Related Experiment Videos
Hydrophobic core substitutions in calbindin D9k: effects on stability and structure
K Julenius1, E Thulin, S Linse
1Physical Chemistry 2, Chemical Center, Lund University, Sweden. Karin.Julenius@fkem2.Ith.se
Biochemistry
|June 24, 1998
Summary
Hydrophobic core mutations significantly alter calbindin D9k protein stability and unfolding cooperativity. These changes correlate with side-chain surface area alterations but preserve native structure and calcium-binding response.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Engineering
Background:
- Calbindin D9k is a four-helix calcium-binding protein.
- Understanding hydrophobic core mutations is crucial for protein stability and function.
Purpose of the Study:
- Investigate the impact of hydrophobic core mutations on calbindin D9k stability and structure.
- Analyze the relationship between mutation-induced changes and protein stability.
- Assess the effects on unfolding cooperativity and native structure.
Main Methods:
- Site-directed mutagenesis to introduce eleven mutations in the hydrophobic core.
- Circular dichroism spectroscopy to monitor urea and thermal induced unfolding.
- NMR and fluorescence spectroscopy to assess native protein structure.
Main Results:
- Mutations significantly affected protein stability, with free energies of unfolding ranging from 6.6 to 27.4 kJ/mol.
- A strong correlation was observed between the change in side-chain surface area and the change in free energy of unfolding (Delta Delta GNU).
- Despite substantial stability changes, native protein structures and calcium-binding responses remained largely intact.
Conclusions:
- Hydrophobic core mutations in calbindin D9k profoundly influence protein stability and unfolding cooperativity.
- The observed stability changes are quantitatively linked to alterations in side-chain surface area.
- Calbindin D9k maintains its native structure and calcium-binding capabilities even with significant hydrophobic core modifications.