Current Protocols in Protein Science最新文献_第8页

Methods for Studying Interactions of Detergents and Lipids with α-Helical and β-Barrel Integral Membrane Proteins 洗涤剂和脂类与α-螺旋和β-桶状整体膜蛋白相互作用的研究方法

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI: 10.1002/0471140864.ps2907s74

S. Saif Hasan, Danas Baniulis, Eiki Yamashita, Mariya V. Zhalnina, Stanislav D. Zakharov, Jason T. Stofleth, William A. Cramer

{"title":"Methods for Studying Interactions of Detergents and Lipids with α-Helical and β-Barrel Integral Membrane Proteins","authors":"S. Saif Hasan, Danas Baniulis, Eiki Yamashita, Mariya V. Zhalnina, Stanislav D. Zakharov, Jason T. Stofleth, William A. Cramer","doi":"10.1002/0471140864.ps2907s74","DOIUrl":"10.1002/0471140864.ps2907s74","url":null,"abstract":"Methods for studying interactions of protein with lipids and detergents are described for representatives of two major classes of membrane proteins: (1) the α-helical hetero-oligomeric integral cytochrome b6f complex of oxygenic photosynthesis from cyanobacteria, and (2) the outer membrane β-barrel proteins BtuB and OmpF from Gram-negative Escherichia coli bacteria. Details are presented on the use of detergents for purification and crystallization of the b6f complex as well as a method for lipid exchange. The positions of detergent and lipid molecules, which define eight potential lipid-binding sites in the b6f complex, are described. Differences in detergent strategies for isolation and crystallization of β-barrel proteins relative to those for oligomeric helical membrane proteins are discussed, and purification and assessment of protein quality by circular dichroism (CD) is presented. Curr. Protoc. Protein Sci. 74:29.7.1-29.7.30. © 2013 by John Wiley & Sons, Inc.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"74 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471140864.ps2907s74","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101512","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Quantitative Analysis of Surface Expression of Membrane Proteins Using Cold-Adapted Proteases 利用冷适应蛋白酶定量分析膜蛋白的表面表达

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI: 10.1002/0471140864.ps0311s73

Faraz Ahmad, Kai Kaila, Peter Blaesse

引用次数: 0

Protein Knockouts in Living Eukaryotes Using deGradFP and Green Fluorescent Protein Fusion Targets 利用降解fp和绿色荧光蛋白融合靶标的真核生物蛋白敲除

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI: 10.1002/0471140864.ps3002s73

Emmanuel Caussinus, Oguz Kanca, Markus Affolter

引用次数: 22

Overview of Affinity Tags for Protein Purification 蛋白质纯化的亲和标签概述

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI: 10.1002/0471140864.ps0909s73

Michelle E. Kimple, Allison L. Brill, Renee L. Pasker

引用次数: 268

Computational Large-Scale Mapping of Protein-Protein Interactions Using Structural Complexes 使用结构复合物的蛋白质-蛋白质相互作用的计算大规模映射

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI: 10.1002/0471140864.ps0309s73

Benjamin Shoemaker, Stefan Wuchty, Anna R. Panchenko

引用次数: 2

BioID: A Screen for Protein-Protein Interactions BioID:蛋白质-蛋白质相互作用的筛选

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI: 10.1002/0471140864.ps1923s74

Kyle J. Roux, Dae In Kim, Brian Burke

引用次数: 201

Site-Specific Protein Labeling with SNAP-Tags 用SNAP-Tags标记位点特异性蛋白质

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI: 10.1002/0471140864.ps3001s73

Nelson B. Cole

{"title":"Site-Specific Protein Labeling with SNAP-Tags","authors":"Nelson B. Cole","doi":"10.1002/0471140864.ps3001s73","DOIUrl":"10.1002/0471140864.ps3001s73","url":null,"abstract":"<div>\u0000 \u0000 Site-specific labeling of cellular proteins with chemical probes is a powerful tool for studying protein function in living cells. A number of small peptide and protein tags have been developed that can be labeled with synthetic probes with high efficiencies and specificities and provide flexibility not available with fluorescent proteins. The SNAP-tag is a modified form of the DNA repair enzyme human O6-alkylguanine-DNA-alkyltransferase, and undergoes a self-labeling reaction to form a covalent bond with O6-benzylguanine (BG) derivatives. BG can be modified with a wide variety of fluorophores and other reporter compounds, generally without affecting the reaction with the SNAP-tag. In this unit, basic strategies for labeling SNAP-tag fusion proteins, both for live cell imaging and for in vitro analysis, are described. This includes a description of a releasable SNAP-tag probe that allows the user to chemically cleave the fluorophore from the labeled SNAP-tag fusion. In vitro labeling of purified SNAP-tag fusions is briefly described. Curr. Protoc. Protein Sci. 73:30.1.1-30.1.16. © 2013 by John Wiley & Sons, Inc.</div>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"73 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/0471140864.ps3001s73","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"32101376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 56

Nonradioactive Analysis of Dynamic Protein Palmitoylation 动态蛋白棕榈酰化的非放射性分析

Current Protocols in Protein Science Pub Date : 2018-02-16 DOI: 10.1002/0471140864.ps1415s73

Brent R. Martin

引用次数: 26

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation 通过sortase介导的转肽酶进行位点特异性蛋白标记

Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.38

John M. Antos, Jessica Ingram, Tao Fang, Novalia Pishesha, Matthias C. Truttmann, Hidde L. Ploegh

{"title":"Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation","authors":"John M. Antos, Jessica Ingram, Tao Fang, Novalia Pishesha, Matthias C. Truttmann, Hidde L. Ploegh","doi":"10.1002/cpps.38","DOIUrl":"10.1002/cpps.38","url":null,"abstract":"Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.38","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35232425","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 121

Mass-Tag Labeling Using Acyl-PEG Exchange for the Determination of Endogenous Protein S-Fatty Acylation 用酰基-聚乙二醇交换质标签法测定内源性蛋白质s-脂肪酰化

Current Protocols in Protein Science Pub Date : 2018-02-13 DOI: 10.1002/cpps.36

Avital Percher, Emmanuelle Thinon, Howard Hang

{"title":"Mass-Tag Labeling Using Acyl-PEG Exchange for the Determination of Endogenous Protein S-Fatty Acylation","authors":"Avital Percher, Emmanuelle Thinon, Howard Hang","doi":"10.1002/cpps.36","DOIUrl":"10.1002/cpps.36","url":null,"abstract":"The covalent coupling of fatty acids to proteins provides an important mechanism of regulation in cells. In eukaryotes, cysteine fatty acylation (S-fatty acylation) has been shown to be critical for protein function in a variety of cellular pathways as well as microbial pathogenesis. While methods developed over the past decade have improved the detection and profiling of S-fatty acylation, these are hampered in their ability to characterize endogenous protein S-fatty acylation levels under physiological conditions. Furthermore, understanding the contribution of specific sites and levels of S-fatty acylation remains a major challenge. To evaluate S-fatty acylation of endogenous proteins as well as to determine the number of S-fatty acylation events, we developed the acyl-PEG exchange (APE) that utilizes cysteine-specific chemistry to exchange S-fatty acylation sites with mass-tags of defined size, which can be readily observed by western blotting. APE provides a readily accessible approach to investigate endogenous S-fatty acylation from any sample source, with high sensitivity and broad applicability that complements the current toolbox of methods for thioester-based post-translational modifications. © 2017 by John Wiley & Sons, Inc.","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.36","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35371490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 29