Studies of Myelin Basic Protein (MBP) using Electron Paramagnetic Resonance (EPR) Spectroscopy
Year: 2009
Type: other
Abstract: Properties of the chemical vicinity of the spin labels within MBP 5. Conclusions and Outlook• Appendix • List of Abbreviations and Symbols• References• Acknowledgements • Eidesstattliche ErklärungMBP clustering on artificial myelin membranes.27 Additional information was gained by NMR experiments, which first underlined the 'unstructured' property of MBP (> 1/3 of the protein remains unordered), but, second, was also able to allocate regions of high mobility or possible αhelicity to the protein sequence.[28][29][30][31] Further crucial information was obtained by standard continuous wave (CW) electron paramagnetic resonance (EPR) measurements based on sitedirected spin labeling (SDSL) of MBP in artificial membrane vesicles.With power saturation measurements, the immunodominant epitope was found to be positioned within a central αhelical region.19 The orientation and penetration depth of the helix into the membrane depend on the charge of the protein.The helical region of MBP C1 (healthy) stays within a more buried position compared to the one of MBP C8 (decreased charge, MS), which can be correlated to the auto-immuno response.Thus EPR spectroscopy already revealed its potential to elucidate MBP structure and to selectively characterize different protein regions of MBP.19 Methods of EPR spectroscopy do not need long-or intermediate-ranged order and can characterize molecular structures up to a range of 8 nanometers and in the pico-to microsecond time scale of rotational reorientation.With sophisticated methods of pulse EPR an even more detailed study of MBP may thus be possible.In this thesis, pulse EPR methods like electron spin echo (ESE) experiments, 2-and 3-pulse electron spin echo envelope modulation (ESEEM) experiments, and hyperfine sublevel correlation (HYSCORE) experiments were performed on MBP C1 in artificial membrane vesicles.Selective nanometer distance measurements using double electron electron resonance (DEER)were performed.This broad arsenal of pulse EPR methods should allow even deeper insights into the interaction of MBP and interaction of MBP with the membrane component.Distance information obtained from DEER data could enable determination of the interaction model and even deliver information about the overall dimensions in case of possible MBP aggregation.This thesis is divided into five chapters.The first two chapters provide the basic knowledge to understand the scientific approach used in this thesis.In Chapter 1, the reader is introduced into the biological system under investigation and the basic EPR theory, as far as it is necessary to understand the performed EPR measurements.In Chapter 2, the reader is provided with additional, detailed information about the sample preparation, the individual measurements procedures, and data analysis.Chapter 3 presents experimental results obtained by the different CW and pulse EPR measurements.Here, first results from the experiments concerned with the uptake and interaction of MBP and Cu 2+ ions are presented, followed by the results obtained for nitroxide spin-labeled MBP in solution and, finally, in artificial membrane vesicles.Chapter 4 delivers a discussion of the results based on the theoretical background, which is formulated in Chapter 1, highlighting correlations between results obtained from the different experiments.In
Source: Max Planck Digital Library
Author: Timo Bund
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W614153339