Functional Assay to Correlate Protein Oligomerization States with Membrane Pore Formation

0534692 - ÚFCH JH 2021 RIV US eng J - Journal Article
Šachl, Radek - Čujová, Sabína - Singh, Vandana - Riegerová, Petra - Kapusta, Peter - Müller, H.-M. - Steringer, J. P. - Hof, Martin - Nickel, W.
Functional Assay to Correlate Protein Oligomerization States with Membrane Pore Formation.
Analytical Chemistry. Roč. 92, č. 22 (2020), s. 14861-14866. ISSN 0003-2700. E-ISSN 1520-6882
R&D Projects: GA ČR(CZ) GC20-01401J; GA ČR(CZ) GX19-26854X
Institutional support: RVO:61388955
Keywords : Peptides and proteins * Oligomerization * Fluorescence, * Oligomers,
OECD category: Physical chemistry
Impact factor: 6.986, year: 2020
Method of publishing: Limited access

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.
Permanent Link: http://hdl.handle.net/11104/0312867