Structural studies of membrane transport proteins [Elektronische Ressource] / von Vinothkumar Kutti Ragunath

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2006
• Nombre de lectures: 22
• Poids de l'ouvrage: 11 Mo

Extrait

Structural Studies of Membrane
Transport Proteins
Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften
vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe Universität
in Frankfurt am Main
von
Vinothkumar Kutti Ragunath
Aus Madurai, Indien
Frankfurt am Main 2005
vom Fachbereich Biochemie, Chemie und Pharmazie der Johann
Wolfgang Goethe Universität als Dissertation angenommen
Dekan:
1. Gutachter:
2. Gutachter:
Datum der Disputation:
TABLE OF CONTENTS
SUMMARY ...................................................................................... 1
1 INTRODUCTION....................................................................... 5
1.1 ORIGIN OF MEMBRANE TRANSPORT........................................... 6
1.2 CHARGE SEPERATION AND DONNAN POTENTIAL..................... 7
1.3 OSMOTIC CONSEQUNECES OF FIXED ANIONS WITHIN THE
CELL................................................................................................. 8
1.4 MEMBRANE PROTEINS ARE a-HELICAL OR b-SHEETS ........... 10
1.5 TRANSPORT OF SOLUTES .......................................................... 11
1.5.1 DIFFUSION AND PERMEATION ......................................................................11
1.5.2 FACILITATED TRANSPORT .............................................................................11
1.5.3 HALLMARKS OF FACILITATED TRANSPORT ...............................................14
1.6 WHY DO WE HAVE CHANNELS AND PUMPS/CARRIERS.......... 14
1.7 IMPORTANCE OF IONS AND ION GRADIENTS........................... 16
1.7.1 SODIUM ..............................................................................................................17
1.7.2 POTASSIUM.......................................................................................................18
1.7.3 ANIONS...............................................................................................................18
1.8 ACTIVE TRANSPORT.................................................................... 19
1.8.1 PRIMARY TRANSPORT....................................................................................20
1.8.1.1 ATPases .........................................................................................................20
1.8.1.2 GROUP TRANSLOCATION..........................................................................21
1.8.1.3 BACTERIORHODOPSIN...............................................................................22
1.8.1.4 ABC TRANSPORTERS .................................................................................23
1.9 SECONDARY TRANSPORT .......................................................... 24
1.9.1 EVOLUTION OF TRANSPORTERS..................................................................28
1.10 APPROACHES FOR STUDYING MEMBRANE PROTEINS....... 33
1.10.1 3D CRYSTALLIZATION AND X-RAY CRYSTALLOGRAPHY.........................33
1.10.2 ELECTRON MICROSCOPY ..............................................................................36
1.10.3 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY..............................37
1.11 LEARNING ABOUT MEMBRANE PROTEINS............................ 41
1.11.1 SODIUM/PROTON ANTIPORTERS..................................................................41
MjNhaP1.......................................................................................................43
1.11.2 SODIUM/SOLUTE SYMPORTER FAMILY.......................................................44
Proline Transporter ......................................................................................45
1.11.3 BCCT family ........................................................................................................45
Carnitine Transporter...................................................................................47
1.11.4 OUTER MEMBRANE PROTEINS .....................................................................47
OmpG ...........................................................................................................48
2 MATERIAL AND METHODS.................................................. 51
2.1 BASIC MOLECULAR BIOLOGICAL TECHNIQUES....................... 52
2.1.1 PREPARATION OF COMPETENT CELLS.......................................................52
2.1.2 DNA TRANSFORMATION .................................................................................52
2.1.3 MAINTENANCE OF BACTERIAL STRAINS.....................................................53
2.2 CELL GROWTH.............................................................................. 53
2.3 MEMBRANE PREPARATION AND SOLUBILIZATION.................. 54
2.4 ESTIMATION OF TOTAL PROTEIN IN MEMBRANES .................. 54
2.5 PROTEIN PURIFICATION.............................................................. 54
2.5.1 AFFINTIY PURIFICATION WITH HIS TAG.......................................................54
2.5.2 GEL FILTRATION...............................................................................................55
2.6 PROTEIN ESTIMATION ................................................................. 55
2.7 POLYACRYLAMIDE GEL ELECTROPHORESIS........................... 55
2.7.1 SDS GEL ELECTROPHORESIS.......................................................................55
2.7.2 BLUE NATIVE GEL ELECTROPHORESIS ......................................................56
2.8 VISUALIZING PROTEIN AFTER ELECTROPHORESIS................ 57
2.8.1 COOMASSIE STAINING....................................................................................57
2.8.2 SILVER STAINING .............................................................................................57
2.8.3 WESTERN BLOTTING.......................................................................................58
2.9 CD SPECTROSCOPY.................................................................... 59
2.10 FLUORESCENCE SPECTROSCOPY ........................................ 59
2.10.1 Tryptophan fluorescence....................................................................................59
2.10.2 Determination of CMC ........................................................................................59
2.10.3 Activity measurements of MjNhaP1...................................................................59
2.11 ESTIMATION OF TOTAL DETERGENT..................................... 60
2.12 MALDI MASS SPECTROMETRY ............................................... 60
2.13 THIN LAYER CHROMATAGRAPHY........................................... 61
2.14 PREPARATION OF LIPID STOCKS ........................................... 61
2.15 DETERMINATION OF ONSET/TOTAL SOLUBILIZATION OF
LIPIDS......................................................................................... 61
2.16 RECONSTITUTION OF PROTEINS ........................................... 62
2.16.1 RECONSTITUTION FOR FUNCTIONAL STUDIES.........................................62
2.16.2 2D CRYSTALLIZATION .....................................................................................62
2.17 ELECTRON MICROSCOPY ....................................................... 62
2.17.1 NEGATIVE STAINING AND SCREENING .......................................................62
2.17.2 PREPARING SPECIMEN FOR CRYO ELECTRON MICROSCOPY..............63
2.17.3 IMAGE COLLECTION ........................................................................................63
2.17.4 IMAGE PROCESSING .......................................................................................63
2.18 GENERATION OF PROJECTION MAP FROM PDB
COORDINATES .......................................................................... 64
2.19 CALCULATION OF MjNhaP1 DIFFERENCE MAP..................... 64
2.20 3D CRYSTALLIZATION .............................................................. 64
3 MjNhaP1 ................................................................................. 65
3.1 RESULTS ....................................................................................... 66
3.1.1 CLONING OF MJ0057 FROM GENOMIC DNA................................................66
3.1.2 EXPRESSION AND PURIFICATION.................................................................66
3.1.3 GEL FILTRATION OF MjNhaP1 ........................................................................68
3.1.4 ACTIVITY ASSAY...............................................................................................69
3.1.5 2D CRYSTALLIZATION .....................................................................................70
3.1.6 DIFFERENTIAL SCREEN OF LIPIDS...............................................................72
3.1.7 CRYO-EM AND CALCULATION OF PROJECTION MAP...............................74
3.1.8 CRYSTALLIZATION AT HIGHER pH................................................................77
3.1.9 DIFFERENCE MAP............................................................................................81
3.1.10 RESAMPLING OF DATA ...................................................................................83
3.1.11 CALCULATION OF DIFFERENCE MAP IN REAL SPACE..............................86
3.1.12 OBSERVATIO