Cell-free synthesis and folding of transmembrane OmpA reveals higher order structures and premature truncations.
We use a cell-free transcription-translation system to monitor the effect of different lipids on the synthesis and folding of the transmembrane domain of the outer membrane protein OmpA from E. coli under physiological conditions. Folding is consistent with previous observations made in vitro at high pH. Synthesis and folding yields are optimal in phosphocholine lipids, particularly in short chain lipids and small vesicles, while lipid rafts do not promote folding compared to the folding in the absence of lipids. Truncated species are observed during translation in the presence of the periplasmic chaperone Skp, which likely binds to the newly synthesized polypeptide chain during cell-free translation and thus prematurely terminate polypeptide chain synthesis. In contrast, folded and unfolded dimers of OmpA correlate negatively with folding yields. This suggests that dimer formation competes with folding and insertion of monomeric OmpA, though folded dimers slowly appear to convert to folded monomers.
Debnath
Dilip K
DK
Center for insoluble protein structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C., Denmark.
Otzen
Daniel E
DE
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D010713:Phosphatidylcholines / Q000737:chemistry; D017510:Protein Folding; D053835:Unilamellar Liposomes / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010713:Phosphatidylcholines; D053835:Unilamellar Liposomes; C072721:Skp protein, E coli; C078167:OMPA outer membrane proteins; C490900:SurA protein, E coli; D019696:Peptidylprolyl Isomerase; C017251:1,2-oleoylphosphatidylcholine