image: A The fluorescence of a fluorophore changes rapidly with its distance to a graphene oxide layer. B The experimental setup of SIFA on TIRF microscopy. C The morphology (upper panel) and the profile of the thickness (lower panel) of a lipid bilayer composed of DOPC/DOPA. D The dependence of the relative intensity on distance in SIFA with different critical distances d0 (Li et al. 2016). E The scheme of LipoFRET. F FRET from one donor to multiple acceptors. G The absorption spectra of TB (blue) and the emission spectra of the donor Alexa Fluor 555-MAL (green). H The dependence of the relative intensity on the distance to the inner surface of the liposome membrane in LipoFRET (Ma et al. 2021)
Credit: Chenguang Yang, Dongfei Ma, Shuxin Hu, Ming Li, Ying Lu
Key Findings:
- IFA: This method utilizes the fluorescence energy transfer between a fluorophore and monolayer graphene oxide to measure the distance between the fluorophore and the lipid bilayer. It allows researchers to track the three-dimensional movement of the protein within the membrane and capture nanoscale changes in insertion depth.
- LipoFRET: This technique relies on FRET between a donor fluorophore and multiple acceptor quenchers encapsulated in liposomes. By analyzing the intensity of the fluorescence, researchers can determine the penetration depth of the protein within the liposome membrane and study curvature-sensitive membrane proteins.
- Applications: The protocol demonstrates the effectiveness of these methods by studying the insertion dynamics of MLKL and α-synuclein, two important membrane proteins involved in cell death and neurodegenerative diseases, respectively.
Significance: The development of SIFA and LipoFRET represents a significant advancement in the field of membrane protein research. These techniques offer valuable tools for understanding the structure and function of membrane proteins, and their potential applications in drug discovery and disease treatment.
The work entitled “Real-time analysis of nanoscale dynamics in membrane protein insertion via single-molecule imaging” was published on Biophysics Reports (published on December, 2024).
Journal
Biophysics Reports
Method of Research
Experimental study
Subject of Research
Cells
Article Title
Real-time analysis of nanoscale dynamics in membrane protein insertion via single-molecule imaging
Article Publication Date
31-Dec-2024