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Functional Assay to Correlate Protein Oligomerization States with Membrane Pore Formation
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SYSNO ASEP 0534692 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Functional Assay to Correlate Protein Oligomerization States with Membrane Pore Formation Author(s) Šachl, Radek (UFCH-W) RID, ORCID
Čujová, Sabína (UFCH-W) ORCID
Singh, Vandana (UFCH-W)
Riegerová, Petra (UFCH-W) ORCID
Kapusta, Peter (UFCH-W) RID, ORCID
Müller, H.-M. (DE)
Steringer, J. P. (DE)
Hof, Martin (UFCH-W) RID, ORCID
Nickel, W. (DE)Source Title Analytical Chemistry. - : American Chemical Society - ISSN 0003-2700
Roč. 92, č. 22 (2020), s. 14861-14866Number of pages 6 s. Language eng - English Country US - United States Keywords Peptides and proteins ; Oligomerization ; Fluorescence, ; Oligomers, Subject RIV CF - Physical ; Theoretical Chemistry OECD category Physical chemistry R&D Projects GC20-01401J GA ČR - Czech Science Foundation (CSF) GX19-26854X GA ČR - Czech Science Foundation (CSF) Method of publishing Limited access Institutional support UFCH-W - RVO:61388955 UT WOS 000592852900002 EID SCOPUS 85096123765 DOI 10.1021/acs.analchem.0c03276 Annotation In-membrane oligomerization is decisive for the function (or dysfunction) of many proteins. Techniques were developed to characterize membrane-inserted oligomers and the hereby obtained oligomerization states were intuitively related to the function of these proteins. However, in many cases, it is unclear whether the obtained oligomerization states are functionally relevant or are merely the consequence of nonspecific aggregation. Using fibroblast growth factor 2 (FGF2) as a model system, we addressed this methodological challenge. FGF2 oligomerizes in a PI(4,5)P2-dependent manner at the inner plasma membrane leaflet. This process results in membrane insertion and the formation of a lipidic membrane pore, the key intermediate in unconventional secretion of FGF2. To tackle the problem of discriminating functional oligomers from irrelevant aggregates, we present a statistical single molecule and single vesicle assay determining the brightness of individually diffusing in-membrane oligomers and correlating their oligomerization state with membrane pore formation. Importantly, time-dependent membrane pore formation was analyzed with an ensemble of single vesicles providing detailed statistics. Our findings demonstrate that quantifying oligomeric states alone does not allow for a deep understanding of the structure–function relationship of membrane-inserted oligomers. Workplace J. Heyrovsky Institute of Physical Chemistry Contact Michaela Knapová, michaela.knapova@jh-inst.cas.cz, Tel.: 266 053 196 Year of Publishing 2021 Electronic address http://hdl.handle.net/11104/0312867
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