Characterization of Aquaporin Z proteoliposome structure and functionality via microscopy and scattering methods

Area of Science:

• Biophysics
• Membrane protein biophysics
• Biomaterials

Background:

• Aquaporins facilitate efficient water transport, but biomimetic systems face performance and stability challenges.
• Reconstituting aquaporins into liposomes is crucial for studying their function in a controlled environment.
• The aquaporin Z (AqpZ) model system is used to investigate protein insertion and water transport.

Purpose of the Study:

• To develop and apply an analytical toolkit for assessing AqpZ reconstitution and functionality in phosphatidylcholine proteoliposomes.
• To evaluate the structural and functional impact of AqpZ incorporation into liposomes.
• To investigate the effect of protein concentration and labeling on AqpZ activity and water permeability.

Main Methods:

• Dynamic light scattering (DLS)
• Cryogenic transmission electron microscopy (Cryo-TEM)
• Confocal microscopy (LSM and STED)
• Stopped-flow light scattering
• Small-angle X-ray scattering (SAXS)

Main Results:

• AqpZ incorporation increased liposome size and hydrophobic bilayer thickness.
• Distinct permeability profiles were observed for AqpZ, AqpZ-GFP, and AqpZ-Atto594.
• Labeled AqpZ variants (AqpZ-GFP, AqpZ-Atto594) showed a loss of function despite successful structural reconstitution.
• Water transport per AqpZ molecule remained constant, with increased reconstitution and permeability correlating with higher AqpZ concentrations.

Conclusions:

• The developed analytical toolkit enables comprehensive assessment of AqpZ reconstitution and function in liposomes.
• Protein labeling strategies can significantly impair aquaporin functionality, posing a challenge for biomimetic applications.
• AqpZ concentration is a key factor influencing liposome properties and water transport efficiency.