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Resolving the native conformation of Escherichia coli OmpA
Author(s) -
Negoda Alexander,
Negoda Elena,
Reusch Rosetta N.
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07823.x
Subject(s) - periplasmic space , dsba , bacterial outer membrane , chemistry , lipid bilayer , crystallography , protein folding , folding (dsp implementation) , covalent bond , native state , stereochemistry , escherichia coli , membrane , biophysics , biochemistry , biology , organic chemistry , electrical engineering , gene , engineering
The native conformation of the 325‐residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow‐pore, two‐domain structure has vied with a large‐pore, single‐domain structure. Our recent studies show that Ser163 and Ser167 of the N‐terminal domain (1–170) are modified in the cytoplasm by covalent attachment of oligo‐( R )‐3‐hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N‐terminal domain to be incorporated into planar lipid bilayers as narrow pores (∼ 80 pS, 1 m KCl, 22 °C). Here, we examined the potential effect(s) of periplasmic modifications on pore structure by comparing OmpA isolated from outer membranes (M‐OmpA) with OmpA isolated from cytoplasmic inclusion bodies (I‐OmpA). Chemical and western blot analysis and 1 H‐NMR showed that segment 264–325 in M‐OmpA, but not in I‐OmpA, is modified by cOHBs. Moreover, a disulfide bond is formed between Cys290 and Cys302 by the periplasmic enzyme DsbA . Planar lipid bilayer studies indicated that narrow pores formed by M‐OmpA undergo a temperature‐induced transition into stable large pores (∼ 450 pS, 1 m KCl, 22 °C) [energy of activation ( E a ) = 33.2 kcal·mol −1 ], but this transition does not occur with I‐OmpA or with M‐OmpA that has been exposed to disulfide bond‐reducing agents. The results suggest that the narrow pore is a folding intermediate, and demonstrate the decisive roles of cOHB‐modification, disulfide bond formation and temperature in folding OmpA into its native large‐pore configuration.