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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":"<p><p>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.</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":"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
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":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":"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
Synthesis, derivatization, and conformational scanning of peptides containing N-Aminoglycine. 含有 N-氨基甘氨酸的肽的合成、衍生化和构象扫描。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-27 DOI: 10.1016/bs.mie.2024.04.018
Syrah K Starnes, Juan R Del Valle
{"title":"Synthesis, derivatization, and conformational scanning of peptides containing N-Aminoglycine.","authors":"Syrah K Starnes, Juan R Del Valle","doi":"10.1016/bs.mie.2024.04.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.04.018","url":null,"abstract":"<p><p>N-alkylated glycine residues are the main constituent of peptoids and peptoid-peptide hybrids that are employed across the biomedical and materials sciences. While the impact of backbone N-alkylation on peptide conformation has been extensively studied, less is known about the effect of N-amination on the secondary structure propensity of glycine. Here, we describe a convenient protocol for the incorporation of N-aminoglycine into host peptides on solid support. Amide-to-hydrazide substitution also affords a nucleophilic handle for further derivatization of the backbone. To demonstrate the utility of late-stage hydrazide modification, we synthesized and evaluated the stability of polyproline II helix and β-hairpin model systems harboring N-aminoglycine derivatives. The described procedures provide facile entry into peptidomimetic libraries for conformational scanning.</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":"141419869","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":"https://doi.org/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":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":"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
Isotopic labelings for mechanistic studies. 用于机理研究的同位素标记。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-07 DOI: 10.1016/bs.mie.2024.01.011
Houchao Xu, Jeroen S Dickschat
{"title":"Isotopic labelings for mechanistic studies.","authors":"Houchao Xu, Jeroen S Dickschat","doi":"10.1016/bs.mie.2024.01.011","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.01.011","url":null,"abstract":"<p><p>The intricate mechanisms in the biosynthesis of terpenes belong to the most challenging problems in natural product chemistry. Methods to address these problems include the structure-based site-directed mutagenesis of terpene synthases, computational approaches, and isotopic labeling experiments. The latter approach has a long tradition in biosynthesis studies and has recently experienced a revival, after genome sequencing enabled rapid access to biosynthetic genes and enzymes. Today, this allows for a combined approach in which isotopically labeled substrates can be incubated with recombinant terpene synthases. These clearly defined reaction setups can give detailed mechanistic insights into the reactions catalyzed by terpene synthases, and recent developments have substantially deepened our understanding of terpene biosynthesis. This chapter will discuss the state of the art and introduce some of the most important methods that make use of isotopic labelings in mechanistic studies on terpene synthases.</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":"141469491","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 density-threshold affinity: Calculating lipid binding affinities from unbiased coarse-grained molecular dynamics simulations. 密度-阈值亲和力：通过无偏粗粒度分子动力学模拟计算脂质结合亲和力。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-04 DOI: 10.1016/bs.mie.2024.03.008
Jesse W Sandberg, Ezry Santiago-McRae, Jahmal Ennis, Grace Brannigan
{"title":"The density-threshold affinity: Calculating lipid binding affinities from unbiased coarse-grained molecular dynamics simulations.","authors":"Jesse W Sandberg, Ezry Santiago-McRae, Jahmal Ennis, Grace Brannigan","doi":"10.1016/bs.mie.2024.03.008","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.008","url":null,"abstract":"<p><p>Many membrane proteins are sensitive to their local lipid environment. As structural methods for membrane proteins have improved, there is growing evidence of direct, specific binding of lipids to protein surfaces. Unfortunately the workhorse of understanding protein-small molecule interactions, the binding affinity for a given site, is experimentally inaccessible for these systems. Coarse-grained molecular dynamics simulations can be used to bridge this gap, and are relatively straightforward to learn. Such simulations allow users to observe spontaneous binding of lipids to membrane proteins and quantify localized densities of individual lipids or lipid fragments. In this chapter we outline a protocol for extracting binding affinities from these localized distributions, known as the \"density threshold affinity.