Electron paramagnetic resonance spectroscopic analyses of membrane transport proteins [Elektronische Ressource] / vorgelegt von Daniel Hilger

Daniel Hilger

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 28
• Poids de l'ouvrage: 22 Mo

Extrait

Electron paramagnetic resonance
spectroscopic analyses of
membrane transport proteins






Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München







vorgelegt von
Daniel Hilger
aus Georgsmarienhütte







München
26. Januar 2010



















GUTACHTER:
1. Prof. Dr. Heinrich Jung
2. Prof. Dr. Dirk Schüler
TAG DER MÜNDLICHEN PRÜFUNG: 04. Mai 2010
II





III

T H M
2009 ILGER ELA NI E HE TR ANSM EMBR AN E DOMAI N BY Publications and manuscripts originating from this thesis
CHAPTER 2
Hilger, D., Böhm, M., Hackmann, A., and Jung, H. (2008) Role of Ser340 and Thr341 in
+transmembrane domain IX of the Na /proline transporter PutP of Escherichia coli in ligand
binding and transport. J. Biol. Chem. 283: 4921-4929.
CHAPTER 3
Raba, M., Baumgartner, T., Hilger, D., Klempahn, K., Härtel, T., Jung, K., and Jung, H.
+(2008) Function of transmembrane domain IX in the Na /proline transporter PutP. J. Mol.
Biol. 382: 884-893.
CHAPTER 4
Hilger, D., Polyhach, Y., Jung, H., and Jeschke, G. (2009) Backbone structure of
+/proline transporter PutP of Escherichia coli. Biophys. J. transmembrane domain IX of the Na
96: 217-225.
CHAPTER 5
Hilger, D., Polyhach, Y., Jung, H., and Jeschke, G. (2009) Modeling of the helix bundle of the
+
Na /proline transporter PutP of Escherichia coli based on EPR distance restraints and
structural homology. Manuscript
CHAPTER 6
Hilger, D., Jung, H., Padan, E., Wegener, C., Vogel, K.-P., Steinhoff, H.-J., and Jeschke, G.
(2005) Assessing oligomerization of membrane proteins by four-pulse DEER: pH-dependent
+ +dimerization of NhaA Na /H antiporter of E. coli. Biophys. J. 89: 1328-1338.
CHAPTER 7
Hilger, D., Polyhach, Y., Padan, E., Jung, H., and Jeschke, G. (2007) High-resolution
+ +structure of a Na /H antiporter dimer obtained by pulsed electron paramagnetic resonance
distance measurements. Biophys. J. 93: 3675-3683.
CHAPTER 8
Jeschke, G., Chechik, V., Ionita, P., Godt, A., Zimmermann, H., Banham, J., Timmel, C. R.,
Hilger, D., and Jung, H. (2006) DeerAnalysis2006 - a comprehensive software package for
analyzing pulsed ELDOR data. Appl. Magn. Reson. 30: 473-498.

Reviews and book chapters (not presented in this thesis)
Jung, H., Pirch, T., and Hilger, D. (2006) Secondary transport of amino acids in prokaryotes.
J. Membr. Biol. 213: 119-133.
Hilger, D., and Jung, H. (2009) Protein chemical and EPR spectroscopic approaches to
monitor membrane protein structure and dynamics. In Bacterial Signaling (Krämer, R., and
Jung, K., eds.) pp. 247-263, Wiley-VCH, Weinheim.

