Dissecting The Mechanism Of Periplasmic Chaperone FkpA At Different Temperatures In Escherichia Coli

?-barrel outer membrane proteins(OMPs)are widely present in the outer membranes of Gram-negative bacteria,mitochondria and chloroplasts.They are involved in a variety of biological processes(e.g.,nutrient uptake and protein secretion).The biogenesis of ?-barrel OMPs in Gram-negative bacteria is a complicated process.First,?-barrel OMPs are translocated across the inner membrane after they are biosynthesized by the ribosome in the cytosol.After translocation,the nascent polypeptides of OMPs are then transported through the periplasmic space by periplasmic quality control factors such as SurA,Skp,DegP,FkpA and/or PpiD.Under the assistance of the BAM complex,?-barrel OMPs are finally folded,assembled and inserted into the outer membrane.The periplasmic quality control factor FkpA of E.coli belongs to the peptide acyl prolyl cis-trans isomerase family(PPIase)and exhibits molecular chaperone activity,however,its biological function in cells is unclear at present.In this study,we investigated the interaction of the periplasmic chaperone FkpA with its substrates and functional partners in Escherichia coli by utilizing chemical crosslinking and site-directed in vivo photo-crosslinking.We constructed a total of 29 unnatural amino acid-incorporated FkpA variant proteins,and the interaction between FkpA and its substrate proteins at different temperatures was analyzed.FkpA-interacting proteins were identified through a combination of denaturing affinity chromatography,SDS-PAGE separation and Q-TOF mass spectrometry analysis.And also,a double-labeled protein antibody screening method was used to verify the presence of FkpA dimers in living cells.The results show that FkpA functions as a dimer in living cells in Escherichia coli and FkpA interacts with its substrates or function partners mainly via the C domain.In addition,the interaction between FkpA and periplasmic chaperone PpiD was discovered in this study,and such interaction seems to be dependent on temperature.It is speculated that FkpA and PpiD function in a cooperative way and FkpA plays a more important role for outer membrane protein biogenesis under heat shock conditions.These results provide useful insights into the function and mechanism of FkpA in living cells.