Einzeltreffer — DigiBib

Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.

Competing Interests: The authors declare no competing interest.

Titel:	Driving integrative structural modeling with serial capture affinity purification.
Autor/in / Beteiligte Person:	Liu, X ; Zhang, Y ; Wen, Z ; Hao, Y ; Banks, CAS ; Lange, JJ ; Slaughter, BD ; Unruh, JR ; Florens, L ; Abmayr, SM ; Workman, JL ; Washburn, MP
Link:	Full Text Finder (Volltext)
Zeitschrift:	Proceedings of the National Academy of Sciences of the United States of America, Jg. 117 (2020-12-15), Heft 50, S. 31861-31870
Veröffentlichung:	Washington, DC : National Academy of Sciences, 2020
Medientyp:	academicJournal
ISSN:	1091-6490 (electronic)
DOI:	10.1073/pnas.2007931117
Schlagwort:	Cell Cycle Proteins genetics Cell Cycle Proteins isolation & purification Cell Cycle Proteins metabolism Co-Repressor Proteins genetics Co-Repressor Proteins isolation & purification Co-Repressor Proteins metabolism Feasibility Studies Fluorescent Dyes chemistry HEK293 Cells Humans Intravital Microscopy Microtubule-Associated Proteins genetics Microtubule-Associated Proteins isolation & purification Microtubule-Associated Proteins metabolism Molecular Imaging methods Molecular Probes chemistry Phosphoproteins genetics Phosphoproteins isolation & purification Phosphoproteins metabolism Protein Binding Recombinant Proteins genetics Recombinant Proteins isolation & purification Recombinant Proteins metabolism Chromatography, Affinity methods Mass Spectrometry methods Models, Molecular
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Language: English [Proc Natl Acad Sci U S A] 2020 Dec 15; Vol. 117 (50), pp. 31861-31870. <i>Date of Electronic Publication: </i>2020 Nov 30. MeSH Terms: Models, Molecular* ; Chromatography, Affinity / methods ; Mass Spectrometry / methods ; Cell Cycle Proteins / genetics ; Cell Cycle Proteins / isolation & purification ; Cell Cycle Proteins / metabolism ; Co-Repressor Proteins / genetics ; Co-Repressor Proteins / isolation & purification ; Co-Repressor Proteins / metabolism ; Feasibility Studies ; Fluorescent Dyes / chemistry ; HEK293 Cells ; Humans ; Intravital Microscopy ; Microtubule-Associated Proteins / genetics ; Microtubule-Associated Proteins / isolation & purification ; Microtubule-Associated Proteins / metabolism ; Molecular Imaging / methods ; Molecular Probes / chemistry ; Phosphoproteins / genetics ; Phosphoproteins / isolation & purification ; Phosphoproteins / metabolism ; Protein Binding ; Recombinant Proteins / genetics ; Recombinant Proteins / isolation & purification ; Recombinant Proteins / metabolism References: Nat Biotechnol. 2003 Nov;21(11):1387-95. (PMID: 14595367) ; Methods Mol Biol. 2008;421:191-209. (PMID: 18826056) ; Mol Cancer Res. 2012 Mar;10(3):326-35. (PMID: 22258766) ; Nat Commun. 2019 Mar 8;10(1):1118. (PMID: 30850613) ; Nat Methods. 2013 Aug;10(8):730-6. (PMID: 23921808) ; Nature. 2002 Jan 10;415(6868):141-7. (PMID: 11805826) ; Oncotarget. 2015 Mar 10;6(7):4773-89. (PMID: 25749382) ; Nucleic Acids Res. 2015 Jul 1;43(W1):W174-81. (PMID: 25883148) ; Nat Commun. 2017 May 19;8:15473. (PMID: 28524877) ; J Pathol. 