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Towards the structure of proteins involved in membrane transport processes : NptA, PulG and SycD/YopB/YopD

Towards the structure of proteins involved in membrane transport processes : NptA, PulG and SycD/YopB/YopD

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SCHÄFER, Karsten, 2009. Towards the structure of proteins involved in membrane transport processes : NptA, PulG and SycD/YopB/YopD [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Schafer2009Towar-6767, title={Towards the structure of proteins involved in membrane transport processes : NptA, PulG and SycD/YopB/YopD}, year={2009}, author={Schäfer, Karsten}, address={Konstanz}, school={Universität Konstanz} }

The work presented here was focussed on the expression, purification and crystallization of proteins that are involved in transport processes across bacterial membranes: NptA from Vibrio cholerae, PulG from Klebsiella oxytoca and SycD/YopB/YopD from Yersinia enterocolitica. Sodium dependent phosphate cotransporters (NaPi II's) have been known for a long time from higher vertebrates where they play an important role in phosphate homoeostasis and bone metabolism. Due to their substantial physiological and medical relevance they are functionally well characterized. Despite the obvious need for a high resolution structure, NaPi II's were so far not amenable to structural investigations since it was not possible to express and purify them in sufficient amounts. Chapter 2 describes the recombinant expression of NptA, a recently discovered sequence and functional homologue from the Gram<sup>-</sup> bacterium V. cholerae. Extensive screenings for suitable solubilization conditions and optimization of the purification yielded ~2 mg His-tagged NptA from 5 l Escherichia coli culture, but only in the form of inhomogeneous, large oligomers/aggregates inappropriate for crystallization. Attempts to prepare these aggregates into a homogeneous solution of mono- or small oligomers failed. During the course of this work, further NaPi II homologues were identified in the genomes of numerous other bacteria. Ten genes were selected from databases and the E. coli optimized sequences were inserted into a pET vector for expression. p<sup>32</sup>-phosphate uptake measurements revealed an increased sodium dependent uptake in E. coli C43 for eight of the constructs, indicating functional expression. Seven transporters showed a clear signal on western blots when tested for their expression. These new expression systems are the basis for further attempts to reach the ultimate goal of a high resolution crystal structure. The pseudopilin PulG is an essential component of the type II pullulanase secretion machinery of the Gram<sup>-</sup> bacterium K. oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins where these residues form a long alpha-helical spine that protrudes from a globular head domain. Chapter 3 presents the crystal structure of truncated PulG lacking the N-terminal hydrophobic region --- the first structure of a type II pseudopilin. The obtained crystals belonged to space group P6<sub>5</sub>22 and diffracted to a 1.6 A resolution. The model was refined to a final R-factor of 16.7% (R<sub>free</sub> = 19.8%). Truncated PulG was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize the type IV pilins, even though sequence similarity is missing in the crystallized head domain. The highly variable loop region with a disulfide bond which is found in the type IV pilins is not present in the PulG structure. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The PulG structure was used to build an atomic model of the pseudopilus assembly by fitting PulG monomers into data obtained by cryo electron microscopy and mass spectrometry analyses of pilus filaments. The complex type III protein secretion machinery of Y. enterocolitica directly injects bacterial effectors into immune cells of mammalian hosts. Two hydrophobic components, YopD and YopB, have one, respectively two predicted transmembrane helices and form the translocation pore in the host cell membrane. Targeting of these translocators depends on the presence of their specific chaperone SycD in the bacterial cytoplasm where it prevents the presecretional aggregation of YopB and YopD. With the goal to determine the three-dimensional structures of the chaperone and its complexes with the translocators, His-tagged SycD was recombinantly expressed in E. coli, either alone or together with YopB and/or YopD as described in chapter 4. Extensive crystallization experiments with the purified His-SycD dimer and a homogeneous His-SycD/YopD complex (most probably composed of 2 + 2 monomers) did not produce crystals --- potentially due to the sensitivity of His-SycD to oxidation at one or more of the four cysteine sites. Thus, different combinations of cysteine versus alanine mutations were introduced in His-SycD. One double mutant His-SycD<sup>(C113A, C164A)</sup> exhibited the same behavior in respect to YopD binding but was not sensitive to oxidation. This mutant is a further promising candidate for crystallization experiments of the chaperone and the chaperone/YopD complex. Versuche zur Strukturbestimmung von Proteinen mit Beteiligung an Membrantransportprozessen : NptA, PulG und SycD/YopB/YopD Schäfer, Karsten Schäfer, Karsten Towards the structure of proteins involved in membrane transport processes : NptA, PulG and SycD/YopB/YopD terms-of-use application/pdf 2011-03-24T17:29:02Z 2009 eng 2011-03-24T17:29:02Z

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