Investigating protein-membrane interactions using native reverse micelles constructed from naturally sourced lipids

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Accurate membrane models are crucial for studying membrane-associated proteins and their interactions.
• Existing models often lack the lipid diversity found in biological membranes, limiting high-resolution studies.
• Native reverse micelles (nRMs) offer a promising alternative for more biologically relevant membrane studies.

Purpose of the Study:

• To develop and characterize native reverse micelles (nRMs) using diverse eukaryotic lipids.
• To assess the ability of nRMs to encapsulate and maintain the structural integrity of proteins.
• To evaluate the utility of nRMs for studying the interactions of peripheral membrane proteins.

Main Methods:

• Formulation of nRMs using soy lecithin, porcine brain lipids, or bovine heart lipids with n-dodecylphosphocholine.
• Characterization of nRMs using dynamic light scattering and 31P-NMR.
• Protein NMR was used to verify protein structure and interactions within nRMs.

Main Results:

• Diverse nRM formulations were successfully developed and characterized.
• Ubiquitin encapsulation showed minimal structural changes, validating the nRM system.
• Encapsulated peripheral membrane proteins (GPx4, PEBP1, FABP4) exhibited expected interactions with the nRM inner surface.

Conclusions:

• The developed nRM formulations provide a biologically relevant context for high-resolution studies of membrane-interacting proteins up to ~21 kDa.
• This approach allows for efficient study of membrane proteins using various lipid extracts.
• The nRM system offers a versatile tool for investigating membrane protein function in specific biological contexts.