalytic properties. We describe how to express, purify, prepare and test two types of pathological amyloid (tau and α-synuclein) and two functional amyloid proteins, namely CsgA from Escherichia coli and FapC from Pseudomonas. We therefore preface the methods section with an introduction to these two examples of functional amyloid and their remarkable structural and kinetic properties and high physical stability, which renders them very attractive for a range of nanotechnological designs, both for structural, medical and catalytic purposes. The simplicity and high surface exposure of the CsgA amyloid is particularly useful for the introduction of new functional properties and we therefore provide a computational protocol to graft active sites from an enzyme of interest into the amyloid structure. We hope that the methods described will inspire other researchers to explore the remarkable opportunities provided by bacterial functional amyloid in biotechnology.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180224","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
Oligo-benzamide-based peptide mimicking tools for modulating biology. 基于寡聚苯甲酰胺的多肽模拟工具，用于调节生物学。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-27 DOI: 10.1016/bs.mie.2024.04.022
Chia-Yuan Chen, Scott Elmore, Ismail Lalami, Henry Neal, Ratna K Vadlamudi, Ganesh V Raj, Jung-Mo Ahn
{"title":"Oligo-benzamide-based peptide mimicking tools for modulating biology.","authors":"Chia-Yuan Chen, Scott Elmore, Ismail Lalami, Henry Neal, Ratna K Vadlamudi, Ganesh V Raj, Jung-Mo Ahn","doi":"10.1016/bs.mie.2024.04.022","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.04.022","url":null,"abstract":"<p><p>The oligo-benzamide scaffold is a rigid organic framework that can hold 2-3 functional groups as O-alkyl substituents on its benzamide units, mirroring their natural arrangement in an α-helix. Oligo-benzamides demonstrated outstanding α-helix mimicry and can be readily synthesized by following high yielding and iterative reaction steps in both solution-phase and solid-phase. A number of oligo-benzamides have been designed to emulate α-helical peptide segments in biologically active proteins and showed strong protein binding, in turn effectively disrupting protein-protein interactions in vitro and in vivo. In this chapter, the design of oligo-benzamides for mimicking α-helices, efficient synthetic routes for producing them, and their biomedical studies showing remarkable potency in inhibiting protein functions are discussed.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141419866","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
Free energy calculations for membrane morphological transformations and insights to physical biology and oncology. 膜形态转化的自由能计算以及对物理生物学和肿瘤学的启示。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-14 DOI: 10.1016/bs.mie.2024.03.028
Kshitiz Parihar, Seung-Hyun Ko, Ryan Bradley, Phillip Taylor, N Ramakrishnan, Tobias Baumgart, Wei Guo, Valerie M Weaver, Paul A Janmey, Ravi Radhakrishnan
{"title":"Free energy calculations for membrane morphological transformations and insights to physical biology and oncology.","authors":"Kshitiz Parihar, Seung-Hyun Ko, Ryan Bradley, Phillip Taylor, N Ramakrishnan, Tobias Baumgart, Wei Guo, Valerie M Weaver, Paul A Janmey, Ravi Radhakrishnan","doi":"10.1016/bs.mie.2024.03.028","DOIUrl":"10.1016/bs.mie.2024.03.028","url":null,"abstract":"<p><p>In this chapter, we aim to bridge basic molecular and cellular principles surrounding membrane curvature generation with rewiring of cellular signals in cancer through multiscale models. We describe a general framework that integrates signaling with other cellular functions like trafficking, cell-cell and cell-matrix adhesion, and motility. The guiding question in our approach is: how does a physical change in cell membrane configuration caused by external stimuli (including those by the extracellular microenvironment) alter trafficking, signaling and subsequent cell fate? We answer this question by constructing a modeling framework based on stochastic spatial continuum models of cell membrane deformations. We apply this framework to explore the link between trafficking, signaling in the tumor microenvironment, and cell fate. At each stage, we aim to connect the results of our predictions with cellular experiments.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11258396/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723923","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
Building complex membranes with Martini 3. 用 Martini 3 构建复杂的膜。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-09 DOI: 10.1016/bs.mie.2024.03.010
Tugba Nur Ozturk, Melanie König, Timothy S Carpenter, Kasper B Pedersen, Tsjerk A Wassenaar, Helgi I Ingólfsson, Siewert J Marrink
{"title":"Building complex membranes with Martini 3.","authors":"Tugba Nur Ozturk, Melanie König, Timothy S Carpenter, Kasper B Pedersen, Tsjerk A Wassenaar, Helgi I Ingólfsson, Siewert J Marrink","doi":"10.1016/bs.mie.2024.03.010","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.010","url":null,"abstract":"<p><p>The Martini model is a popular force field for coarse-grained simulations. Membranes have always been at the center of its development, with the latest version, Martini 3, showing great promise in capturing more and more realistic behavior. In this chapter we provide a step-by-step tutorial on how to construct starting configurations, run initial simulations and perform dedicated analysis for membrane-based systems of increasing complexity, including leaflet asymmetry, curvature gradients and embedding of membrane proteins.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723919","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
Preface. 序言
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 DOI: 10.1016/S0076-6879(24)00284-2
Tobias Baumgart, Markus Deserno
{"title":"Preface.","authors":"Tobias Baumgart, Markus Deserno","doi":"10.1016/S0076-6879(24)00284-2","DOIUrl":"https://doi.org/10.1016/S0076-6879(24)00284-2","url":null,"abstract":"","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723930","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
Experimental considerations for precise RNA-mediated insertion of transgenes. RNA 介导的转基因精确插入的实验考虑因素。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-10 DOI: 10.1016/bs.mie.2024.07.007
Sarah M Palm, Briana Van Treeck, Kathleen Collins
{"title":"Experimental considerations for precise RNA-mediated insertion of transgenes.","authors":"Sarah M Palm, Briana Van Treeck, Kathleen Collins","doi":"10.1016/bs.mie.2024.07.007","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.007","url":null,"abstract":"<p><p>Precise RNA-mediated insertion of transgenes (PRINT) is a pioneering method for site-specific, safe-harbor transgene supplementation of the human genome that harnesses a eukaryotic retroelement protein and relies solely on the delivery of RNA. Here we outline important considerations in the design of the two required RNAs, details for the production and transfection of these RNAs to cells, and read-outs for successful transgene addition. Throughout, tips and key concepts are laid out to enable general use of this method.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400653","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 assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays. 茄属植物和玉米中的 9-顺式环氧类胡萝卜素二加氧酶（NCED）的体外测定和抑制作用。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-06 DOI: 10.1016/bs.mie.2024.05.012
Peter J Harrison, Jake Chandler, Andrew J Thompson, Timothy D H Bugg
{"title":"In vitro assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays.","authors":"Peter J Harrison, Jake Chandler, Andrew J Thompson, Timothy D H Bugg","doi":"10.1016/bs.mie.2024.05.012","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.012","url":null,"abstract":"<p><p>The article reports methods for the expression and assay of 9-cis-epoxycarotenoid cleavage dioxygenase (NCED), an enzyme involved in the biosynthesis of phytohormone abscisic acid in plants. A method for the preparation of the unstable substrate 9'-cis-neoxanthin from fresh spinach is described. The inhibition of Solanum lycopersicum NCED by a series of aryl hydroxamic acid inhibitors is illustrated, and inhibitors D2 and D4 are assayed against NCED isozymes from Zea mays.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291298","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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