Biophysical and biochemical characterisation of the SMR proteins Hsmr and EmrE [Elektronische Ressource] / Ines Lehner

Ines Jilani

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2011
• Nombre de lectures: 84
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Biophysical and biochemical
characterisation of the SMR Proteins
Hsmr and EmrE










Dissertation zur Erlangung des Doktorgrades der
Naturwissenschaften

vorgelegt dem Fachbereich Biochemie, Chemie, Pharmazie
Institut für Biophysikalische Chemie
Johann Wolfgang Goethe Universität Frankfurt am Main
Zentrum für Biomolekulare Magnetische Resonanz Spektroskopie













von Ines Lehner
Frankfurt am Main 2008






D30





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





Dekan: Prof. Dr. Schwalbe

Gutachter: Prof. Dr. Glaubitz
Prof. Dr. Tampé

Datum der Disputation:




















II Acknowledgments

Acknowledgments

I would like to thank my supervisor Prof. C. Glaubitz for the opportunity to conduct my
Ph.D. thesis research in his laboratory. In particular, I am grateful for the provision of
excellent laboratory conditions, support and enthusiasm for the project.

I am thankful to the members of the Glaubitz group past and present. In particular I am
indebted to Dr. Jakob Lopez and Daniel Basting for fruitful discussions, help with NMR
experiments and python scripts.

I would like to thank Dr. Frank Bernhard, Professor Dötsch and the members of the
Dötsch group for providing me with several plasmids and cell free extract.

I am indebted to my cooperation partners Björn Meyer, Dr. Karla Werner, Nina
Morgner, Dr. Winfried Haase and Dr. Vitali Vogel. Additionally I would like to thank
Dr. Chris Lu for advice regarding AUC experiments and Max Stadler for help with
solution state NMR experiments.

Prof. S. Schuldiner is acknowledged for providing several SMR protein plasmids to the
laboratory of Professor C. Glaubitz and Theofanis Manolikas is acknowledged for
providing the EmrE E25A mutant.

Finally I would like to acknowledge the financial support of this thesis provided by the
Sonderforschungsbereich 628 “Functional Membrane Proteomics” der Deutschen
Forschungsgemeinschaft.

II Abbreviations


Abbreviations

ABC adenosine triphosphate binding cassette
ac acriflavine
ADP adenosine diphosphate
APS ammonium persulfate
ATP adenosine triphosphate
AUC analytical ultracentrifugation
OG n-ß-octyl-D-glucopyranoside
bla beta lactamase
BMRB Biological Magnetic Resonance Bank
BNPAGE Blue native PAGE
BSA bovine serum albumin
bR bacteriorhodopsin
bz benzalkonium
CCCP carbonyl cyanide 3-chlorophenylhydrazone
CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid
chl chloramphenicol
CL cardiolipin
cmc critical micellar concentration
COSY correlation spectroscopy
CP cross polarisation
CSA chemical shift anisotropy
CV column volume
DDM n-Dodecyl-ß-D-maltoside
DHBs Matrix (2,5-dihydroxy benzoic acid:2-hydroxy-5-methoxy benzoic acid = 10:1
DHPC 1,2-diheptanoyl-sn-glycero-3-phosphocholine
DMPC 1,2-Dimyristoyl-sn-phosphatidylcholine
DMT drug/metabolite transporter
DOPC 1,2-Dioleoyl-sn-phosphatidylcholine
®
DPC Fos-ß-choline 12
DQ double quantum
DQSQ double quantum single quantum
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
EM electron microscopy
EPR electron paramagnetic resonance
ery erythromycin
etbr ethidium bromide
FM feeding mix
FPLC fast protein liquid chromatography
FTIR Fourier transform infrared
GPCRs G-protein coupled receptors
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HR high resolution
HRP horseradish peroxidase
HSQC heteronuclear single quantum coherence
IC concentration at which 50 % inhibition is observed 50
IEX ion exchange chromatography
IMAC immobilised metal affinity chromatography
INEPT insensitive nuclei enhanced by polarisation transfer
I Abbreviations


