Springs and Speeds in Free Energy Reconstruction from Irreversible Single-Molecule Pulling Experiments†

2008 ◽  
Vol 112 (19) ◽  
pp. 5892-5897 ◽  
Author(s):  
David D. L. Minh ◽  
J. Andrew McCammon
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Energy Reconstruction

scholarly journals Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor

2018 ◽  
Vol 115 (46) ◽  
pp. 11688-11693 ◽  
Author(s):  
Thomas Suren ◽  
Daniel Rutz ◽  
Patrick Mößmer ◽  
Ulrich Merkel ◽  
Johannes Buchner ◽  
...  
Keyword(s):  
Free Energy ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Load ◽  
Force Spectroscopy ◽  
Folding Pathway ◽  
Hormone Binding ◽  
Single Molecule Force Spectroscopy

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a “lid” structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of 41 kBT (24 kcal/mol). The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional 12 kBT of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s−1. From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

scholarly journals Slow domain reconfiguration causes power-law kinetics in a two-state enzyme

2018 ◽  
Vol 115 (3) ◽  
pp. 513-518 ◽  
Author(s):  
Iris Grossman-Haham ◽  
Gabriel Rosenblum ◽  
Trishool Namani ◽  
Hagen Hofmann
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Power Law ◽  
Energy Landscape ◽  
Rate Equations ◽  
Free Energy Landscape ◽  
Closed Domain ◽  
Single Molecule Fret ◽  
Power Law Kinetics ◽  
Interdomain Interactions

Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.

scholarly journals Free Energy of Membrane Protein Unfolding Derived from Single-Molecule Force Measurements

2007 ◽  
Vol 93 (3) ◽  
pp. 930-937 ◽  
Author(s):  
Johannes Preiner ◽  
Harald Janovjak ◽  
Christian Rankl ◽  
Helene Knaus ◽  
David A. Cisneros ◽  
...  
Keyword(s):  
Free Energy ◽  
Membrane Protein ◽  
Single Molecule ◽  
Protein Unfolding ◽  
Force Measurements

scholarly journals Extraction of Free Energy Parameters from Dynamic Single-Molecule Force-Extension Measurements

2012 ◽  
Vol 102 (3) ◽  
pp. 174a
Author(s):  
Dario Meluzzi ◽  
Arijit Maitra ◽  
Gaurav Arya
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Force Extension ◽  
Energy Parameters
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