Barriers to Folding of the Transmembrane Domain of the Escherichia coli Autotransporter Adhesin Involved in Diffuse Adherence
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- 作者：Jesper E. Mogensen ; Damini Tapadar ; M. Alexander Schmidt ; and Daniel E. Otzen
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：March 22, 2005
- 年：2005
- 卷：44
- 期：11
- 页码：4533 - 4545
- 全文大小：297K
- 年卷期：v.44,no.11(March 22, 2005)
- ISSN：1520-4995
文摘
Adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers thediffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 aminoacid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid 
-domain integrated in theouter membrane. The -domain consists of two parts: the 1-domain, which is predicted to form two-strands on the bacterial cell surface, and the 2-domain, which constitutes the transmembrane domain.We here present a detailed biophysical analysis of the AIDA -domain addressing its refolding propertiesand its different conformational states and their stability. We find that the 2-domain in solution can foldonly when the 1-domain is present and only with 50% efficiency. However, 100% refolding of the 2-domain, with or without the 1-domain, can be achieved in the presence of a solid support. Folding canonly take place above the cmc of the detergent used, but the refolded state is retained if diluted below thecmc, revealing a kinetic barrier to dissociation of the detergent molecules from the folded protein. Refoldingattempts of the 2-domain in the absence of a solid support result in the formation of an oligomericmisfolded state both in the absence and in the presence of detergent. Despite being misfolded, thesestates unfold cooperatively with a Tm 
70 
C. The refolded protein in the nonionic detergentoctylpolyoxyethylene (oPOE) can only be thermally unfolded in the presence of SDS. The linear relationshipbetween SDS mole fraction and unfolding temperature, Tm, predicts a Tm of 112.9 ± 1.2 C for the 2-domain and 132.7 ± 12.2 C for the entire -domain in pure oPOE. Thus, the 1-domain also stabilizesthe 2-domain. In conclusion, our data show that the in vitro refolding of the AIDA -domain is criticallydependent on a solid support, suggesting that in vivo specific biological factors may assist in folding theprotein correctly into the outer membrane to avoid the formation of stably misfolded conformations.
-domain integrated in theouter membrane. The -domain consists of two parts: the 1-domain, which is predicted to form two-strands on the bacterial cell surface, and the 2-domain, which constitutes the transmembrane domain.We here present a detailed biophysical analysis of the AIDA -domain addressing its refolding propertiesand its different conformational states and their stability. We find that the 2-domain in solution can foldonly when the 1-domain is present and only with 50% efficiency. However, 100% refolding of the 2-domain, with or without the 1-domain, can be achieved in the presence of a solid support. Folding canonly take place above the cmc of the detergent used, but the refolded state is retained if diluted below thecmc, revealing a kinetic barrier to dissociation of the detergent molecules from the folded protein. Refoldingattempts of the 2-domain in the absence of a solid support result in the formation of an oligomericmisfolded state both in the absence and in the presence of detergent. Despite being misfolded, thesestates unfold cooperatively with a Tm 70 C. The refolded protein in the nonionic detergentoctylpolyoxyethylene (oPOE) can only be thermally unfolded in the presence of SDS. The linear relationshipbetween SDS mole fraction and unfolding temperature, Tm, predicts a Tm of 112.9 ± 1.2 C for the 2-domain and 132.7 ± 12.2 C for the entire -domain in pure oPOE. Thus, the 1-domain also stabilizesthe 2-domain. In conclusion, our data show that the in vitro refolding of the AIDA -domain is criticallydependent on a solid support, suggesting that in vivo specific biological factors may assist in folding theprotein correctly into the outer membrane to avoid the formation of stably misfolded conformations.