This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator. The analysis of outer membrane protein folding in an E.coli mutant defective in lipid export was the focal point of this study. Following biosynthesis in the cytoplasm, proteins destined for the E.coli outer membrane (OM) are transported across the inner membrane (IM) by the Sec machinery. Once in the periplasm, they are folded into their correct three dimensional structures by a number of molecular chaperones. Here, we present data on the folding of outer membrane proteins OmpA, OmpC and OmpF in the temperature-sensitive mutant of MsbA that is defective in export of LPS and phospholipids to the OM under nonpermissive conditions. W3110 (wildtype) and WD2 (MsbA mutant) were grown at both permissive (30 C) and nonpermissive (44 C) temperatures and analyzed for levels of unfolded OmpA, OmpC, and OmpF using pulse-chase labeling and immunoprecipitation. We found that there is detectable increase in unfolded OmpA in MsbA mutant WD2 during growth under nonpermissive conditions. We were able to detect OmpC and OmpF as well, but the levels of unfolded OmpC and OmpF were not high enough to detect a significant difference between strains. Further study involving more purified antibodies and modified technique is required to conclude the significance of LPS in the folding of OmpC and OmpF. Through this study we can safely conclude that proper folding of outer membrane protein OmpA in the periplasm requires LPS and or phospholipids.
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