2016 Aug;239(4):459-72. (PMID: 27171498) ; Biochem Biophys Res Commun. 2005 Sep 23;335(2):343-50. (PMID: 16098913) ; Mol Cell Proteomics. 2015 Apr;14(4):1137-47. (PMID: 25648531) ; Nat Methods. 2006 Feb;3(2):83-9. (PMID: 16432516) ; Genes Dev. 2014 Mar 15;28(6):622-36. (PMID: 24589551) ; Nat Chem Biol. 2017 Jul;13(7):750-756. (PMID: 28504676) ; Cell Rep. 2020 Apr 14;31(2):107516. (PMID: 32294434) ; J Biol Chem. 2017 Dec 22;292(51):20808-20817. (PMID: 29061846) ; J Med Chem. 2019 Oct 24;62(20):9008-9025. (PMID: 31550156) ; J Cell Physiol. 2019 Jun;234(6):8963-8974. (PMID: 30317618) ; J Cell Biochem. 2019 Sep;120(9):14562-14572. (PMID: 31017716) ; Methods Mol Biol. 2007;356:195-208. (PMID: 16988404) ; BMC Bioinformatics. 2008 Jan 23;9:40. (PMID: 18215316) ; Nat Biotechnol. 2003 Jan;21(1):86-9. (PMID: 12469133) ; ChemMedChem. 2016 Oct 19;11(20):2327-2338. (PMID: 27634332) ; Nucleic Acids Res. 2016 May 19;44(9):e88. (PMID: 26893353) ; Mol Cell Proteomics. 2011 Jan;10(1):M110.002212. (PMID: 20736410) ; Trends Biochem Sci. 2016 Jan;41(1):20-32. (PMID: 26654279) ; J Biol Chem. 2015 Jun 12;290(24):15030-41. (PMID: 25878247) ; J Mol Biol. 2016 Feb 22;428(4):720-725. (PMID: 26410586) ; Nat Protoc. 2018 Mar;13(3):478-494. (PMID: 29419816) ; Biochem Biophys Res Commun. 2019 Apr 9;511(3):498-503. (PMID: 30803761) ; J Proteome Res. 2012 Feb 3;11(2):564-75. (PMID: 22149079) ; Elife. 2018 Mar 16;7:. (PMID: 29547122) ; Nucleic Acids Res. 2019 Jan 8;47(D1):D581-D589. (PMID: 30407591) ; Mol Cell Proteomics. 2018 Jul;17(7):1432-1447. (PMID: 29599190) ; Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):17954-9. (PMID: 23077255) ; Bioinformatics. 2015 Oct 1;31(19):3222-4. (PMID: 26026169) ; Nat Methods. 2017 Jan;14(1):61-64. (PMID: 27892958) ; J Med Chem. 2019 Oct 24;62(20):8996-9007. (PMID: 31260300) ; J Exp Clin Cancer Res. 2018 May 9;37(1):100. (PMID: 29743122) ; Nat Protoc. 2018 Dec;13(12):2964-2990. (PMID: 30446747) ; PLoS One. 2013 Jul 22;8(7):e69764. (PMID: 23894536) ; EMBO Rep. 2011 Oct 28;12(11):1160-6. (PMID: 21960006) ; Oncotarget. 2017 Apr 25;8(17):28939-28958. (PMID: 28423652) ; Structure. 2018 Jun 5;26(6):894-904.e2. (PMID: 29657133) ; J Mol Biol. 2017 Feb 3;429(3):399-407. (PMID: 27939290) ; Nat Biotechnol. 1999 Oct;17(10):1030-2. (PMID: 10504710) ; J Biol Chem. 2007 Jan 5;282(1):647-56. (PMID: 17082182) ; PLoS Pathog. 2014 Sep 11;10(9):e1004343. (PMID: 25211330) Grant Information: R01 GM112639 United States GM NIGMS NIH HHS; R35 GM118068 United States GM NIGMS NIH HHS Contributed Indexing: Keywords: chromatin; cross-linking mass spectrometry; epigenetics; integrative structural modeling; quantitative imaging Substance Nomenclature: 0 (Cell Cycle Proteins) ; 0 (Co-Repressor Proteins) ; 0 (Fluorescent Dyes) ; 0 (Microtubule-Associated Proteins) ; 0 (Molecular Probes) ; 0 (Phosphoproteins) ; 0 (Recombinant Proteins) ; 0 (SPINDOC protein, human) ; 0 (spindlin) Entry Date(s): Date Created: 20201201 Date Completed: 20210201 Latest Revision: 20210514 Update Code: 20240513 PubMed Central ID: PMC7749342

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Driving integrative structural modeling with serial capture affinity purification.