Related Experiment Video
Updated: Jul 9, 2026
10:23
Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules
Published on: April 25, 2025
Axis-dependent anisotropy in protein unfolding from integrated nonequilibrium single-molecule experiments, analysis,
Rene A Nome1, Jason Ming Zhao, Wouter D Hoff
1Department of Chemistry, Institute for Biophysical Dynamics, 929 East 57th Street, University of Chicago, Chicago, IL 60637, USA.
Summary
Protein unfolding mechanisms were studied using nonequilibrium techniques. Results reveal distinct unfolding pathways and challenge the idea that cooperative unfolding is universal in protein stability.
Area of Science:
- Biophysics
- Computational Biology
- Protein Dynamics
Background:
- Protein stability is crucial for biological function.
- Understanding protein unfolding mechanisms is key to deciphering protein dynamics and misfolding diseases.
- Nonequilibrium techniques offer powerful tools to probe complex energetic landscapes.
Purpose of the Study:
- To elucidate protein unfolding mechanisms by mapping energy landscapes.
- To investigate energetic and mechanistic anisotropy in protein unfolding.
- To challenge the universality of cooperative unfolding in protein stability.
Main Methods:
- Integrating experimental and theoretical nonequilibrium techniques.
- Single-molecule force-extension experiments on photoactive yellow protein along defined axes.
- Nonequilibrium statistical mechanical analysis and atomistic simulations.
- Steered molecular dynamics (SMD) simulations and Jarzynski-Hummer-Szabo (JHS) analysis.
Main Results:
- Energetic and mechanistic anisotropy observed in protein unfolding along different pulling axes.
- One axis showed a transition-state-like unfolding with simultaneous hydrogen bond breakage.
- The other axis exhibited a noncooperative unfolding with a constant force profile.
- High agreement between SMD simulations and JHS analysis of experimental data.
Conclusions:
- Protein unfolding pathways are anisotropic and depend on the pulling axis.
- Cooperative unfolding is not a universal feature of protein stability.
- The study reveals a rich energy landscape extending to the fully unfolded state.
Related Concept Videos
01:25Protein Folding
Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation, critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.
Protein Structure Is Critical to Its Biological Function
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing...
Protein Structure Is Critical to Its Biological Function
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing...
01:22Protein Folding
Overview
01:22Protein Folding
Overview
03:00Molecular Chaperones and Protein Folding
The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...
The...