Tuning membrane protein mobility by confinement into nanodomains

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• High-speed atomic force microscopy (HS-AFM) enables visualization of single soluble proteins.
• Studying single membrane proteins with HS-AFM is challenging due to unsuitable supports and high protein mobility.

Purpose of the Study:

• To develop a novel platform for studying membrane proteins using HS-AFM.
• To overcome limitations of existing methods for visualizing membrane protein dynamics and conformation.

Main Methods:

• Utilized streptavidin crystals grown on mica-supported lipid bilayers as porous membrane supports.
• Incorporated biotinylated lipids into membranes for protein attachment.
• Employed glutaraldehyde-cross-linking of streptavidin to control transmembrane protein lateral mobility.
• Applied HS-AFM for high-resolution imaging of membrane proteins.

Main Results:

• Demonstrated the utility of the streptavidin crystal platform with SecYEG and GlpF.
• Successfully tuned the lateral mobility of transmembrane proteins for HS-AFM imaging.
• Achieved sub-molecular resolution imaging of GlpF (aquaglyceroporin).
• Observed the binding of the motor protein SecA to SecYEG (protein translocation channel).

Conclusions:

• The developed platform overcomes key limitations in studying membrane proteins with HS-AFM.
• Enables detailed investigation of protein conformation and interactions in a controlled environment.
• Advances the capability of HS-AFM for structural and functional analysis of membrane protein complexes.