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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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Quantitative FRET Förster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

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Measuring PETase enzyme kinetics by single-molecule microscopy.

Yuwei Zhang¹, William O Hancock²

¹Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania.

Biophysical Journal

|September 20, 2024

Summary

This summary is machine-generated.

Researchers developed a new single-molecule assay to study PETase enzymes, crucial for degrading plastic pollution. This method reveals enzyme binding dynamics, aiding the engineering of more effective plastic-degrading enzymes.

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Area of Science:

Biochemistry
Environmental Science
Materials Science

Background:

Polyethylene terephthalate (PET) is a major source of microplastics pollution.
Microbial and enzymatic degradation of PET is a key strategy to mitigate pollution.
Existing bulk assays struggle to characterize PETase enzyme kinetics on insoluble PET.

Purpose of the Study:

To develop a single-molecule microscopy assay for quantifying PETase enzyme kinetics.
To provide a more detailed mechanistic understanding of PETase-substrate interactions.
To aid in the engineering of improved PETase enzymes for plastic degradation.

Main Methods:

Developed a single-molecule microscopy assay using quantum dot-labeled PETase.
Quantified enzyme landing rates and binding durations on surface-immobilized PET films.
Analyzed binding data using biexponential fitting to distinguish active and nonspecific binding.

Main Results:

Wild-type PETase showed distinct populations of active (2.7 s) and nonspecific binding.
A hyperactive mutant (S238F/W159H) exhibited faster apparent on-rate and slower off-rate.
The single-molecule assay provides a more mechanistic view of PETase activity compared to bulk assays.

Conclusions:

The single-molecule assay offers a powerful tool for characterizing PETase kinetics.
Understanding binding dynamics is crucial for engineering more robust and active PETases.
This approach can accelerate efforts to combat microplastics pollution through enhanced enzymatic degradation.