title: Effect of crowding by Ficolls on OmpA and OmpT refolding and membrane insertion.
abstract: Folding of outer membrane proteins (OMPs) has been studied extensively in vitro. However, most of these studies have been conducted in dilute buffer solution, which is different from the crowded environment in the cell periplasm, where the folding and membrane insertion of OMPs actually occur. Using OmpA and OmpT as model proteins and Ficoll 70 as the crowding agent, here we investigated the effect of the macromolecular crowding condition on OMP membrane insertion. We found that the presence of Ficoll 70 significantly slowed down the rate of membrane insertion of OmpA while had little effect on those of OmpT. To investigate if the soluble domain of OmpA slowed down membrane insertion in the presence of the crowding agent, we created a truncated OmpA construct that contains only the transmembrane domain (OmpA171). In the absence of crowding agent, OmpA171 refolded at a similar rate as OmpA, although with decreased efficiency. However, under the crowding condition, OmpA171 refolded significantly faster than OmpA. Our results suggest that the periplasmic domain slows down the rate, while improves the efficiency, of OmpA folding and membrane insertion under the crowding condition. Such an effect was not obvious when refolding was studied in buffer solution in the absence of crowding.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
authors.forename: Cui
authors.initials: C
authors.lastname: Ye
authors.forename: Qian
authors.initials: Q
authors.lastname: Chai
authors.forename: Meng
authors.initials: M
authors.lastname: Zhong
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D048168:Escherichia coli K12 / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005362:Ficoll / Q000737:chemistry* / Q000494:pharmacology; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058849:Protein Refolding / Q000187:drug effects; D013997:Time Factors
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C499588:ompT protein, E coli; C078167:OMPA outer membrane proteins; D005362:Ficoll; D010447:Peptide Hydrolases