Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate.
Escherichia coli outer membrane protein A (OmpA) is a well-established model for the study of membrane assembly. Previous studies have shown that the essential sequence for outer membrane localization, known as the sorting signal, is contained in a segment of the eighth beta-strand, residues 163-171. Sequential digestion of OmpA, purified from outer membranes or inclusion bodies with cyanogen bromide and Staphylococcus aureus GluC, yielded peptides 162-174(LSLGVSYRFGQGE). Western blot and chemical assays indicated that the peptide was covalently modified by oligo-(R)-3-hydroxybutyrate (cOHB), a flexible, amphipathic oligoester. MALDI/MS was consistent with modification of peptides 162-174 by up to ten R-3-hydroxybutyrate (HB) residues. Western blot analysis of mutants of the peptide, using anti-OHB IgG, indicated that cOHB modification was not inhibited by the single mutations S163G, S167G, Y168F, R169N or R169D; however, cOHB was not detected on peptides containing the double mutations S163G:S167G S163G:V166G, L162G:S167G, and L164G:S167G. MALDI/MS/MS of double mutant S163G:S167G confirmed the absence of cOHB-modification. The results suggest that cOHB may be attached to one or both serines, and point to the importance of the flanking hydrophobic residues. Modification by cOHB may play a role in outer membrane targeting and assembly of OmpA.
Xian
Mo
M
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Fuerst
Michelle M
MM
Shabalin
Yuri
Y
Reusch
Rosetta N
RN
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D006885:Hydroxybutyrates / Q000737:chemistry*; D011091:Polyesters / Q000737:chemistry*; D021382:Protein Sorting Signals; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000094122:Polyhydroxybutyrates
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D006885:Hydroxybutyrates; D011091:Polyesters; D021382:Protein Sorting Signals; C078167:OMPA outer membrane proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates