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Publié par | universitat_ulm |
Publié le | 01 janvier 2005 |
Nombre de lectures | 19 |
Poids de l'ouvrage | 5 Mo |
Extrait
Protein Folding Dynamics
Single-Molecule Studies of
Ribonuclease HI on Biocompatible Surfaces
Dissertation
zur Erlangung des Doktorgrades Dr. rer. nat.
der Fakultät für Naturwissenschaften
der Universität Ulm
vorgelegt von
Elza Kuzmenkina
geborene Amirgoulova
aus Narofominsk, Russland
Ulm, 2005 Universität Ulm, Abteilung Biophysik
Oberer Eselsberg 1
D-89069, Ulm
Amtierender Dekan: Prof. Dr. K.-D. Spindler
1. Berichterstatter: Prof. Dr. G. U. Nienhaus
2. Berichterstatter: Prof. Dr. M. Pietralla
Tag der Promotion: 10. November 2005 Contents
1 Introduction ................................................................................................................... 4
1.1 Why study protein folding? .................................................................................... 4
1.2 Why study folding on the single-molecule level? .................................................. 6
1.3 FRET as a spectroscopic ruler to study biomolecules............................................ 7
1.4 Why we need to immobilize proteins on biocompatible surfaces.......................... 9
1.4.1 Protein-based surfaces ................................................................................... 11
1.4.2 PEG-based surfaces ....................................................................................... 11
1.5 Model proteins...................................................................................................... 12
1.5.1 Ribonuclease HI ............................................................................................ 12
1.5.2 eqFP611......................................................................................................... 15
2 Materials and methods................................................................................................. 18
2.1 Ensemble absorption and fluorescence spectroscopy........................................... 18
2.1.1 Setup .............................................................................................................. 18
2.1.2 Concentration measurements......................................................................... 18
2.2 Single-molecule fluorescence microscopy ........................................................... 18
2.2.1 Setup 18
2.2.2 Sandwich cell................................................................................................. 22
2.2.3 Software......................................................................................................... 22
2.2.4 Measurements protocols................................................................................ 22
2.2.4.1 Imaging................................................................................................... 22
2.2.4.2 Recording of fluorescence time traces.................................................... 22
2.2.5 Intensity corrections for the analysis............................................................. 23
2.2.5.1 Images..................................................................................................... 24
2.2.5.2 Traces 24
2.2.6 Analysis of FRET efficiency changes from single molecule traces.............. 24
2.2.7 Correlation functions ..................................................................................... 26
2.3 Buffers and solutions............................................................................................ 28
2.4 RNase H biochemical procedures ........................................................................ 28
2.4.1 Construction of the double-cysteine mutant of RNase H.............................. 28
2.4.1.1 Primers for site-directed mutagenesis and plasmid sequencing ............. 28
2.4.1.2 Site-directed mutagenesis....................................................................... 29
2.4.1.3 Competent cells ...................................................................................... 30
2.4.1.4 Transformation of E. coli with a mutated plasmid ................................. 30
2.4.1.5 Saving and characterization of colonies ................................................. 31
2.4.2 RNase H expression and purification ............................................................ 31
2.4.2.1 Bacteria growth....................................................................................... 31
2.4.2.2 Purification of RNase H ......................................................................... 31
2.4.3 Activity assay ................................................................................................ 32
2.4.3.1 RNA-DNA hybrid .................................................................................. 32
2.4.3.2 Enzyme kinetics in dilute solution ......................................................... 32
2.4.3.3 Measurement of activity 33
2.4.4 Label conjugation .......................................................................................... 33
2.4.4.1 For testing specific/unspecific adsorption of RNase H onto surfaces.... 33
2.4.4.2 For FRET measurements........................................................................ 35
2.5 eqFP611 biochemical procedures......................................................................... 35
2.6 Preparation of glass surfaces ................................................................................ 35
2.6.1 Cleaning and aminosilanization of glass coverslips...................................... 35
2.6.2 Protein-based surfaces ................................................................................... 36
2.6.3 Linear PEG surfaces ...................................................................................... 36
12.6.4 Cross-linked PEG surface.............................................................................. 36
2.6.5 Immobilization of biotinylated target molecules on surfaces........................ 37
2.7 Linear extrapolation method for protein denaturation.......................................... 37
2.8. Interpretation of FRET efficiency values ............................................................ 38
3 Results ......................................................................................................................... 41
3.1 Resistance of the surfaces to unspecific protein adsorption................................. 41
3.1.1 Protein-based surfaces ................................................................................... 42
3.1.2 PEG-based surfaces ....................................................................................... 42
3.2 Protein structure on biocompatible surfaces......................................................... 44
3.2.1 Folding/unfolding of immobilized RNase H................................................. 44
3.2.2 Fluorescence brightness of eqFP611 ............................................................. 47
3.3 Irreversible denaturation of eqFP611 with GdmCl .............................................. 48
3.4 Thermodynamics and kinetics of RNase H folding/unfolding............................. 49
3.4.1 Free energies of the folded and unfolded states ............................................ 49
3.4.2 Sizes of the folded and unfolded states 53
3.4.3 Reorientation times of the dyes attached to RNase H ................................... 54
3.4.4 Rates of conformational changes................................................................... 57
3.4.4.1 Transitions between states...................................................................... 57
3.4.4.2 Reconfiguration of the unfolded protein chain....................................... 59
3.4.5 Modeling dynamic heterogeneity of the unfolded state ................................ 60
3.4.5.1 Data......................................................................................................... 60
3.4.5.2 Model...................................................................................................... 61
3.4.5.3 Results .................................................................................................... 62
3.4.6 Modeling the expansion of the unfolded state............................................... 62
3.4.6.1 Data 62
3.4.6.2 Model 63
3.4.6.3 Results 65
3.5 Enzymatic function of RNase H on cross-linked PEG surfaces........................... 66
4 Discussion.................................................................................................................... 69
4.1 Biocompatible surfaces......................................................................................... 69
4.1.1 Surface architecture and resistance to protein adsorption ............................. 69
4.1.1.1 Protein-based surfaces ............................................................................ 69
4.1.1.2 PEG-based surfaces ..........................................................................