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Biochemistry. 2005 Mar 22;44(11):4533-45.

Barriers to folding of the transmembrane domain of the Escherichia coli autotransporter adhesin involved in diffuse adherence.

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Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.


Adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid beta-domain integrated in the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predicted to form two beta-strands on the bacterial cell surface, and the beta(2)-domain, which constitutes the transmembrane domain. We here present a detailed biophysical analysis of the AIDA beta-domain addressing its refolding properties and its different conformational states and their stability. We find that the beta(2)-domain in solution can fold only when the beta(1)-domain is present and only with 50% efficiency. However, 100% refolding of the beta(2)-domain, with or without the beta(1)-domain, can be achieved in the presence of a solid support. Folding can only take place above the cmc of the detergent used, but the refolded state is retained if diluted below the cmc, revealing a kinetic barrier to dissociation of the detergent molecules from the folded protein. Refolding attempts of the beta(2)-domain in the absence of a solid support result in the formation of an oligomeric misfolded state both in the absence and in the presence of detergent. Despite being misfolded, these states unfold cooperatively with a T(m) approximately 70 degrees C. The refolded protein in the nonionic detergent octylpolyoxyethylene (oPOE) can only be thermally unfolded in the presence of SDS. The linear relationship between SDS mole fraction and unfolding temperature, T(m), predicts a T(m) of 112.9 +/- 1.2 degrees C for the beta(2)-domain and 132.7 +/- 12.2 degrees C for the entire beta-domain in pure oPOE. Thus, the beta(1)-domain also stabilizes the beta(2)-domain. In conclusion, our data show that the in vitro refolding of the AIDA beta-domain is critically dependent on a solid support, suggesting that in vivo specific biological factors may assist in folding the protein correctly into the outer membrane to avoid the formation of stably misfolded conformations.

[Indexed for MEDLINE]

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