The aquaporin sidedness revisited.

Area of Science:

• Structural Biology
• Biophysics
• Membrane Protein Research

Background:

• Aquaporins are transmembrane proteins crucial for water transport and osmoregulation in living organisms.
• Aquaporins typically form tetrameric assemblies, with each subunit containing an aqueous pore and internal sequence repeats.
• Determining the precise orientation (sidedness) of aquaporin subunits is challenging due to their structural symmetry.

Purpose of the Study:

• To resolve the sidedness of Aquaporin Z (AqpZ) from Escherichia coli.
• To establish a reliable method for determining the orientation of transmembrane proteins using electron microscopy.
• To compare the structural features of AqpZ with human erythrocyte aquaporin-1 (AQP1).

Main Methods:

• Reconstitution of AqpZ into highly ordered two-dimensional crystals.
• Freeze-drying and metal-shadowing of crystals for electron microscopy.
• Utilizing crystals with both metal-coated and uncoated regions for surface relief and density map reconstruction.
• Cross-correlation analysis to align atomic force microscopy (AFM) topographs with projection maps.
• Comparison of AqpZ structure with the known 3D structure of AQP1.

Main Results:

• Successful reconstruction of AqpZ surface relief and projection maps from the same crystal.
• Unambiguous alignment of AqpZ topography with its density maps and subsequently with AQP1 structure.
• Direct interpretation of AqpZ topographical features through comparison with AQP1's 3D structure.

Conclusions:

• The sidedness originally proposed for aquaporin density maps was inverted.
• This study provides a robust method for determining transmembrane protein orientation.
• Structural insights into AqpZ contribute to understanding water channel function and evolution.