Protein reconstitution into giant unilamellar vesicles from native nanodiscs.

Abstract

The reconstitution of purified membrane proteins into lipid bilayers represents a tedious challenge as it relies mainly on empirically optimized purification protocols tailored to the target protein, including laborious detergent selection. The situation is even more complex when the protein's function or structural stability depends on its lipid environment. Using nanodisc-forming amphiphilic polymers helps circumnavigate these problems by allowing the extracted protein to stay in its native lipid environment. Yet, thus far, nanodiscs are of limited use for transport studies due to the problematic protein transfer to vesicles or planar bilayers. Here we exploited the recently introduced glycopolymer Glyco-DIBMA to purify integral membrane proteins such as the voltage-gated potassium ion channel KvAP, the voltage-gated sodium ion channel NavMs, the bacterial translocon complex SecYEG, the eukaryotic serotonin-gated cation channel 5Ht3A, as well as the phospholipid translocating scramblase TMEM16. We used fluorescence correlation spectroscopy to quantify the abundance of the labeled proteins in nanodiscs and optimize protein transfer into giant unilamellar vesicles (GUVs). We thus could achieve protein densities of roughly one copy per diffraction-limited focal area or ∼5400 copies in a GUV with a diameter of ∼20 µm. Such protein density is sufficient for visualizing the reconstituted proteins in vesicular membranes by conventional fluorescence microscopy. The proteins retain functionality, as our GUV influx and efflux studies demonstrated. This project was supported by a grant from the FWF (Austrian Science Fund): P34826.