Molecular Orientation Determination in Nanodiscs at the Single-Molecule Level

Abstract

The function of membrane-bound proteins often depends on their interactions with the lipid bilayer. Bulk absorption-based linear dichroism has been historically used to investigate molecular orientations in the phospholipid bilayer but cannot resolve the actual distribution of molecules embedded in the membrane and is often limited by a poor signal-to-noise ratio. Here, we present single-molecule orientation determination by fluorescence-detected linear dichroism visualization in Nanodisc grids or SOLVING, to determine the molecular orientation of molecules assembled into nanoscale lipid bilayers. We provide a proof-of-concept by using SOLVING to quantitate the orientation distribution of two commonly used fluorescent dyes, DiO and BODIPY, in 10 nm Nanodiscs. Besides confirming the mean orientation determined by bulk absorption measurement, SOLVING provides the actual distribution of orientations and promises to provide key molecular insights into the topology and interactions of multiprotein complexes, such as those observed in intracellular signal transduction.

Ultrafast energy transfer between lipid-linked chromophores and plant light-harvesting complex II

“…Note that nanodiscs were not required in the absence of LHCII, so, for sample (iii) liposomes were used for simplicity formed by following a typical sonication method. 21 Chromophores linked to lipids have been shown to align in the same manner within both liposomes and nanodiscs, 62,63 and thus we expect TR-lipid orientation to be similar in both samples.…”

Multiple mechanisms of self-association of chemokine receptors CXCR4 and CCR5 demonstrated by deep mutagenesis

“…No. 48393230) were cleaned for single-molecule experiments as described previously ( 52 ). The washed coverslips were stored in molecular grade water, air-dried, and flamed for a few seconds.…”

Multiple mechanisms of self-association of chemokine receptors CXCR4 and CCR5 demonstrated by deep mutagenesis

“…No. 48393230) were cleaned for single molecule experiments as described previously (Camp et al, 2020). The washed coverslips were stored in molecular grade water, air-dried, and flamed for a few seconds.…”