IPTG isopropyl β-D-1-thiogalactopyranoside
K dissociation constant d
K partition coefficient D
K dissociation constant of an inhibitor i
K Micheaelis-Menten constant m
LB Luria Bertani
LE lysophosphatidylethanolamine
LILBID laser induced liquid bead ion desorption
LMPG 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)]
LPPG 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)]
MALDI matrix assisted laser desorption
MAS magic angle spinning
MATE multidrug and toxic compound extrusion
Mcs multiple cloning site
MET multidrug endosomal transporter
MFS major facilitator superfamily
M methionine oxidation ox
MS mass spectrometry
2+
MV methylviologen
MW molecular weight
NG N-nonyl-β-D-glucoside
Ni-NTA nickel-nitrilotriacetic acid
NMR nuclear magnetic resonance
NOESY nuclear overhauser enhancement spectroscopy
OG n-ß-octyl-D-glucopyranoside
ori origin
PA phosphatidic acid
PAGE polyacrylamide gel electrophoresis
PDC protein detergent complex
PDSD proton driven spin diffusion
PE phosphatidylethanolamine
PG phosphatidylglycerol
PGP-me phosphatidylglycerolmethylphosphate
PGS phosphatidylglycerosulfate
pI isoelectric point
PMF proton motif force
PML purple membrane lipids
POPC 1-palmitoyl-2-Oleoyl-sn-phosphatidylethanolamine
POPE 1-palmitoyl-2-Oleoyl-sn-phosphatidylethanolamine
POPG 1-palmitoyl-2-Oleoyl-sn-phosphatidylglycerol
ppm parts per million
PS phosphatidylserine
PSMR paired small multidrug resistance
rbs ribosomal binding site
RND resistance nodulation cell division
RM reaction mix
R Stokes radius s
RT room temperature
R6G rhodamine 6G
SDS sodium dodecyl sulphate
SEC size exclusion chromatography
II Abbreviations


SMF sodium motif force
SMP small multidrug pumps
SMR small multidrug resistance
S/N signal to noise
S-TGD-1 3-HSO-galactose1,6-mannose1,2-glucose1,1-sn-2,3-diphytanylglycerol.
SUG suppressor of groEL mutations protein
TCA trichloroacetic acid
TEMED tetramethylethylendiamin
tet tetracycline
TFA trifluoroacetic acid
TFE tetrafluoroethylene
TGD-1 galactose1,6-mannose1,2-glucose1,1-sn-2,3-diphytanylglycerol
TMSP 3-(trimethylsilyl)propionic acid
TOF time of flight
+
TPB tetraphenyl boron
+
TPP tetraphenyl phosphonium
tri trimethoprim
TROSY transverse relaxation optimized spectroscopy
TV total volume
UV ultraviolet
van vancomycin
VV void volume
YT yeast tryptophan

III List of Figures


List of Figures

Chapter 1 - Introduction
Figure 1 Bacterial defence mechanisms 2
Figure 2 Schematic representation of the efflux mechanisms described 3
for ABC multidrug transporter
Figure 3 Sequence alignment of selected SMR proteins using ClustalW 4
Figure 4 Schematic drawing of proposed alternate access mechanism for 7
substrate transport as proposed by Fleishman et al. and
modified according to recent findings by Basting et al.
Figure 5 Schematic EmrE and Hsmr topology diagrams 8
Figure 6 Selection of substrates of SMR proteins 9
Figure 7 Archaeal lipids 11
13
Figure 8 Glycine C spectra simulated at different MAS speeds 14
Figure 9 Overview of protein states investigated and methods used 17

Chapter 2 – Materials and Methods
Figure 10 pT7-7 vector as originally designed 20
Figure 11 pET21a(+) vector map 22
Figure 12 Schematic diagram of a HSQC pulse sequence 35
Figure 13 Schematic diagram of NOESY experiment 35
Figure 14 Schematic diagram of PDSD experiment 36
Figure 15 Schematic diagram of DQSQ experiment 36
Figure 16 Schematic diagram of the quadruple echo experiment 37

Chapter 3 – Cell free expression
Figure 17 Schematic representation of the semi-continuous E. coli based 38
cell free expression system with coupled transcripton and
translation
Figure 18 Choices for in vitro SMR protein expression 39
Figure 19 Screening SMR protein production by cell free expression in 40
precipitation mode
Figure 20 Affinity purification of solubilised Hsmr and evaluation of 42
optimal elution method
Figure 21 Soluble expression of SMR proteins in cell free expression 43
systems

Chapter 4 - Large scale in vivo Hsmr preparation and optimization of
detergent and reconstitution conditions
Figure 22 Overview of Hsmr sample preparation optimization performed 45
Figure 23 Hsmr expression yield in mg protein / L culture medium 46
Figure 24 Anion exchange chromatography of Hsmr after IMAC 49
Figure 25 Gel filtration of Hsmr to improve sample homogeneity, assess 50
molecular weight of Hsmr + micelle and test th