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Contents
Abbreviations ... V Symbols ... IX
I.Biological context ... 1
Membrane transport and antibiotic resistance ... 3
1. General features of membrane transport ... 3
1.1. Transport mechanism of membrane transporters ... 3
2. Membrane transporters are involved in antibiotic resistance ... 4
2.1. Origin of antibiotic resistance ... 4
2.2. Mechanisms of acquired resistance ... 5
2.3. Role of multidrug transporters in multidrug resistant bacteria ... 5
3. Major Facilitator Superfamily transporters ... 6
3.1. Structural signatures of the MFS transporters ... 7
3.2. Transport mechanism of the MFS transporters ... 8
3.3. Multidrug transport within the MFS ... 9
LmrP from Lactococcus lactis: a tractable model to understand secondary multidrug transport in MFS .... 11
1. A model to illustrate MDR among MFS ... 11
2. Substrate spectrum and binding mechanism ... 11
2.1. LmrP crystal structure gives insight into substrate binding and multidrug recognition ... 12
3. Transport energetics ... 13
4. Roles of key residues ... 13
5. Protonation drives the conformational changes ... 14
6. Transport mechanism ... 16
7. Lipids modulate the function and structure of LmrP ... 18
8. Did we unravel all LmrP mysteries? ... 20
How to study LmrP in a biologically relevant system ... 21
1. Nanodiscs ... 21
2. Proteoliposomes ... 22
3. Summary of the different systems used in this thesis ... 22
II.Aims of the work ... 23
III.smFRET to follow the conformational changes of LmrP ... 27
Theoretical introduction ... 29
1. FRET: a molecular ruler ... 29
1.1. Basic theory of energy transfer ... 29
2. Single-molecule FRET to study the structural dynamics of proteins ... 31
2.1. Ensemble versus single-molecule detection ... 31
2.2. Proteins must be labelled for smFRET measurements ... 32
2.3. In solution-based burst analysis with confocal microscope ... 32
2.4. Getting the most out of a single experiment with MFD-PIE ... 33
2.5. Description of the dual-colour MFD-PIE setup ... 36
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3. smFRET vs DEER ... 40
Preparation and optimisation of LmrP samples for smFRET measurements ... 41
1. Design of the double cysteine mutants and selection of the distance reporters ... 41
1.1. Evaluation of the distance reporters ... 42
1.2. Molecular biology: construction of the double cysteine mutants ... 45
2. Transport activity assay ... 46
3. Expression, purification and labelling of LmrP for smFRET measurements ... 47
4. Reconstitution of LmrP in nanodiscs ... 48
5. How to increase the quality of the data? ... 49
Conformational dynamics of LmrP with smFRET measurements ... 50
1. smFRET to follow the conformational rearrangements of LmrP ... 50
1.1. Effect of the pH on LmrP conformational changes ... 50
1.2. Role of the D68 residue ... 51
1.3. Effect of the lipids on LmrP conformational changes ... 52
1.4. Effect of ligands on LmrP conformational changes ... 52
2. smFRET to characterise the kinetics of the transitions of LmrP ... 59
2.1. Hoechst modifies the kinetics of the cytosolic side of LmrP… ... 59
2.2. …But not of the extracellular one ... 60
2.3. Effect of the other ligands on the kinetics of the transitions ... 61
2.4. Effects of the lipids on the kinetics of the transitions... 66
Partial discussion ... 67
IV.Methodological development for studying LmrP under transport conditions ... 69
Theory of proteoliposome formation ... 71
1. Choice of the lipidic composition ... 71
2. The different types of liposomes ... 72
3. Preparation of unilamellar liposomes ... 72
4. Reconstitution of membrane proteins in liposomes ... 73
4.1. Solubilisation of the liposomes ... 74
4.2. Protein insertion ... 74
4.3. Detergent removal ... 75
5. Proteoliposome characterisation ... 76
pH-gradient impositions and LmrP reconstitution in proteoliposomes through the literature ... 77
1. Imposition of a pH gradient across the membrane of proteoliposomes ... 77
2. LmrP reconstitution in proteoliposomes ... 79
Optimisations to reconstitute LmrP in proteoliposomes and establish a transport assay ... 80
1. Improvement of the proteoliposome tightness ... 80
1.1. Lipid composition... 80
1.2. Detergent used to solubilise the liposomes ... 83
1.3. LmrP concentration ... 83
1.4. Residual presence of detergent ... 84
