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Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1
Published on: September 18, 2019
Hidden complexity in the isomerization dynamics of Holliday junctions
Changbong Hyeon1, Jinwoo Lee, Jeseong Yoon
1School of Computational Sciences, Korea Institute for Advanced Study, Seoul 130-722, Korea. hyeoncb@kias.re.kr
Nature Chemistry
|October 24, 2012
Summary
Biomolecules can adopt multiple functional states. This study quantifies heterogeneous dynamics in Holliday junctions using single-molecule fluorescence resonance energy transfer (smFRET), revealing distinct, kinetically isolated conformational clusters.
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Biomolecules may possess multiple functionally competent folded states due to complex energy landscapes.
- Recent single-molecule experiments reveal molecule-to-molecule variations in enzyme and ribozyme folding dynamics, but lack quantitative analysis and structural insights.
- Holliday junctions are DNA structures with potential for complex folding dynamics.
Purpose of the Study:
- To develop a quantitative method for analyzing heterogeneous dynamics in biomolecules.
- To investigate the isomerization dynamics of Holliday junctions using single-molecule fluorescence resonance energy transfer (smFRET).
- To understand the structural origins of observed dynamic heterogeneity.
Main Methods:
- Application of concepts from glass physics to analyze single-molecule data.
- Utilizing complementary clustering analysis on smFRET data.
- Probing isomerization dynamics over extended observation times (approximately 40 seconds).
Main Results:
- Demonstrated that the ergodicity of Holliday junction dynamics is broken.
- Showed that the conformational space of Holliday junctions is partitioned into kinetically disconnected clusters.
- Identified internal multiloops of varying sizes and flexibilities, stabilized by Mg(2+) ions, as the likely cause of persistent heterogeneity.
Conclusions:
- Holliday junction dynamics exhibit persistent heterogeneity, characterized by kinetically disconnected conformational states.
- The observed heterogeneity is attributed to structural features like multiloops influenced by magnesium ions.
- Experimental manipulation with Mg(2+) pulses confirmed the ability to interconvert between different dynamic patterns, supporting the proposed structural basis.
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