Methods in enzymology最新文献_第8页

STED super-resolution microscopy of mitochondrial translocases. 线粒体转运酶的 STED 超分辨率显微镜。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-21 DOI: 10.1016/bs.mie.2024.07.052

Sarah V Schweighofer, Kaushik Inamdar, Daniel C Jans, Stefan Jakobs

{"title":"STED super-resolution microscopy of mitochondrial translocases.","authors":"Sarah V Schweighofer, Kaushik Inamdar, Daniel C Jans, Stefan Jakobs","doi":"10.1016/bs.mie.2024.07.052","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.052","url":null,"abstract":"The mitochondrial translocases of the outer membrane (TOM) and of the inner membrane (TIM) act together to facilitate the import of nuclear-encoded proteins across the mitochondrial membranes. Stimulated Emission Depletion (STED) super-resolution microscopy enables the in situ imaging of such complexes in single cells at sub-diffraction resolution. STED microscopy requires only conventional sample preparation techniques and provides super-resolved raw data without the need for further image processing. In this chapter, we provide a detailed example protocol for STED microscopy of TOM20 and mitochondrial DNA in fixed mammalian cells. The protocol includes instructions on sample preparation for immunolabeling, including cell line selection, fixation, permeabilization, blocking, labeling and mounting, but also recommendations for sample and microscope performance evaluation. The protocol is supplemented by considerations on key factors that influence the quality of the final image and also includes some considerations for the analysis of the acquired images. While the protocol described here is aimed at imaging TOM20 and DNA, it contains all the information for an immediate adaptation to other cellular targets.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"299-327"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564667","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 protein phosphorylation at the mitochondrial protein import machinery by PhosTag electrophoresis. 通过 PhosTag 电泳监测线粒体蛋白质导入机制的蛋白质磷酸化。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-11 DOI: 10.1016/bs.mie.2024.07.063

Fatimah Lami Imam, Chris Meisinger, Adinarayana Marada

引用次数: 0

The effect of hydrostatic pressure on lipid membrane lateral structure. 静水压力对脂膜横向结构的影响。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-16 DOI: 10.1016/bs.mie.2024.03.019

Nicola L C McCarthy, Chi L Chan, Giulia E C M Mignini Urdaneta, Yifei Liao, Robert V Law, Oscar Ces, John M Seddon, Nicholas J Brooks

引用次数: 0

Using lipid binding proteins and advanced microscopy to study lipid domains. 利用脂质结合蛋白和先进的显微镜研究脂质结构域。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-10 DOI: 10.1016/bs.mie.2024.03.026

Nario Tomishige, Kohta Takahashi, Brigitte Pollet, Ludovic Richert, Yves Mély, Toshihide Kobayashi

引用次数: 0

Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes. 固体支撑脂质双层膜上由薄片蛋白介导的生物分子凝聚物的荧光成像。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-23 DOI: 10.1016/bs.mie.2024.04.007

Karthik B Narayan, Laura Baeyens, Honey Priya James, Aparna Swain, Tobias Baumgart

{"title":"Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes.","authors":"Karthik B Narayan, Laura Baeyens, Honey Priya James, Aparna Swain, Tobias Baumgart","doi":"10.1016/bs.mie.2024.04.007","DOIUrl":"10.1016/bs.mie.2024.04.007","url":null,"abstract":"Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"33-48"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545033","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

Quantifying uncertainty in trans-membrane stresses and moments in simulation. 在模拟中量化跨膜应力和力矩的不确定性。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-05-17 DOI: 10.1016/bs.mie.2024.04.008

Samuel L Foley, Markus Deserno

引用次数: 0

Determining the esterase activity of peptides and peptide assemblies. 确定肽和肽组合物的酯酶活性。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-20 DOI: 10.1016/bs.mie.2024.02.002

Patrizia Janković, Daniela Kalafatovic

{"title":"Determining the esterase activity of peptides and peptide assemblies.","authors":"Patrizia Janković, Daniela Kalafatovic","doi":"10.1016/bs.mie.2024.02.002","DOIUrl":"10.1016/bs.mie.2024.02.002","url":null,"abstract":"Catalytic peptides are gaining attention as alternatives to enzymes, especially in industrial applications. Recent advances in peptide design have improved their catalytic efficiency with approaches such as self-assembly and metal ion complexation. However, the fundamental principles governing peptide catalysis at the sequence level are still being explored. Ester hydrolysis, a well-studied reaction, serves as a widely employed method to evaluate the catalytic potential of peptides. The standard colorimetric reaction involving para-nitrophenyl acetate hydrolysis acts as a benchmark assay, providing a straightforward and efficient screening method for rapidly identifying potential catalysts. However, maintaining standardized conditions is crucial for reproducible results, given that factors such as pH, temperature, and substrate concentration can introduce unwanted variability. This necessity becomes particularly pronounced when working with peptides, which often exhibit slower reaction rates compared to enzymes, making even minor variations significantly influential on the final outcome. In this context, we present a refined protocol for assessing the catalytic activity of peptides and peptide assemblies, addressing critical considerations for reproducibility and accuracy.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"697 ","pages":"423-433"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180207","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

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":"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 Co2+ and Cu2+ 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/KM = 0.08 M/s. The protocols mentioned in this chapter meticulously cover the design, synthesis, self-assembly and enzyme kinetics.","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

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":"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.","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