Probing single biomolecules in solution using the anti-Brownian electrokinetic (ABEL) trap

Area of Science:

• Biophysics
• Single-molecule biophysics
• Biochemistry

Background:

• Single-molecule fluorescence measurements provide insights into biomolecular diversity and dynamics.
• Solution-phase measurements are ideal for native biomolecular environments but limited by diffusion (approx. 1 ms observation time).
• Surface immobilization can perturb biomolecules, introducing artifacts and heterogeneity.

Purpose of the Study:

• To develop a method for significantly prolonging observation times of single biomolecules in solution.
• To enable detailed studies of biomolecular photodynamics and enzymatic properties in their native environment.
• To overcome the limitations of diffusion in solution-phase single-molecule assays.

Main Methods:

• Development of the anti-Brownian electrokinetic (ABEL) trap.
• Integration of high-sensitivity single-molecule fluorescence microscopy, real-time feedback control, and electrokinetic flow in a microfluidic chamber.
• Application to study allophycocyanin, TRiC chaperonin, G protein-coupled receptors, and nitrate reductase.

Main Results:

• The ABEL trap successfully prolonged observation times for single biomolecules in aqueous solution.
• Observed rich dynamics in allophycocyanin's emission brightness and excited state lifetime, indicating state changes.
• Studied ATP binding stoichiometry of TRiC, revealing deviations from standard cooperativity models; observed conformational changes in GPCRs upon agonist binding; determined rate constants for nitrate reductase's enzymatic cycle.

Conclusions:

• The ABEL trap is a powerful tool for extracting detailed information about single biomolecules in solution.
• It allows for the study of complex dynamics and properties previously inaccessible due to diffusion limitations.
• Demonstrated broad applicability across various biomolecular systems, including photosynthetic proteins, enzymes, and receptors.