Three-dimensional structure of the glycine-betaine transporter BetP by cryo electron crystallography [Elektronische Ressource] / von Ching-Ju Tsai

Ching-Ju Tsai

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 37
• Langue: Deutsch
• Poids de l'ouvrage: 21 Mo

Extrait

Three-dimensional structure of
the glycine-betaine transporter BetP
by cryo electron crystallography



Dissertation
Zur Erlangung des Doktorgrades der Naturwissenschaften

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



von
Ching-Ju Tsai
aus Taipei, Taiwan

Frankfurt am Main 2008















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


Dekan:

1. Gutachter: Prof. Dr. Bernd Ludwig

2. Gutachter: Prof. Dr. Werner Kühlbrandt

Datum der Disputation: 08. Dezember, 2008




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Table of Contents
Abstract.......................................................................................................................6
Zusammenfassung...................................................................................................9
Abbreviations...........................................................................................................14
1. Introduction......................................................................................................16
1.1. Membrane permeability....................................................................16
1.2. Osmotic adaptation in bacteria........................................................17
1.2.1. The glycine-betaine transporter BetP ....................................19
1.2.2. Interaction between BetP and lipids.......................................23
1.3. Structure determination of membrane proteins ............................25
1.3.1. Electron microscopy and electron crystallography ..............27
1.3.2. 2D crystallization .......................................................................29
1.3.3. Data collection and processing ...............................................34
1.4. Overview of this thesis......................................................................34
2. Materials and Methods..................................................................................36
2.1. Protein expression and membrane preparation ...........................36
2.1.1. Materials and reagents.............................................................36
2.1.2. Plasmid preparation and transformation................................36
2.1.3. Culture growth and protein expression..................................37
2.1.4. Membrane preparation37
2.2. Protein purification and quality analysis ........................................37
2.2.1. Materials and reagents.............................................................37
2.2.2. Membrane solubilization ..........................................................38
2.2.3. Protein purification ....................................................................38
2.2.4. SDS polyacrylamide gel electrophoresis...............................38
2.2.5. Western blotting.........................................................................39
2.3. C. glutamicum lipid preparation.......................................................39
2.3.1. Material .......................................................................................39
2.3.2. Culture growth............................................................................40
2.3.3. Lipid preparation, extraction, and lipid polarization..............40
2.3.4. Lipid analysis..............................................................................41
2.4. 2D Crystallization42
2.4.1. Materials and reagents.............................................................42
2.4.2. Incubation ...................................................................................42
2.4.3. Detergent removal.....................................................................43
2.5. Electron microscopy..........................................................................43
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2.5.1. Data collection of negatively stained samples......................43
2.5.2. Cryo data collection ..................................................................44
2.6. Data Processing ................................................................................45
2.6.1. Single image processing..........................................................45
2.6.2. Tilt geometry refinement...........................................................46
2.6.3. Handedness determination......................................................46
2.6.4. Data merging..............................................................................47
2.6.5. 3D map reconstruction .............................................................47
2.6.6. Non-crystallographic symmetry of the 3D map ....................48
3. Results...............................................................................................................49
3.1. Characterization of BetP ΔC45 ........................................................49
3.2. 2D Crystallization49
3.2.1. Detergents, lipids, and temperature .......................................49
3.2.2. Dialysis conditions.....................................................................51
3.3. 2D crystals of BetP ΔC45 in E. coli lipids and bovine CL ............55
3.3.1. Projection structure from a negatively stained crystal.........55
3.3.2. Mirror-symmetric projection structure ....................................56
3.3.3. Deconvolution by expanding the unit cell ..............................58
3.3.4. A very rare case: p22 2 crystals............................................61 1 1
3.4. 2D crystals of BetP ΔC45 in C. glutamicum lipids.........................65
3.4.1. Projection structure of sheet-like crystals..............................66
3.4.2. Projection structure of tubular crystals...................................68
3.5. Projection structure of BetP ΔC45 in PG 16:0-18:1......................70
3.6. Lipid analysis by TLC and mass spectrometry.............................72
3.6.1. Preliminary analysis by thin-layer chromatography .............73
3.6.2. Mass Spectrometric analysis by multiple precursor ion
scanning......................................................................................75
3.7. 3D map and NCS averaging............................................................79
3.7.1. Data collection of tilted crystals...............................................79
3.7.2. Handedness determination......................................................81
3.7.3. 3D data merging ........................................................................82
3.7.4. 3D map........................................................................................88
4. Discussion........................................................................................................99
4.1. Evaluation of protein sample quality ..............................................99
4.2. 2D crystallization .............................................................................100
4.2.1. Optimization of crystallization conditions.............................100
4.2.2. Tubular 2D crystals .................................................................101
4.2.3. Morphology and resolution ....................................................102
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4.3. Role of different lipids in crystallization........................................102
4.3.1. Pseudo crystals in E. coli lipids/bovine CL mixture............102
4.3.2. Well-ordered crystals in PG 16:0-18:1 and in C.
glutamicum lipids.....................................................................105
4.4. BetP ΔC45 and lipids.......................................................................106
4.4.1. Role of lipids in BetP ΔC45 crystallization............................106
4.4.2. Role of cardiolipin in osmoadaptation..................................108
4.4.3. Chill activation in 2D crystallization ......................................108
4.4.4. Functional lipids for crystallization ........................................109
4.5. 3D structure of BetP ΔC45..............................................................110
4.5.1. Comparison of BetP ΔC45 and BetP wild type....................110
4.5.2. Asymmetric trimer of BetP ΔC45 ...........................................112
4.5.3. Tentative identification of the cytoplasmic membrane
surface ......................................................................................114
4.5.4. Different monomer conformations ........................................116
4.5.5. Oligomeric state and transport function of BetP.................119
4.6. Prospects..........................................................................................121
4.6.1. Interaction between BetP and the substrate.......................121
4.6.2. Function of the N-terminus ....................................................122
5. Appendix....................................................................................................