Characterization of the PTR peptide transporter family of Escherichia coli [Elektronische Ressource] / Daniel Harder

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Ernährungsphysiologie



Characterization of the PTR peptide transporter family of
Escherichia coli



Daniel Harder



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.




Vorsitzender: Univ.-Prof. Dr. M. Klingenspor

Prüfer der Dissertation:
1. Univ.-Prof. Dr. H. Daniel
2. Dr. S. Scherer



Die Dissertation wurde am 18.12.2008 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 20.04.2009 angenommen.







































































Even the longest journey begins with a single step.
(Lao-tzu)


Table of Contents


TABLE OF CONTENTS


1. SUMMARY.........................................................................................................................1
ZUSAMMENFASSUNG ........................................................................................................ 2

2. INTRODUCTION ................................................................................................................. 3
2.1. Peptide transport..................................................................................................... 3
2.1.1. The PTR family.................................................................................................... 3
2.1.2. Mammalian PEPT1 and PEPT2 .......................................................................... 6
2.1.3. Peptide transport in E. coli .................................................................................. 8
2.2. Protein structure of PTR transporters...................................................................... 8

3. AIM OF THE THESIS ......................................................................................................... 11

4. RESULTS........................................................................................................................ 13

4.1. Sequence analysis................................................................................................ 13
4.1.1. Homology search in E. coli................................................................................ 13
4.1.2. Transmembrane domain structure prediction of the PTR family members ....... 16
4.1.3. Genetic localization of the PTR members of E. coli .......................................... 17

4.2. Overexpression in E. coli....................................................................................... 19

2+4.3. Purification of proteins by Ni affinity chromatography......................................... 21

4.4. Functional characterization................................................................................... 24
4.4.1. In vivo uptake experiments................................................................................ 24
4.4.1.1. DtpA - transport characteristics and substrate specificity...................... 25
4.4.1.2. DtpB - transport charact 32
4.4.1.3. DtpC - transport charactecificity 36
4.4.1.4. DtpD - transport characteristics and substrate sp...................... 42
4.4.2. In vitro uptake studies ....................................................................................... 47
4.4.2.1. Transport in membrane vesicles ........................................................... 48
4.4.2.2. Uptake into proteoliposomes................................................................. 49
4.4.2.3. Electrical measurements employing proteoliposomes........................... 50
4.4.3. Analysis of KO-strains 51
4.4.4. Growth curves................................................................................................... 53
4.4.4.1. Growth curves - alafosfalin .................................................................... 53
4.4.4.2. Growth curves - chloramphenicol .......................................................... 54

4.5. Expression of the transporters in Xenopus oocytes.............................................. 55

4.6. Structural characterization..................................................................................... 58
4.6.1. Crosslink experiments....................................................................................... 58
4.6.2. Gel filtration....................................................................................................... 61
4.6.3. Dynamic light scattering ....................................................................................62
4.6.4. Circular dichroism spectrometry........................................................................ 63
4.6.5. MALDI-TOF mass spectr 63
4.6.6. Protein crystallization........................................................................................ 65
4.6.6.1. 2D crystallization................................................................................... 65
4.6.6.2. 3D 67


Table of Contents



5. DISCUSSION ................................................................................................................... 69
5.1. Sequence analysis................................................................................................ 69
5.2. Overexpression..................................................................................................... 70
5.3. Purification............................................................................................................ 71
5.4. Functional characterization: substrate specificity.................................................. 71
5.5. characterization: transport mode......................................................... 76
5.6. Studies with deletion mutant E. coli lines.............................................................. 77
5.7. Growth curves....................................................................................................... 78
5.8. Protein structural information ................................................................................ 79

6. OUTLOOK....................................................................................................................... 83

7. MATERIALS AND METHODS............................................................................................. 85
7.1. Protein sequence analysis .................................................................................... 85
7.2. Statistical data analysis and calculation................................................................ 85
7.3. Cloning.................................................................................................................. 85
7.4. Overexpression in E. coli ...................................................................................... 87
7.5. Growth experiments.............................................................................................. 87
7.6. Western blot analysis............................................................................................ 88
7.7. Transport assays with β-Ala-Lys(AMCA) .............................................................. 88
7.8. ssays with radiolabeled tracer substrates........................................... 89
7.9. ssays with KO-strains ......................................................................... 90
2+7.10. Purification by Ni affinity chromatography .......................................................... 91
7.11. Reconstitution into proteoliposomes ..................................................................... 92
7.12. Uptake in cytochrome c oxidase energized proteoliposomes............................... 92
2 one7.13. Electrical measurements with the SURFE R setup 93
7.14. Expression in Xenopus oocytes............................................................................ 94
7.14.1. RNA production by in vitro transcription............................................................ 95
7.14.2. Two-electrode voltage clamp 96
7.14.3. Lysis of oocytes for Western blot ...................................................................... 96
7.14.4. Immunohistochemistry ...................................................................................... 96
7.14.5. Radiolabeled uptake in Xenopus oocytes......................................................... 97
7.15. Chemical crosslink experiments ........................................................................... 97
7.16. Gel filtration....................................