Multicolor subunit counting method to decipher membrane receptor stoichiometry

Abstract: Membrane proteins are important components of cell signaling, membrane transport, etc. These proteins are mostly multimeric complexes and they often interact with different biomolecules. Determining the stoichiometry of these membrane proteins can help us to understand how these proteins function. Western blot, electrophysiology, FRET, etc. are the most used methods for determining protein stoichiometry. In the past few years, single-molecule imaging provided an opportunity to study proteins at the single molecule level and low protein density. The subunit counting method is a single-molecule imaging method that uses bleaching step counts from fluorophores to count subunits of a protein. Although robust, the method is still restricted to one color only (mEGFP is widely used because of its high photo-stability) due to the inapplicability of some other fluorophores (e.g. mCherry can be used to study colocalization. However, their bleaching step counting is difficult due to low photo-stability).

In this research, our first aim was to establish the multicolor subunit counting method using bleaching step counts of different suitable fluorophores to determine protein stoichiometry. We successfully established two multicolor subunit counting methods using SNAP-tag and YFP. We used SNAP-tag intracellularly and counted its bleaching steps to predict protein subunit composition. We observed fluorescence probability of the SNAP-tag is ~70% and its labeling efficiency is ~50%. YFP is in a different spectral range than GFP. It is photostable compared to blue and red fluorescent proteins. Here, we represent a principle to unmix the GFP and YFP spectra so that YFP bleaching steps can be used to predict the subunit composition of a protein. We used GFP variant mEGFP and the most stable YFP variant mGold for our study. We observed that the fluorescence probability of mGold is ~80%. We validated our established methods using NMDA receptor (GluN1/GluN3A) as the positive control. Secondly, we used the SNAP-tag/mEGFP multicolor subunit counting method to investigate two unresolved questions. First, we studied the possible molecular mechanism behind the apparent non-fluorescence of a fraction of fluorophores when expressed in oocytes. We found that there is a ~10% probability that the fluorophores are cleaved off after the C-terminal domain of a receptor causing their apparent non-fluorescence. Secondly, we studied the assembly of GluA1/GluA2 heteromers of AMPA receptors and investigated the molecular mechanism behind the 2:2 stoichiometry preference. We observed that no structural domain of AMPA receptor subunits is solely responsible for the 2:2 preference

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2022

Classification and Topics

• Keyword: Rezeptor

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2022
• Creator: Mukhopadhyay, Antara
• Contributor: Ulbrich, Maximilian ; Reiff, Dierk Frithjof ; Di Ventura, Barbara ; Lillemeier, Björn F. ; Klinik für Innere Medizin IV. Freiburg im Breisgau ; Albert-Ludwigs-Universität Freiburg. Fakultät für Biologie

Further information

• DOI: 10.6094/UNIFR/232231
• URN: urn:nbn:de:bsz:25-freidok-2322312
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 15.08.2025, 7:37 AM CEST