X-ray structure of the Na_1hn+-coupled glycine-betaine symporter BetP from Corynebacterium glutamicum [Elektronische Ressource] / von Susanne Ressl

Tiago

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 53
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

+X-ray Structure of the Na -coupled
Glycine-Betaine Symporter BetP from
Corynebacterium glutamicum




Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften

vorgelegt im Fachbereich 14 Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe Universität
in Frankfurt am Main


von
Susanne Ressl
aus Vímperk
Tschechische Republik


Frankfurt am Main (2009)

(D30)

Diese Arbeit wurde in der Abteilung Strukturbiologie des Max-Planck-Instituts für
Biophysik in Frankfurt am Main durchgeführt und vom Fachbereich 14 Biochemie,
Chemie und Pharmazie der Johann Wolfgang Goethe Universität Frankfurt als
Dissertation angenommen.


Diese Arbeit wurde von Dr. Christine Ziegler und Dr. Anke Terwisscha van
Scheltinga betreut.








Dekan: Prof. Dr. Dieter Steinhilber
1. Gutachter: Prof. Clemens Glaubitz
2. Gutachter: Prof. Werner Kühlbrandt

Datum der Disputation:



Ein Gelehrter in seinem Laboratorium ist nicht nur ein Techniker;
er steht auch vor den Naturgesetzen
wie ein Kind vor der Märchenwelt.
†Marie Curie, *07.11.1867 - 04.07.1934









* Pro moje milí rodi!e *


Parts of this thesis have been published in the following articles:

Ressl, S., Terwisscha van Scheltinga, A.C., Vonrhein, C., Ott, V. & Ziegler, C.:
Molecular basis of transport and regulation in the Na+/betaine symporter
BetP. (2009) Nature, 458, 47-52











All relevant coordinates and structure factors have been deposited in the RCSB
Protein Data Bank under the access code:

2WIT Crystal structure of the sodium-coupled glycine-betaine symporter
BetP from Corynebacterium glutamicum with bound substrate



TABLE OF CONTENTS

ABSTRACT...................................................................................................................................................1
ZUSAMMENFASSUNG...........................3
ABBREVIATIONS.....................................................................................................................................9
1. Introduction........11
1.1. Active transport of solutes across a membrane....12
1.2. Fluctuations in the environment – osmotic stress12
+1.3. The Na -coupled glycine-betaine BetP from Corynebacterium glutamicum ...........................13
1.3.1. Electrochemical properties of BetP................................................................................................14
1.3.2. Osmoregulatory properties of BetP...............................14
1.3.3. Osmosensing properties of BetP....16
1.3.4. Structural studies on BetP................................................................................................................20
1.4. Aim of this work ........................................................21
2. Materials...............................................22
2.1. Detergents and phospholipids .................................................................................................22
2.2. Bacteria and plasmids................23
3. Methods...............................................................................................................24
3.1. Production, purification and characterisation of BetP!N29EEE44/45/46AAA ...24 StrepII
3.1.1. Competent cells and plasmid DNA transformation....................................................................24
3.1.2. Cell growth and betP gene expression.............................24
3.1.3. Production of selenomethionine BetA ..........................................................25
3.1.4. Membrane preparation and solubilisation.....................................................25
3.1.5. Isolation of BetA with StrepTactin®-affinity chromatography.................26
3.1.6. Size exclusion chromatography.......................................................................................................27
3.1.7. Concentrating protein samples........27
3.1.8. Protein concentration estimation....27
3.1.9. SDS-polyacrylamide gel electrophoresis ........................................................................................28
3.1.10. Blue and clear native gel electrophoresis.....................28
3.1.11. Staining of polyacrylamide gels .....................................................................................................29
3.1.12. Western blotting and immuno-detection29
3.1.13. Two-dimensional thin-layer-chromatography............30
3.2. Crystallography...........................................................................................31
3.2.1. Crystal lattices and symmetry ..........................................................................31
3.2.2. Macromolecular crystallography.....32
3.2.3. Crystal mounting and cryo protection ...........................................................35
3.3. X-ray structure analysis .............................................................................36
3.3.1. Theory of X-ray diffraction................................36
3.3.2. Reciprocal space and the Ewald construction..............39
3.3.3. Temperature factors..........................................................41
3.3.4. The Patterson function.....................................................................................42
3.3.5. Data collection...................................43
3.3.6. Indexing, scaling and data reduction..............................44
3.3.7. Correction for diffraction data anisotropy ....................................................46
3.3.8. Phase determination by anomalous scattering..............................................49
3.3.9. Non-crystallographic symmetry ......................................................................56
3.3.10. Density modification.......................................................57
3.3.11. Model building and electron density maps..................................................60
3.3.12. Macromolecular refinement...........................................61
3.3.13. Structure validation.........................67
3.3.14. Graphical presentation of protein structures..............70
3.3.15. Structure comparison......................................................................................................................70
4. Expression, purification and crystallisation....................72
4.1. Expression and purification of BetA......................72
4.2. Expression, purification and crystallisation of SeMet-BetA................................................76
4.3. Clear-native- and blue-native-PAGE of BetA and SeMet-BetA........79
4.4. Lipid and detergent content by thin-layer chromatography................82
I
4.5. Crystallisation of BetA and SeMet-BetA................................................................................83
4.6. Discussion ...................................................................89
5. Crystallographic data collection, processing and statistics...........................92
5.1. Native BetA.................................................................92
5.2. SeMet-BetA.97
5.3. Discussion .................................................................101
6. Phasing...............................................................................106
6.1. Phasing with Xenon.................................................107
6.2. Phasing with Ta Br................113 6 12
6.3. Discussion .................................................................................................................................117
7. Structure determination of SeMet-BetA.......................121
7.1. Phase determination by SAD with SeMet............121
7.2. Re-phasing, density modification and anisotropy correction............................................125
7.3. Model building and refinement..............................................................................................129
7.4. Structure validation of the BetP model................135
7.5. Discussion .................................................................141
8. Structure of BetP..............................144
8.1. Protomer structure...................................................................................................................145
8.2. Trimer architecture..................149
8.3. Substrate binding......................153
8.3.1. The intracellular gate at Trp377 ....................................................................................................156
8.4. Putative sodium binding site..159
8.5. Cation-! interactions in BetP.162
8.6. Interactions of the C-terminus of protomers A, B and C .................................................165
8.7. Resting state of BetP ...............................................................................168
8.8. Atomic activation model of BetP..........................169
8.9. Structural comparison with other transporters having the inverted-repeat motif .........177
8.10. Symmetry