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Updated: Jan 1, 2026
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Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy
Published on: April 28, 2011
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Membrane-Protein Unfolding Intermediates Detected with Enhanced Precision Using a Zigzag Force Ramp
David R Jacobson1, Lyle Uyetake1, Thomas T Perkins2
1JILA, National Institute of Standards and Technology and University of Colorado, Boulder, Colorado.
Biophysical Journal
|December 29, 2019
Summary
This study introduces a novel zigzag force ramp technique using atomic force microscopy to better resolve membrane protein unfolding intermediates. This method reveals more states and longer dwell times, enhancing our understanding of protein stability.
Area of Science:
- Biophysics
- Structural Biology
- Membrane Protein Dynamics
Background:
- Quantifying membrane protein energetics in lipid bilayers is crucial but challenging.
- Atomic force microscopy (AFM) has been used for protein unfolding, but resolving closely spaced intermediates is difficult due to limited trajectory data and noise.
- Previous methods struggled with brief dwell times and instrumental noise, hindering structural assignment of unfolding intermediates.
Purpose of the Study:
- To develop a new atomic force microscopy (AFM) technique for enhanced resolution of membrane protein unfolding intermediates.
- To overcome limitations of traditional constant-velocity force ramps in capturing transient states.
- To investigate the unfolding pathway of bacteriorhodopsin (bR) with its retinal intact.
Main Methods:
- Developed a "zigzag" force ramp protocol using AFM with alternating positive and negative loading rates.
- Applied the zigzag protocol to bacteriorhodopsin (bR) with a site-specific enzymatic cleavage between helices E and F.
- Pulled the E helix using a site-specific, covalent attachment, keeping the retinal associated with the protein during unfolding.
Main Results:
- Zigzag unfolding trajectories revealed 40% more states and longer dwell times compared to constant-velocity methods.
- Identified 31 intermediates during the unfolding of five helices of EF-cleaved bR.
- Resolved a previously reported intermediate into two distinct states and identified a novel intermediate interacting with retinal.
Conclusions:
- The zigzag force ramp technique significantly improves the resolution and dwell times for observing membrane protein unfolding intermediates.
- This enhanced resolution allows for more precise characterization of protein folding pathways and interactions.
- The findings provide new insights into the mechanical stability and functional mechanisms of membrane proteins like bacteriorhodopsin.
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