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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Direct Single Molecule Imaging of Enhanced Enzyme Diffusion.

Mengqi Xu¹, Jennifer L Ross¹, Lyanne Valdez²

¹Department of Physics, University of Massachusetts, Amherst, Massachusetts 01003, USA.

Physical Review Letters

|October 22, 2019

Summary

Enzymes diffuse faster with their substrates, a phenomenon known as enhanced diffusion. Direct single-molecule imaging confirms this, revealing higher diffusion increases than previously reported using fluorescence correlation spectroscopy (FCS).

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Area of Science:

Biochemistry and Biophysics
Enzymology
Single-molecule biophysics

Background:

Recent studies suggest enzymes exhibit enhanced diffusion in the presence of their substrates.
Fluorescence correlation spectroscopy (FCS) has been the primary method for measuring this enhanced diffusion.
Concerns regarding artifacts in FCS measurements due to environmental sensitivity have raised questions about previous findings.

Purpose of the Study:

To validate enhanced enzyme diffusion using a complementary, direct single-molecule imaging technique.
To investigate the influence of enzyme concentration on substrate-induced diffusion enhancement.
To determine if enzyme oligomerization state changes during catalytic turnover.

Main Methods:

Direct single-molecule imaging to track individual enzyme diffusion trajectories in solution.
Analysis of enzyme trajectories to directly calculate diffusion coefficients, minimizing environmental artifacts.
Experiments conducted with varying total enzyme concentrations and in the presence/absence of substrate.

Main Results:

Direct single-molecule imaging confirms enhanced enzyme diffusion in the presence of substrate.
The observed relative increase in enzyme diffusion is greater than previously reported with FCS.
Enhanced diffusion is independent of total enzyme concentration, and substrate presence does not alter enzyme oligomerization state.

Conclusions:

Direct single-molecule imaging provides a robust method for studying enzyme mobility, overcoming limitations of FCS.
Enzyme diffusion is significantly enhanced by substrate presence, with a magnitude potentially underestimated by previous methods.
Enzyme catalytic activity does not appear to involve changes in oligomerization state or be affected by total enzyme concentration regarding diffusion enhancement.