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Unfolding study of a trimeric membrane protein AcrB
Cui Ye1, Zhaoshuai Wang, Wei Lu
1Department of Chemistry, University of Kentucky, Lexington, Kentucky, 40506.
Insights
Investigating the folding of Escherichia coli inner membrane protein AcrB, researchers found that while individual monomers can refold secondary structures, trimer re-association limits the overall folding process for this complex protein.
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.
Abstract:
The folding of a multi-domain trimeric α-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop , from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB.
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