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The Functional And Mechanistic Study Of BamA In Outer Membrane Protein Biogenesis

Posted on:2020-12-01Degree:DoctorType:Dissertation
Country:ChinaCandidate:X D MaFull Text:PDF
GTID:1480306185496214Subject:Biochemistry and Molecular Biology
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Unlike the a-helical integrated membrane proteins that can be found in almost every known cell membrane,the b-barrel integrated membrane proteins can be only found in the outer membrane of Gram-negative bacteria,mitochondria and chloroplasts.The b-barrel integrated membrane proteins belong to Omp85/TPS(outer-membrane protein of 85 kDa /two-partner secretion)superfamily.Members of Omp85/TPS superfamily are folded and inserted into outer membrane by utilizing a universal BAM(b-barrel assembly machinery)complex,whose core component is BamA.Recent researches have manifested the significance of BAM complex in membrane integrity,function and cell survival.However,the mechanism remains unknown.In this paper,we dedicated our efforts to the research of the POTRA domains of BamA from Haemophilus influenzae.We successfully aquired the crystal structures of POTRA1-4,POTRA3-5 and POTRA4 by means of X-ray crystallography,respectively.Based on superimposition,SAXS,MD simulation and BLI,it was found that POTRA1-5had significant flexibility that was important for BAM machinery function.In the meantime,we verified that POTRA domains from N-to C-terminus showed increased binding activity towards substrates of OMPs by using ELISA.The mutants targeting the relatively conservative a2 and b2 of POTRA5 were also determined to impair the binding activity towards substrates.Futhermore,the complementary assay and biofilm formation assay presented that poly-Ala mutation of all a2 helices inhibited the viability of bacteria and biofilm formation of mutants.In summary,we speculated that the relatively conservative a2-b2 edges formed a spiraled cavity as the putative OMP travelling pathway and the POTRA flexibility would play a role in OMP biogenesis.Our results offer a new insight into the mechanism of the flexibility-mediated substrates recruitment of POTRA.
Keywords/Search Tags:BAM complex, POTRA domains, substrate recruitment, POTRA flexibility
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