Atomic force microscopy of native purple membrane.

Area of Science:

• Biophysics
• Structural Biology
• Microscopy

Background:

• Purple membrane (PM) contains bacteriorhodopsin (BR), a light-driven proton pump crucial for cellular energy.
• Understanding BR structure and dynamics is key to elucidating its function.
• AFM offers high-resolution imaging of biological surfaces in native-like conditions.

Purpose of the Study:

• To classify major conformations of native bacteriorhodopsin (BR) surfaces in purple membrane (PM).
• To map the variability and flexibility of polypeptide loops connecting transmembrane alpha-helices within BR.
• To investigate the influence of crystal packing on BR loop dynamics and inter-molecular interactions.

Main Methods:

• Atomic Force Microscopy (AFM) for subnanometer resolution imaging of PM in buffer solution.
• Single molecule AFM imaging to analyze individual BR molecules and their conformations.
• Single molecule force-spectroscopy to probe interactions between BR molecules and within secondary structure elements.

Main Results:

• AFM enabled classification of major BR surface conformations and mapping of polypeptide loop variability.
• Differences in loop position, variability, and flexibility were observed between trigonal and orthorhombic PM crystal forms.
• Schiff base bond cleavage led to trigonal PM crystal disassembly, which was reversible upon regeneration.

Conclusions:

• AFM provides detailed insights into BR structure and dynamics within the PM.
• Polypeptide loop characteristics are influenced by BR packing arrangements in the lipid bilayer.
• Combined AFM and force-spectroscopy offer a powerful approach to study molecular interactions in biological systems.