IV
Contributions to publications and manuscripts presented in this thesis
CHAPTER 2
M. Böhm constructed the mutants, performed the transport analyses, and determined the
kinetic parameters of the PutP derivatives in intact cells. D. Hilger analyzed the expression of
the mutants, generated the double-Cys mutants, and established the in situ cross-linking
approach. A. Hackmann carried out the accessibility analyses. H. Jung performed the
analyses of the ion dependence of proline uptake and determined the kinetic parameters of
the PutP derivatives in proteoliposomes. He wrote the manuscript and discussed the results
with D. Hilger.
CHAPTER 3
M. Raba, T. Baumgartner, and K. Klempahn constructed the mutants. M. Raba, T.
Baumgartner, K. Klempahn, and T. Härtel performed the transport measurements. M. Raba
and T. Baumgartner analyzed the expression of the mutants, determined the kinetic
parameters of the PutP derivatives, and performed the accessibility analyses. D. Hilger
analyzed the influence of ligands on MTSET labeling. He conducted the experiments
performed by M. Raba, T. Baumgartner, T. Härtel, and K. Klempahn. H. Jung wrote the
manuscript and discussed the results with D. Hilger and M. Raba.
CHAPTER 4
D. Hilger constructed the mutants. He prepared the samples and performed the functional
analyses and DEER measurements. Y. Polyhach analyzed the data, established the
modeling approach, and generated the rotamer libraries together with G. Jeschke. H. Jung
developed the concept of the study together with G. Jeschke Y. Polyhach, and D. Hilger. G.
Jeschke wrote the manuscript together with D. Hilger, Y. Polyhach, and H. Jung.
CHAPTER 5
D. Hilger constructed the mutants. He prepared the samples and performed the functional
analyses and EPR measurements. Y. Polyhach analyzed the data and established the
modeling approach together with G. Jeschke. H. Jung developed the concept of the study
together with D. Hilger, G. Jeschke, and Y. Polyhach. D. Hilger wrote the manuscript with G.
Jeschke.
CHAPTER 6
E. Padan provided the mutants. D. Hilger prepared the samples and performed the DEER
measurements. He carried out the CW EPR power saturation measurements and analyzed
the data together with K.-P. Vogel. C. Wegener performed the room- and low-temperature
V
CW EPR measurements. H. Jung developed the concept of the study together with H.-J.
Steinhoff, G. Jeschke, and E. Padan. G. Jeschke wrote the manuscript with D. Hilger, H.-J.
Steinhoff, and H. Jung.
CHAPTER 7
E. Padan provided the mutants. D. Hilger prepared the samples and performed the DEER
measurements. Y. Polyhach analyzed the data, established the approach, and generated the
rotamer libraries together with G. Jeschke. H. Jung developed the concept of the study
together with G. Jeschke and E. Padan. G. Jeschke wrote the manuscript with D. Hilger, Y.
Polyhach, and H. Jung.
CHAPTER 8
G. Jeschke designed research together with V. Chechik, A. Godt, C. Timmel, and H. Jung.
He analyzed the data, developed the software and wrote the manuscript. P. Ionita
synthesized spin-labeled gold nanoparticles. H. Zimmermann synthesized deuterated shape-
persistent biradical model compounds. A. Godt synthesized shape-persistent biradical model
compounds. J. Banham analyzed data and performed DEER measurements. D. Hilger
prepared spin-labeled proteins and performed DEER measurements.



I hereby confirm the above statements:




Daniel Hilger Prof. Dr. Heinrich Jung


VI
INDEX
INDEX

ABBREVIATIONS .................................................................................................................................. XI
SUMMARY............. 1
ZUSAMMENFASSUNG ............................................................................................................................ 3
CHAPTER 1
Introduction........ 6
1.1. Diversity of transport systems .................................................................................................. 6
1.2. Diversity and function of secondary transporters ...................................................................... 7
1.3. Coupling ions of secondary transporter systems ...................................................................... 8
1.4. Mechanism of secondary transport .......................................................................................... 9
1.5. Structure of secondary transporters ....................................................................................... 12
1.5.1. Structure of transmembrane domains ............................................................................. 12
1.5.2. Transmembrane helix packing and structural symmetry .................................................. 13
1.5.3. Quaternary structure ...................................................................................................... 15
1.6. Site-directed spin labeling and EPR spectroscopy on membrane proteins.............................. 17
+
1.7. The Na /proline symporter PutP of E. coli .............................................................................. 20
+ +
1.8. The Na /H antiporter NhaA of E. coli .................................................................................... 24
1.9. Scope of this work ................................................................................................................. 30
1.10. References .......................................................................................................................... 31
CHAPTER 2
+
Role of Ser340 and Thr341 in transmembrane domain IX of the Na /proline transporter PutP
of Escherichia coli in ligand binding and transport ..................................................................... 44
2.1. Abstract................................................................................................................................. 44
2.2. Introduction ........................................................................................................................... 44
2.3. Materials and Methods .......................................................................................................... 45
2.3.1