EPR spectroscopic investigation of membrane protein structure and folding on light harvesting complex LHCIIb [Elektronische Ressource] / Aleksei Viktorovich Volkov

Volkov

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2008
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

V196r S160r S123r V90r S59r S52r S3r Reference 2 Reference 1 EPR Spectroscopic Investigation of Membrane Protein Structure and Folding on Light Harvesting Complex LHCIIb 0.4 0.9 0.35 0.8 0.3 0.7 0.25 0.6 0.50.2 0.4 0.15 0.3 0.1 0.2 0.05 0.1 0 0 Mutants 6 2.8x10 V196r RR TX unfolded 6 2.4x10 6 2.0x10 6 1.6x10 6 1.2x10 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 2sin (β/2) 1.0 DEER kinetics iterhelix DEER kinetics helix 0.8 ESEEM kinetics T kinetics 1 0.6 0.4 0.2 0.0 +0.2 0 500 1000 1500 2000 2500 3000 Folding time / s Aleksei Viktorovich Volkov D77 (Dissertation Universität Mainz) Π DD2O2OD2O C 1/τ / 1/s 2  EPR Spectroscopic Investigation of Membrane Protein Structure and Folding on Light Harvesting Complex LHCIIb Dissertation zur Erlangung des Grades “Doktor der Naturwissenschaften” in Promotionsfach Chemie am Fachbereich Chemie, Pharmazie und Geowissenschaften der Johannes Gutenberg+Universität in Mainz Aleksei Volkov geboren in Tallinn Mainz, den 23.06.2008  Measure what is measurable, and make measurable what is not so. Galileo Galilei To my wife  Contents 1. INTRODUCTION....................................................................................................................... 1 2. THEORY.................................................................................................................................... 5 2.1. INTRODUCTION TO EPR SPECTROSCOPY............................................................................... 5 2.1.1. CW EPR spectroscopy ............................................................................................... 9 2.1.1.1. CW progressive power saturation measurements ...........................................................12 2.1.2. Pulse EPR spectroscopy .......................................................................................... 13 2.1.2.1 Electron spin echo (ESE) experiment ...............................................................................16 2.1.2.2. ESEEM experiment .........................................................................................................17 2.1.2.3. Electron relaxation and experiments to measure relaxation ............................................19 2.1.2.4. The DEER experiment as a tool for distance measurements ..........................................24 2.2. LHCII STRUCTURE AND FUNCTION...................................................................................... 28 2.3. PROTEIN FOLDING .............................................................................................................. 29 3. EXPERIMENTAL PART ......................................................................................................... 31 3.1. PROTEIN PREPARATION....................................................................................................... 31 3.1.1. LHCIIb monomers..................................................................................................... 31 3.1.1.1. LHCIIb trimers..................................................................................................................36 3.1.1.2. Deuterium exchange experiments ...................................................................................39 3.1.2. Reference samples................................................................................................... 39 3.1.3. Cryoprotection of LHCIIb samples............................................................................ 40 3.1.4. LHCIIb samples for kinetic measurements............................................................... 40 3.1.5. Sample concentration............................................................................................... 43 3.2. EPR .................................................................................................................................. 44 3.2.1. Experimental procedure ........................................................................................... 44 3.2.1.1. X+ Band CW measurements ...........................................................................................44 3.2.1.2. X+ Band CW progressive power saturation measurements .............................................44 3.2.1.3. X+band pulse measurements..........................................................................................45 3.2.1.4. W+band pulse measurements.........................................................................................47 3.2.2 Data analysis ............................................................................................................. 48 3.2.2.1. X+ Band CW measurements ...........................................................................................48 3.2.2.2. X+ Band CW progressive power saturation measurements .............................................48 3.2.2.3. Relaxation........................................................................................................................49 3.2.2.4. X+ Band ESEEM .............................................................................................................50 3.2.2.5. X+ Band DEER.................................................................................................................51  4. RESULTS................................................................................................................................ 54 4.1. LHCIIB STRUCTURE INVESTIGATION WITH EPR.................................................................... 54 4.1.1. CW EPR spectroscopy ............................................................................................. 54 4.1.2. CW progressive power saturation measurements ................................................... 57 4.1.3. ESEEM ..................................................................................................................... 59 4.1.4. Relaxation................................................................................................................. 65 4.1.5. DEER........................................................................................................................ 76 4.1.5.1. New monomer mutants....................................................................................................76 4.1.5.2. Reproducibility of DEER experiments..............................................................................83 4.1.5.3. New trimers......................................................................................................................84 4.1.5.4. Change of LHCIIb structure as a function of micelle composition....................................86 4.1.6. Comparison between methods................................................................................. 90 4.1.7. New information about LHCIIb structure .................................................................. 97 4.2. INVESTIGATION OF LHCIIB FOLDING WITH EPR.................................................................... 99 4.2.1. Characterization of the samples for kinetic measurements...................................... 99 4.2.1.1. Mutants not suitable for protein folding investigations. ..................................................105 4.2.1.2. Changes of protein geometry as a function of micelle composition in samples used for kinetic measurements.................................................................................................................107 4.2.1.3. Changes of protein samples for kinetic measurements in liquid nitrogen. .....................109 4.2.2. CW spectroscopy for LHCIIb folding studies.......................................................... 110 4.2.3. ESEEM for LHCIIb folding studies.......................................................................... 112 4.2.3.1. ESEEM on unfolded LHCIIb ..........................................................................................112 4.2.3.2. Folding kinetics determination with ESEEM ..................................................................115 4.2.4. Relaxation measurements for LHCIIb folding studies ............................................ 121 4.2.4.1. Folding kinetics determination with relaxation time measurements ...............................129 4.2.5. DEER for LHCIIb folding studies ............................................................................ 131 4.2.5.1. DEER on unfolded LHCIIb.............................................................................................131 4.2.5.2. Folding kinetics determination with DEER .....................................................................138 4.2.6. New information about LHCIIb folding.................................................................... 151 5. CONCLUSION AND OUTLOOK .......................................................................................... 155 6. REFERENCES.......................................................................