Browsing by Subject "Heterologe Genexpression"

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Tth-IM60, eine Membraninsertase aus Thermus thermophilus
(2011) Meyer, Susanne H.; Kuhn, Andreas
The evolutionarily conserved YidC/Oxa1/Alb3 family of proteins catalyzes the insertion of integral membrane proteins in bacteria, mitochondria, and chloroplasts. In this work Tth-IM60 from Thermus thermophilus was identified as a member of this family. The function and structure of the protein was analysed in detail. Complementation studies in a Escherichia coli YidC-depletion strain show a functional replacement of the essential YidC by Tth-IM60 in vivo. A heterologous expression of the his-tagged Tth-IM60 protein was achieved in the E. coli strain C43 and pMS as a plasmid vector. It was shown that Tth-IM60 protein is located in the inner membrane probably in a dimeric state. After purification the protein tends to oligomerize in a higher, but very stable oligomeric state. The Tth-IM60 oligomeric protein was stable for 50 days at least. By size-exclusion chromatography the zwitterionic detergent LDAO and a buffer containing 20 mM TrisHCl pH 8,5, 500 mM NaCl and 10 % glycerol were identified as the best conditions for purification and stability. With this buffer, Tth-IM60 was purified as a dimer via its C-terminal histag by two Ni-IMACs (immobilized metal affinity chromatography) and a size-exclusion chromatography. A final protein concentration up to 10 mg/ml was feasible. The purified Tth-IM60 protein was used for functional and structural studies. A weak binding of Tth-IM60 to the first periplasmic loop of SecF as shown by pulldown assays, suggests a Sec-dependent function of Tth-IM60. In addition, the essential Sec-independent function of Tth-IM60 was demonstrated by the translocation of the YidC substrate Pf3 coat into Tth-IM60 proteoliposomes. Moreover, the results of the single molecule spectroscopy measurements implicate that the translocation of Pf3 coat proteins occurs with a fast kinetics within 5 minutes. The secondary structure of the Tth-IM60 protein was analysed by circular dichroism spectroscopy: 49 ? 55 % alpha-helical, 13 - 14 % beta-sheet, 14 ? 17 % beta-turns and 18 - 21% unordered structures were calculated. Tth-IM60 comprises more alpha-helical structures, but less beta-sheets than YidC, probably because of the first periplasmic loop of Tth-IM60 being shorter. The melting point of Tth-IM60 was determined to 68 °C, which is 10 degrees higher than the melting point of YidC. Furthermore, the Tth-IM60 protein was crystallized and X-ray analysis was performed. However, due to the low resolution the structure of the Tth-IM60 protein could not be determined so far. The second part of this thesis concerns the ?translocating chain-associated membrane? (TRAM) protein. The TRAM protein is involved in the insertion of integral membrane proteins of the endoplasmic reticulum (ER) together with the Sec61-complex. In contrast to the Sec-components, no homologous YidC protein exists in the ER-membrane. Therefore, it was postulated that TRAM and YidC could have functional similarities. For functional studies the TRAM protein from X. laevis was expressed in E. coli as a fusion protein with a N-terminal MBP (maltose-binding protein) and a C-terminal histag. TRAM was purified via two different affinity matrices: Ni sepharose and an amylose resin. It was possible to reconstitute the fusion protein into liposomes. However a translocation of the YidC-substrate Pf3 coat into these proteoliposomes was not detectable. In addition, complementation studies with a YidC-depletion strain did not show that the essential YidC function can be replaced by TRAM in vivo.