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Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT
A H Laszlo1, I M Derrrington1, J H Gundlach1
1University of Washington, Seattle, WA, United States.
Methods in Enzymology
|January 8, 2017
Summary
Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) track enzyme movement on DNA with unprecedented precision. This method reveals enzyme dynamics at the single-nucleotide level, offering new insights into molecular mechanisms.
Area of Science:
- Biophysics
- Molecular Biology
- Biotechnology
Background:
- Nanopore technology is advancing single-molecule studies of enzymes.
- Existing methods lack the spatiotemporal precision to observe enzyme dynamics in real-time.
Purpose of the Study:
- To introduce the Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) technique.
- To demonstrate SPRNT's capability in analyzing enzyme activity at high resolution.
Main Methods:
- Loading an enzyme onto DNA/RNA and threading it into a protein nanopore (MspA).
- Measuring ion current changes through the nanopore as DNA translocates, controlled by enzyme activity.
- Utilizing picometer spatial resolution and millisecond temporal resolution for real-time analysis.
Main Results:
- SPRNT achieved 40pm spatial resolution and millisecond timescale measurements.
- Observed distinct ATP-dependent and ATP-independent substates during helicase Hel308 activity.
- Provided real-time DNA sequence information within the enzyme complex.
Conclusions:
- SPRNT offers a low-cost, high-precision single-molecule method for studying enzyme dynamics.
- The technique enables the observation of previously unresolvable enzyme movements in real-time.
- SPRNT significantly advances the field of enzyme mechanism studies.
