Unfolding study of a trimeric membrane protein AcrB

Area of Science:

• Structural Biology
• Biochemistry
• Membrane Protein Folding

Background:

• Escherichia coli inner membrane protein AcrB is a multi-domain, trimeric α-helical protein with both transmembrane and periplasmic domains.
• Understanding the folding and unfolding mechanisms of such complex membrane proteins is crucial for deciphering their function and potential therapeutic targeting.

Purpose of the Study:

• To investigate the folding and unfolding pathways of the trimeric α-helical membrane protein, AcrB.
• To elucidate the contributions of different domains to the overall unfolding process and the role of trimer association in refolding.

Main Methods:

• Protein unfolding was monitored using intrinsic fluorescence and circular dichroism (CD) spectroscopy in the presence of sodium dodecyl sulfate (SDS) and urea.
• A triple mutant was created to differentiate domain contributions to the unfolding signal.
• Refolding of a monomeric mutant (AcrBΔloop) from an unfolded state was assessed.

Main Results:

• SDS denaturation followed a two-state unfolding model, with signals primarily originating from the soluble domain.
• Trimer association strength had minimal impact on the unfolding profile, indicating trimer dissociation is not rate-limiting.
• Unfolding was irreversible under experimental conditions; refolding of secondary structure in monomers was achievable, but complete refolding and re-association were limited.

Conclusions:

• AcrB unfolding initiates with local structural rearrangements.
• While individual monomer refolding is possible, the re-association of the trimer is a significant bottleneck for achieving fully folded wild-type AcrB.
• The study provides insights into the complex folding dynamics of multi-domain membrane proteins.