1.5. Residual presence of chloroform ... 86
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2.1. Monitoring substrate transport by fluorescence ... 86
2.2. Monitoring proton transport with a pH-sensitive fluorescent probe ... 87
3. pH gradient imposition ... 87
3.1. LmrP co-reconstitution with bacteriorhodopsin ... 88
3.2. Dilution in a buffer with a different pH than the internal pH ... 91
Technical conclusions & perspectives ... 97
1. Importance of the pH gradient ... 97
2. Detection of LmrP transport activity ... 97
3. Proteoliposome characterisation ... 97
4. How to bring the samples to smFRET? ... 98
V.Conclusions... 99
VI.Perspectives ... 103
1. Thorough study of the influence of different ligands on LmrP conformational dynamics ... 105
2. Study the influence of the lipids on LmrP conformational dynamics ... 105
2.1. Nanodiscs ... 105
2.2. Proteoliposomes ... 106
2.3. SMALPs ... 106
3. Investigate LmrP dynamics and kinetics on other time scales ... 106
4. Investigate LmrP conformational dynamics in the presence of a proton gradient ... 107
4.1. Optimising LmrP reconstitution ... 108
5. Establish quantitative transport assays for LmrP ... 108
VII.Methods ... 109
LmrP expression and purification ... 111
1. Bacterial strain and plasmid ... 111
2. Design and construction of the double cysteine mutants to be labelled for smFRET ... 111
3. Preparation of electrocompetent L. lactis NZ9000 cells and transformation ... 112
4. Culture of L. lactis and overexpression of LmrP ... 112
5. Preparation of inside-out membrane vesicles ... 113
6. Transport activity assays ... 113
7. LmrP purification ... 113
smFRET measurements ... 115
1. LmrP labelling for smFRET ... 115
1.1. Properties of the probes ... 115
2. LmrP reconstitution in nanodiscs ... 115
3. MSP1E3D1 production and purification ... 116
4. Sample preparation for smFRET measurements ... 117
4.1. LmrP solubilised in detergent ... 117
4.2. LmrP reconstituted in nanodiscs ... 117
5. Confocal multiparameter setup ... 118
6. Data analysis ... 118
6.1. Burst identification ... 118
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6.3. Description of the correction factors for an accurate determination of E and S. ... 119
6.4. Multi-parameter analysis from single-molecule bursts ... 120
6.5. Three-state photon distribution analysis ... 120
7. Software ... 121
LmrP reconstitution in proteoliposomes ... 122
1. Reconstitution of LmrP and bacteriorhodopsin in proteoliposomes ... 122
1.1. Liposome preparation ... 122 1.2. Bacteriorhodopsin reconstitution ... 122 1.3. LmrP reconstitution ... 122 1.4. Fluorescence assays ... 122 2. LmrP reconstitution in proteoliposomes ... 123 2.1. Liposome preparation ... 123 2.2. LmrP reconstitution ... 123
2.3. Fluorescence transport assays ... 124
3. Proton leakage assays... 125
4. Pyranine calibration ... 125
5. Sucrose gradient ... 125
VIII.Appendix ... 127
Biological context– additional information ... 129
1. MdfA X-ray structures give insight on its transport mechanism ... 129
smFRET– additional information ... 131
1. How to measure FRET efficiency ... 131
2. TCSPC principles ... 131
3. Dynamics characterisation with the static FRET line and correlation spectroscopy ... 132
4. Transport assays of the double cysteine mutants ... 135
5. Labelling optimisations ... 136
6. Nanodisc measurements – optimisations ... 139
7. Instability issues ... 139
8. Multiparameter analysis of the double-labelled populations ... 140
9. Results from the PDA fits ... 147
10. Dynamics between the inward-closed and high proximity ratio states ... 149
11. PDA fits ... 149
LmrP reconstitution in proteoliposomes – additional information ... 154
1. Methods for detergent removal ... 154
2. Liposomes scattering measurements ... 154
3. Pyranine calibration curves ... 155
4. Hoechst 33342 protonation states ... 156
5. Hoechst fluorescence is pH dependent ... 156
6. BR-LmrP proteoliposomes: detergent choice optimisations ... 156
7. Valinomycin increases proton permeability ... 158
8. Additional controls ... 159
9. Unreproducible effect of valinomycin on the BR-generated proton gradient ... 160
Methods - additional information ... 161
1. smFRET dye properties ... 161
2. Strategy for the construction of LmrP double cysteine mutants ... 161
3. List of primers used to introduce the cysteine mutations ... 162
