Manipulating protein conformations by single-molecule AFM-FRET nanoscopy

Area of Science:

• Biophysics
• Nanotechnology
• Molecular Biology

Background:

• Understanding protein conformational changes is crucial for deciphering biological functions.
• Existing single-molecule techniques have limitations in manipulating and simultaneously measuring protein dynamics.

Purpose of the Study:

• To develop a single-molecule AFM-FRET nanoscopy approach for targeted protein manipulation and conformational analysis.
• To investigate the mechanical manipulation of protein conformation and its effect on enzyme activity.

Main Methods:

• Combines atomic force microscopy (AFM) for mechanical manipulation and fluorescence resonance energy transfer (FRET) for conformational monitoring.
• Developed a nanoscopy approach to target and manipulate individual dye-labeled proteins.
• Recorded single-molecule FRET time trajectories and AFM force pulling curves.

Main Results:

• Successfully demonstrated the pinpointing and mechanical manipulation of a single, dye-labeled protein (HPPK enzyme).
• Simultaneously monitored conformational changes using single-molecule FRET during AFM force pulling.
• Observed protein conformational changes induced by specific AFM mechanical force application.

Conclusions:

• The developed AFM-FRET nanoscopy is effective for manipulating single-molecule protein conformation.
• This technique provides a powerful tool for studying enzyme mechanics and dynamics at the single-molecule level.