A Next-Generation qPlus-Sensor-Based AFM Setup: Resolving Archaeal S-Layer Protein Structures in Air and Liquid

Area of Science:

• Microbiology
• Biophysics
• Materials Science

Background:

• Surface-layer (S-layer) proteins are the outermost cell envelope component in many archaea and bacteria.
• These proteins self-assemble into ordered two-dimensional quasicrystalline lattices.
• Understanding S-layer protein structure is crucial for cell biology and nanotechnology.

Purpose of the Study:

• To investigate the self-assembled structure of S-layer proteins from Pyrobaculum aerophilium.
• To compare atomic force microscopy (AFM) imaging with transmission electron microscopy (TEM) under different conditions.
• To develop improved AFM techniques for visualizing S-layer proteins.

Main Methods:

• Extraction and isolation of S-layer proteins from Pyrobaculum aerophilium.
• Atomic force microscopy (AFM) using a qPlus sensor, a next-generation liquid cell, and novel sapphire tips.
• Frequency-modulation AFM (FM-AFM) in both liquid and ambient conditions.
• Comparison with transmission electron microscopy (TEM) under vacuum.

Main Results:

• Initial AFM scans revealed residual detergent (sodium dodecyl sulfate, SDS) layers, obscuring the S-layer structure.
• TEM imaging did not show SDS, highlighting differences in sample preparation requirements.
• Optimized sample preparation and AFM techniques allowed visualization of the S-layer proteins' crystalline structure.
• Successful imaging was achieved in both air and liquid environments.

Conclusions:

• Advanced AFM, with improved sample preparation and instrumentation, can resolve the fine structure of S-layer proteins.
• The study demonstrates the capability of AFM to image S-layer lattices in biologically relevant liquid environments.
• This work provides a foundation for further structural and functional studies of S-layer proteins using AFM.