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Atomic force microscopy (AFM) kymograph analysis reveals single-molecule protein dynamics. While robust to lateral drift, rotational drift can cause artifacts, especially for proteins with complex motion, highlighting the importance of vertical measurements.

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Area of Science:

  • Biophysics
  • Single-molecule biophysics
  • Membrane protein dynamics

Background:

  • Kymograph analysis enhances temporal resolution in biological atomic force microscopy (AFM).
  • It is valuable for studying single-molecule protein dynamics under near-native conditions.
  • Limitations exist due to protein geometry and instrumental drift.

Purpose of the Study:

  • To investigate the conformational dynamics of sparse membrane proteins using AFM kymograph analysis.
  • To compare AFM kymograph analysis for proteins with predominantly vertical (SecDF) versus combined vertical and lateral motion (Pgp).
  • To analyze experimental issues like translational and rotational drift and evaluate transition detection algorithms.

Main Methods:

  • AFM kymograph analysis of membrane proteins (SecDF and Pgp).
  • Kymograph simulations to evaluate conformational transition detection.
  • Comparison of state detection algorithms, including infinite hidden Markov models.

Main Results:

  • Kymograph analysis is largely robust to lateral drift; minor displacements do not significantly impact transition detection or dwell time.
  • Rotational drift can induce artifactual transitions in proteins exhibiting azimuthal height dependence (e.g., Pgp).
  • Vertical height measurements are generally superior to width measurements for membrane proteins.

Conclusions:

  • AFM kymography is a valuable tool for membrane biophysics, particularly for studying sparse protein distributions.
  • Understanding and mitigating rotational drift is crucial for accurate analysis of complex protein dynamics.
  • Improvements in hardware and software will further enhance the utility of AFM kymography.