13.4 Single-molecule kinetics and microscopy techniques

Single-molecule kinetics unveils the hidden world of individual molecules. By studying reactions at the molecular level, we gain insights into heterogeneity, rare events, and dynamic processes that ensemble measurements often miss.

Advanced microscopy techniques like TIRF and AFM enable us to observe and manipulate single molecules. These tools reveal multiple reaction pathways, rate distributions, and conformational changes, providing a deeper understanding of molecular behavior and function.

Single-Molecule Kinetics

Principles of single-molecule kinetics

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• Involves studying behavior and dynamics of individual molecules reveals heterogeneity in reaction rates and pathways that may be hidden in ensemble-averaged measurements
• Investigates enzyme catalysis and conformational changes (protein folding and unfolding dynamics)
• Examines molecular motors and their mechanistic properties (kinesin, myosin)
• Probes receptor-ligand interactions and binding kinetics (antigen-antibody, drug-target)

Microscopy for single-molecule studies

• Fluorescence microscopy
  • Total Internal Reflection Fluorescence (TIRF) microscopy utilizes evanescent wave to selectively excite fluorophores near the surface reduces background fluorescence and improves signal-to-noise ratio
  • Confocal microscopy uses a pinhole to eliminate out-of-focus light enables high-resolution imaging and single-molecule detection
• Atomic Force Microscopy (AFM) measures the force between a sharp probe and the sample surface provides topographical and mechanical information about individual molecules
• Optical tweezers use focused laser beams to trap and manipulate individual molecules allow for the application of precise forces and measurement of molecular responses

Insights from single-molecule techniques

• Reveal presence of multiple reaction pathways different molecules may follow distinct routes to reach the same product helps identify rate-limiting steps and intermediates in complex reactions
• Observe heterogeneity in reaction rates individual molecules may exhibit different rate constants for the same reaction provides information about the distribution of kinetic properties within a population
• Monitor conformational dynamics and fluctuations in real-time allows for the observation of rare or transient states helps elucidate the relationship between molecular structure and function

Analysis of single-molecule data

1. Dwell time analysis measures the duration of specific molecular states or events provides information about the kinetics of transitions between states
2. Fluorescence intensity trajectories plot the fluorescence intensity of individual molecules over time allows for the identification of discrete states and their lifetimes
3. Fluorescence Resonance Energy Transfer (FRET) efficiency measures the distance-dependent energy transfer between donor and acceptor fluorophores provides information about conformational changes and molecular interactions
4. Statistical analysis of single-molecule data requires appropriate statistical methods to account for the stochastic nature of single-molecule events includes techniques such as hidden Markov modeling and maximum likelihood estimation