TAILIEUCHUNG - Báo cáo khoa học: Resolving the native conformation ofEscherichia coli OmpA

The native conformation of the 325-residue outer membrane protein A (OmpA) of Escherichia colihas been a matter of contention. A narrow-pore, two-domain structure has vied with a large-pore, single-domain struc-ture. 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 | ỊFEBS Journal Resolving the native conformation of Escherichia coli OmpA Alexander Negoda Elena Negoda and Rosetta N. Reusch Department of Microbiology and Molecular Genetics Michigan State University East Lansing MI USA Keywords cOHB-modification disulfide bond outer membrane protein protein folding protein targeting Correspondence R. N. Reusch Department of Microbiology and Molecular Genetics Michigan State University East Lansing MI 48824 USA Fax 1 517 353 8957 Tel 1 517 884 5388 E-mail rnreusch@ Received 7 July 2010 revised 17 August 2010 accepted 20 August 2010 doi The native conformation of the 325-residue outer membrane protein A OmpA of Escherichia coli has been a matter of contention. A narrowpore 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 1H-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 Ea 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

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