Electrophysiological characterization of cation coupled symporters and investigation of physicochemical surface processes with a solid-supported membrane [Elektronische Ressource] / von Juan-José García-Celma

Juanjo Guillem

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Electrophysiological Characterization of Cation
Coupled Symporters and Investigation of
Physicochemical Surface Processes with a
Solid-Supported Membrane



Dissertation
Zur Erlangung des Doktorgrades
Der Naturwissenschaften



Vorgelegt beim Fachbereich Biochemie, Chemie, Pharmazie
der Johann Wolfgang Goethe ─ Universität
in Frankfurt am Main


von
Juan José García Celma
Aus Tortosa ─ Tarragona (Spanien)


Frankfurt 2009
(D30)




Vom Fachbereich Biochemie, Chemie, Pharmazie der
Johann Wolfgang Goethe ─ Universität als Dissertation angenommen
















Dekan : Prof. Dr. D. Steinhilber
1. Gutachter: Prof. Dr. J. Wachtveitl
2. Gutachter: Prof. Dr. K. Fendler
Datum der Disputation: TABLE OF CONTENTS
Table of Contents

Table of Contents .............................................................................. i
Table of Figures................................................................................ v
Abbreviations................................................................................... vi
1. Introduction 1
1.1. Translocation through Membranes.........................................................................1
1.1.1. Passive Transport: Diffusion ........................................................................1
1.1.2. Facilitated Transport: Accelerated Diffusion ................................................1
1.1.3. Active Transport: Transport against a Concentration Gradient....................1
1.2. The Relevant Role of the Transporters ..................................................................2
1.3. Ion-Gradient-Driven Permeases.............................................................................2
1.4. Why SSM-based Electrophysiology? .....................................................................3
2. Materials and Methods................................................................. 6
2.1. Film-forming solutions of the SSM..........................................................................6
2.2. Preparation of the Sensor Chips ............................................................................6
2.3. SSM Set-up ............................................................................................................6
2.4. Addition of the Protein Sample...............................................................................7
2.5. Flow Protocol..........................................................................................................8
2.6. Reconstitution of Transporters into Liposomes ......................................................9
3. Analysis of the Transient Currents........................................... 13
3.1. The Measured Output Current does not directly correspond to the Current
generated by the Transporter......................................................................................13
3.2. The Measuring Device..........................................................................................13
3.2.1. Continuous Electrogenic Transport Activity ...............................................16
3.2.2. Initial Charge Displacements .....................................................................17
3.3. Analysis of the Charge Translocated in an Ion Concentration Jump....................18
3.4. The Time Resolution. ...........................................................................................19
i TABLE OF CONTENTS
4. Specific Anion and Cation Binding to Lipid Membranes........ 20
4.1 Introduction............................................................................................................20
4.2 Results...................................................................................................................22
4.2.1. Transient Currents generated by Ion Concentration Jumps ......................22
4.2.2. Dependence of the Translocated Charge on the Nature of the Applied
Cation or Anion. ...................................................................................................23
4.2.3. Interaction of the Different Cations and Anions with Different Lipid
Headgroups. ........................................................................................................23
4.2.4. Concentration Dependence of the Translocated Charge...........................24
4.2.5. Uncompensated Salt Concentration Jumps...............................................25
4.3. Discussion ............................................................................................................26
4.3.1. Chaotropic Anions are attracted to the Lipid Interface...............................26
4.3.2. Kosmotropic Cations are attracted to the Lipid Interface. ..........................26
4.3.3. PC Membranes can be Anion or Cation Selective.....................................26
4.3.4. Headgroup Charges make a Difference. ...................................................27
4.3.5. Interaction Mechanism of Anions and Cations with a Lipid Interface.........27
5. Rapid Activation of MelB ........................................................... 29
5.1. Introduction...........................................................................................................29
5.2. Results..................................................................................................................31
5.2.1. Calculation of Delay and Concentration Rise Time in an Analytical
Approximation. .....................................................................................................31
5.2.2. Experimental Determination of the Concentration Rise Time. ...................32
5.2.3. Influence of the Flow Rate and the Diverter–SSM Distance on the Delay
t and the Concentration Rise Time τ...................................................................32 0
5.2.4. Measurement and Analysis of Charge Translocation in MelB. ..................34
5.3. Discussion ............................................................................................................37
5.3.1. A New Method for the Experimental Determination of the Time Course
of Substrate Surface Concentrations.37
5.3.2. Rapid Solution Exchange at the SSM using a Wall Jet Geometry.............37
5.3.3. The Rapid Electrogenic Conformational Transition in MelB.......................38
ii TABLE OF CONTENTS
6. Electrophysiological Characterization of LacY ....................... 39
6.1. Introduction...........................................................................................................39
6.2 Results...................................................................................................................40
+6.2.1. Downhill Sugar/H Symport generates Transient Currents........................40
6.2.2. Varying Lipid to Protein Ratios...................................................................40
6.2.3. Effect of pH. ...............................................................................................41
6.2.4. Transient Currents in Mutants....................................................................42
6.3. Discussion ............................................................................................................44
6.3.1. Wild-type LacY...........................................................................................44
6.3.2. LacY Mutants. ............................................................................................44
6.3.3. Electrogenic Steps in the LacY Reaction Cycle.........................................45
7. General Discussion and Perspectives ..................................... 48
7.1. Applications and Technical Developments...........................................................48
7.1.1. SSM-Based Electrophysiology...................................................................48
7.1.2. Improvements in the Time Resolution .......................................................48
7.1.3. Specific Interactions between Ions and Lipid Membranes.........................49
7.2. Electrophysiological Characterization of Bacterial Secondary Active
Transporters ................................................................................................................49
7.2.1. Positioning the Major Electrogenic Step ....................................................49
7.2.2. The Role of the Orientation........................................................................50
7.3. Perspectives.........................................................................................................50
8. References .................................................................................. 53
9. Summaries 62
9.1. Summary ..............................................................................................................62
9.1.1. Specific Interaction between Ions and Lipid Headgroups ..................