Properties and crystal structure of a beta-barrel folding mutant

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• The rat intestinal fatty acid binding protein (IFABP) is a beta-barrel protein involved in lipid metabolism.
• Protein stability is crucial for function, and mutations can alter it in unpredictable ways.
• A V60N mutation was predicted to enhance IFABP stability via a novel hydrogen bond.

Purpose of the Study:

• To investigate the effect of the V60N mutation on the stability and folding kinetics of IFABP.
• To reconcile the predicted stability enhancement with experimental observations.

Main Methods:

• Equilibrium denaturation studies using Circular Dichroism (CD) and fluorescence spectroscopy.
• Kinetic analysis of protein unfolding and refolding.
• Structural analysis of the V60N mutant protein.

Main Results:

• The V60N mutant exhibited decreased stability compared to wild-type IFABP, contradicting initial predictions.
• Both mutant and wild-type proteins showed reversible, two-state folding transitions.
• The V60N mutant displayed significantly faster rates of denaturation and renaturation.
• Structural analysis revealed minimal changes, but differences in solvent accessible surface and crystallographic displacement factors suggested entropic destabilization.

Conclusions:

• The V60N mutation leads to entropic destabilization of IFABP, overriding the predicted enthalpic stabilization from the new hydrogen bond.
• Folding intermediates lack secondary structure, and the mutant exhibits complex folding kinetics with an additional slow folding phase.
• Experimental evidence contradicts computational prediction, highlighting the complexity of predicting protein stability changes.