\" The density threshold affinity uses an adaptive and flexible definition of site occupancy that alleviates the need to distinguish between \"bound'' lipids and bulk lipids that are simply diffusing through the site. Furthermore, the method allows \"bead-level\" resolution that is suitable for the case where lipids share binding sites, and circumvents ambiguities about a relevant reference state. This approach provides a convenient and straightforward method for comparing affinities of a single lipid species for multiple sites, multiple lipids for a single site, and/or a single lipid species modeled using multiple forcefields.</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":"141723933","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
Modeling the mechanochemical feedback for membrane-protein interactions using a continuum mesh model. 利用连续网格模型建立膜蛋白相互作用的机械化学反馈模型。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-15 DOI: 10.1016/bs.mie.2024.03.016
Christopher T Lee, Padmini Rangamani
{"title":"Modeling the mechanochemical feedback for membrane-protein interactions using a continuum mesh model.","authors":"Christopher T Lee, Padmini Rangamani","doi":"10.1016/bs.mie.2024.03.016","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.016","url":null,"abstract":"<p><p>The Helfrich free energy is widely used to model the generation of membrane curvature due to different physical and chemical components. The governing equations resulting from the energy minimization procedure are a system of coupled higher order partial differential equations. Simulations of membrane deformation for obtaining quantitative comparisons against experimental observations require computational schemes that will allow us to solve these equations without restrictions to axisymmetric coordinates. Here, we describe one such tool that we developed in our group based on discrete differential geometry to solve these equations along with examples.</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":"141723926","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 fluorine transfer catalysed by an engineered nonheme iron enzyme. 工程非血红素铁酶催化的自由基氟转移。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-10 DOI: 10.1016/bs.mie.2024.03.004
Qun Zhao, Zhenhong Chen, Jinyan Rui, Xiongyi Huang
{"title":"Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.","authors":"Qun Zhao, Zhenhong Chen, Jinyan Rui, Xiongyi Huang","doi":"10.1016/bs.mie.2024.03.004","DOIUrl":"10.1016/bs.mie.2024.03.004","url":null,"abstract":"<p><p>Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp<sup>3</sup>)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.</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/PMC11232670/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140868444","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
Quantification of membrane geometry and protein sorting on cell membrane protrusions using fluorescence microscopy. 利用荧光显微镜对细胞膜突起的膜几何形状和蛋白质分类进行量化。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-16 DOI: 10.1016/bs.mie.2024.01.023
Shilong Yang, Zheng Shi
{"title":"Quantification of membrane geometry and protein sorting on cell membrane protrusions using fluorescence microscopy.","authors":"Shilong Yang, Zheng Shi","doi":"10.1016/bs.mie.2024.01.023","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.01.023","url":null,"abstract":"<p><p>Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences 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":"141545053","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
Thickness determination of hydroperoxidized lipid bilayers from medium-resolution cryo-TEM images. 从中等分辨率的低温 TEM 图像中确定氢过氧化脂质双分子层的厚度。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-23 DOI: 10.1016/bs.mie.2024.02.008
Eulalie Lafarge, Carlos M Marques, Marc Schmutz, Pierre Muller, André P Schroder
{"title":"Thickness determination of hydroperoxidized lipid bilayers from medium-resolution cryo-TEM images.","authors":"Eulalie Lafarge, Carlos M Marques, Marc Schmutz, Pierre Muller, André P Schroder","doi":"10.1016/bs.mie.2024.02.008","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.02.008","url":null,"abstract":"<p><p>As the primary products of lipid oxidation, lipid hydroperoxides constitute an important class of lipids generated by aerobic metabolism. However, despite several years of effort, the structure of the hydroperoxidized bilayer has not yet been observed under electron microscopy. Here we use a 200 kV Cryo-TEM to image small unilamellar vesicles (SUVs) made (i) of pure POPC or SOPC, (ii) of their pure hydroperoxidized form, and (iii) of their equimolar mixtures. We show that the challenges posed by the determination of the thickness of the hydroperoxidized bilayers under these observation conditions can be addressed by an image analysis method that we developed and describe here.</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":"141545061","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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