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Updated: Jul 3, 2026
13:26
Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Published on: September 13, 2014
Direct analysis of cooperativity in multisubunit allosteric proteins
Nitzan Zandany1, Maya Ovadia, Irit Orr
1Department of Life Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
Summary
This study introduces a novel method to directly analyze protein cooperativity by examining subunit interactions. The findings provide evidence for a concerted activation mechanism in voltage-activated potassium channels (Kv).
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Allosteric regulation is crucial for cellular processes, often involving ligand-induced conformational changes in multimeric proteins.
- Cooperativity in protein function arises from changes in intersubunit interactions during ligand binding.
- Existing mutagenesis methods for homooligomeric proteins offer indirect insights into allosteric mechanisms.
Purpose of the Study:
- To develop a general strategy for the direct analysis of cooperativity in multisubunit proteins.
- To differentiate between concerted and sequential conformational transitions underlying protein function.
- To investigate the role of high-order intersubunit interactions in protein cooperativity.
Main Methods:
- Combining measurements of all possible combinations of mutated subunits.
- Employing high-order double-mutant cycle-coupling analysis to assess intersubunit interaction hierarchy.
- Applying the strategy to voltage-activated potassium channel (Kv) proteins.
Main Results:
- The developed strategy allows for direct analysis of cooperativity and intersubunit interactions.
- High-order intersubunit coupling patterns can distinguish between concerted and sequential conformational transitions.
- Compelling evidence for a concerted, all-or-none activation gate opening mechanism in Kv channel pore domains was obtained.
Conclusions:
- This work presents a powerful new approach for dissecting allosteric mechanisms in multisubunit proteins.
- The findings elucidate the specific activation mechanism of voltage-activated potassium channels.
- Direct analysis of high-order intersubunit interactions provides unprecedented detail on protein cooperativity.
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