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Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules
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Thermodynamic signatures in macromolecular interactions involving conformational flexibility
Biological Chemistry
|July 9, 2014
Summary
Investigating trypsin variants revealed that protein flexibility significantly impacts binding energetics. Differences in enthalpy and entropy drive interactions, with solvent effects playing a key role in binding affinities.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Dynamics
Background:
- Macromolecular interactions are governed by complex energetics, especially when protein flexibility is a factor.
- Understanding these energetics is crucial for fields ranging from drug discovery to enzyme engineering.
Purpose of the Study:
- To analyze the enthalpic and entropic contributions to binding energetics in closely related trypsin variants.
- To investigate the role of protein plasticity in modulating these contributions during interactions with L45K-eglin C.
Main Methods:
- Utilized isothermal titration calorimetry to measure binding energetics (enthalpy and entropy).
- Employed X-ray crystallography to determine the structures of protein-inhibitor complexes.
- Analyzed differences in plasticity among four trypsin variants.
Main Results:
- Observed significant differences in released heat (enthalpy) among trypsin variants, despite minimal variation in binding affinities.
- Demonstrated enthalpy-entropy compensation, a common principle in molecular recognition.
- Found that the most flexible trypsin variant's binding was predominantly enthalpically driven, with negligible entropic change.
- Structural analysis revealed minimal differences in protein-inhibitor contacts across variants.
Conclusions:
- Protein flexibility, specifically disorder, significantly influences the energetic contributions (enthalpy and entropy) to binding.
- Enthalpy-entropy compensation is a key feature in the binding of these trypsin variants.
- Solvent effects are a major determinant of binding affinities, compensating for subtle changes in protein structure and flexibility.
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