Structure-function analysis of membrane proteins by infrared spectroscopy [Elektronische Ressource] : porin OmpF, porin OmpG and betaine transporter BetP / von Filiz Korkmaz

Ifb

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 37
• Poids de l'ouvrage: 10 Mo

Extrait

Structure-Function Analysis of Membrane Proteins
by Infrared Spectroscopy: Porin OmpF,
Porin OmpG and Betaine Transporter BetP




Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften


vorgelegt beim Fachbereich Physik
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main


von
Filiz Korkmaz
aus Amsterdam, The Netherlands


Frankfurt am Main, January 2009
(D30)




















vom Fachbereich der Physik der
Johann Wolfgang Goethe-Universität als Dissertation angenommen.





Dekan: Prof. Dr. D. Rischke

Gutachter: Prof. Dr. W. Mäntele
Prof. Dr. B. Ludwig
Prof. Dr. Reinhard Krämer

Datum der Disputation:…………………………………




























































List of Contents

1. INTRODUCTION................................................................................................ - 3 -
1.1 Protein Structure ........................................................................................... - 3 -
1.2 Protein Folding .............................................................................................. - 6 -
1.3 Membrane Proteins ....................................................................................... - 7 -
1.3.1 Porins ...................................................................................................... - 8 -
Porin from Paracoccus denitrificans .............................................................. - 9 -
OmpG from Eschericia coli.......................................................................... - 11 -
1.3.2 Membrane Transport Proteins............................................................... - 12 -
BetP from Corynebacterium glutamicum..................................................... - 13 -
1.4 Protein Structure Determination .................................................................. - 16 -
1.4.1 FTIR Spectroscopy in Protein Research ............................................... - 17 -
Spectrometer............................................................................................... - 19 -
FTIR Signatures of a Protein ....................................................................... - 23 -
1.5 Objectives of This Study.............................................................................. - 24 -
2. MATERIALS AND METHODS.......................................................................... - 27 -
2.1 Materials......................................................................................................... - 27 -
2.1.1 Chemicals.............................................................................................. - 27 -
2.1.2 Plasmid.................................................................................................. - 28 -
2.1.3 PDB Structure ....................................................................................... - 28 -
2.1.4 Culture Medium ..................................................................................... - 28 -
2.1.5 Buffers................................................................................................... - 28 -
2.1.6 Antibiotics - 29 -
2.2 Methods....................................................................................................... - 29 -
2.2.1 Competent Cell Preparation and Transformation of E. coli cells ........... - 29 -
2.2.2 Biochemistry of Porin............................................................................. - 30 -
Expression - 30 -
Purification - 30 -
Protein Quantification .................................................................................. - 32 -
Protein Quality Detection by SDS-PAGE..................................................... - 32 -
Reconstitution of Porin into Liposomes ....................................................... - 33 -
2.2.3 OmpG from E. coli................................................................................. - 34 -
2.2.4 BetP from C. glutamicum....................................................................... - 35 -
2.2.5 Spectroscopic Techniques .................................................................... - 36 -
I FTIR Transmission Spectroscopy ............................................................... - 36 -
2H O vs. H O Buffer..................................................................................... - 37 - 2 2
Protein Structure Analysis by FTIR ............................................................. - 38 -
I- Second Derivative ................................................................................. - 38 -
II- Fourier Self Deconvolution (FSD)......................................................... - 39 -
III- Curve Fitting........................................................................................ - 41 -
FTIR-ATR Spectroscopy ............................................................................. - 43 -
I- H/D Exchange with ATR-FTIR Flow-Through Cell ................................ - 44 -
II- Difference Spectra................................................................................ - 47 -
3. RESULTS AND DISCUSSION......................................................................... - 49 -
3.1 Interaction of P. denitrificans Porin & OmpG from E.coli with Lipids ........... - 49 -
3.2 OmpG-WT and the Mutants OmpG-CYS, OmpG-ALA and OmpG- ∆L6...... - 65 -
3.2.1 Structural Characteristics ...................................................................... - 65 -
3.2.2 Thermal Stability of OmpG WT and Mutants ......................................... - 77 -
3.2.3 H/D Exchange Profile of OmpG............................................................. - 84 -
3.2.4 In situ Opening/Closing of the Channel ................................................. - 91 -
3.3 Structure of BetP WT and Mutants.............................................................. - 99 -
3.3.1 Temperature Dependent Structure Stability ........................................ - 111 -
3.3.2 H/D Exchange Profile of BetP - 119 -
4. CONCLUSION ............................................................................................... - 137 -
5. ZUSAMMENFASSUNG.................................................................................. - 141 -
6. REFERENCES - 145 -
8. ACKNOWLEDGMENTS................................................................................. - 151 -
1. Introduction
1. INTRODUCTION
1.1 Protein Structure
Proteins are involved in every process within living cells. Proteins perform a large variety of
functions such as providing protecting functions, participating in transport processes,
catalysis of all biochemical reactions within the cell and are basic substances of antibodies
and certain hormones. Proteins constitute more than half of the dry weight of a cell. They are
organic compounds formed by a linear chain of amino acids (Fig. 1.1). 20 different amino
acids are used in different combinations to produce proteins in organisms. The sequence, in
which the amino acids are connected via peptide bonds to each other, is determined by the
gene sequence. Correct functionality of a protein depends on its structure and correct
conformation. Therefore understanding the structure, function of proteins and conditions for
functionality has been the focus of many scientific researches. Finding a cure for the
illnesses, like cancer, caused by nonfunctional or malfunctioning proteins depends solely on
such studies.
All amino acids share some common features like they have a α-carbon to which a carboxyl,
an amino and a variable side chain group (R group) are attached. The structure of an amino
acid and the linkage of two amino acids are seen in Figure 1.1. A chain of amino acids
therefore has a free carboxyl group on one end, called C-terminal, and has a free amino
group on the other end of the chain, called N-terminal. The repeated carbon-nitrogen-oxygen
pattern is called the main chain or protein backbone. Amino acids attain different chemical
characteristics depending on the side chain group attached to the α-carbon. The side chain
group is represented by “R” in the figure. The amino acid is then classified as
charged/uncharged, neutral, polar and hydrophobic/hydrophilic depending on the chemical
property of the side chain.

- 3 - Amino acid 1 Amino acid 2
R 1 O H R 2 O H
+H C CαN
+H C CαN
-H O
-H H O
H
Amino group Carboxyl
group
H O 2
R 1 O H
R 2
+ O H C α CN
H C α CN
H
-Peptide bond O H
H

Figure 1. 1 The structure of two amino acids with different R groups are shown at the top. The peptide
bond formation between the C- and N-atoms from the two amino acids is shown at the <