Unfolding study of a trimeric membrane protein AcrB

Cui Ye, Zhaoshuai Wang, Wei Lu, Yinan Wei

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


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, AcrB Δloop, 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.

Original languageEnglish
Pages (from-to)897-905
Number of pages9
JournalProtein Science
Issue number7
StatePublished - Jul 2014


  • Membrane protein
  • Multi-domain
  • Oligomer
  • Protein folding

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


Dive into the research topics of 'Unfolding study of a trimeric membrane protein AcrB'. Together they form a unique fingerprint.

Cite this