Analyse der molekularen Dynamik der Nukleotid-bindenden Domäne des ABC-Transporters HlyB mittels NMR
Zusammenfassung der Projektergebnisse
The aim of the project was the analysis of the molecular dynamics of the NBD of the ABC transporter by liquid NMR. We recorded the spectra of the 15N-labeled NBD and started the sequence assignment procedure. However, we realized soon that the concentration of the protein was not sufficient to obtain the assignment. Therefore we followed different approaches to solve this problem. This included an intensive screen for conditions that would stabilize the protein, 3D-HNCA spectra, cell-free expression and selective labeling. However, none of the approaches was successful. Subsequently, we triple-labeled the protein, but for unknown reason stability was reduced and the concentrations at which the NBD remained in the solution was even lower. Thus, we could obtain an assignment that would have enabled us to extract the molecular dynamics of the protein in presence of different nucleotides or the substrate. In parallel, we developed an easy-to-use assay that allowed us to optimize the detergent concentration of samples of full-length HlyB. However, to this end no conditions could be established that would enable us to record liquid NMR spectra of this membrane protein. Also analysis of the substrate of the ABC transporter, HlyA, revealed a dual function of the C-terminus in secretion and proper folding of HlyA.
Projektbezogene Publikationen (Auswahl)
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(2009) "ABC transporters - a smart example of a molecular machinerie" in ABC transporters in microorganisms, Caister Academic Press, 1-34
T. Jumpertz, I. B. Holland & L. Schmitt
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(2010) Mutations affecting the extreme C-terminus of E. coli haemolysin A reduce haemolytic activity by altering the folding of the toxin. Microbiology 156 2495-2505
T. Jumpertz, C. Chervaux, K. Racher, M. Zouhair, M. A. Blight, I. B. Holland & L. Schmitt
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(2011) High throughput evaluation of the critical micelle concentration of detergents. Anal. Biochem. 408 64-70
T. Jumpertz, B. Tschapek, N. Infed, S. H. J. Smits, R. Ernst & L. Schmitt
