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Quantifying Instrumental Artifacts in Folding Kinetics Measured by Single-Molecule Force Spectroscopy.
Krishna Neupane1, Michael T Woodside2
1Department of Physics, University of Alberta, Edmonton, Alberta, Canada.
Biophysical Journal
|July 3, 2016
Summary
Optical tweezers measurements of single biological molecule kinetics show minimal errors from instrument artifacts. Typical force spectroscopy experiments accurately reflect molecular dynamics, guiding experimental design for reliable kinetic measurements.
Area of Science:
- Biophysics
- Molecular Biology
- Biochemistry
Background:
- Force spectroscopy is a key technique for measuring single biological molecule kinetics.
- Instrumental factors like probes and linkers can influence observed molecular behavior.
- Understanding these influences is crucial for accurate kinetic measurements.
Purpose of the Study:
- To quantify the errors in kinetic measurements of molecular folding using optical tweezers.
- To assess the impact of probe and linker properties on observed kinetics.
- To provide guidance for experimental design in force spectroscopy.
Main Methods:
- Applied theoretical models of probe-linker effects to optical tweezer measurements.
- Analyzed constant-force measurements of DNA hairpin folding.
- Examined transition path measurements using constant trap position and high trap stiffness.
Main Results:
- Instrument artifacts contributed only ~20% error to folding rates in constant-force measurements.
- Measurements of transition paths were found to be within the low-artifact limit.
- Typical optical trap measurements accurately reflect the intrinsic dynamics of molecules.
Conclusions:
- Optical tweezer measurements of molecular kinetics are generally reliable.
- Experimental design choices significantly impact the accuracy of kinetic measurements.
- The study offers practical insights for optimizing force spectroscopy experiments.
