EPR and NMR spectroscopy of spin-labeled proteins
Finiguerra, M.G.
Citation
Finiguerra, M. G. (2011, September 28). EPR and NMR spectroscopy of spin- labeled proteins. Retrieved from https://hdl.handle.net/1887/17881
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/17881
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EPR and NMR Spectroscopy of Spin-Labeled Proteins
Proefschrift
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof. mr. P.F. van der Heijden,
volgens besluit van het College voor Promoties te verdedigen op
op woensdag 28 september 2011 klokke 13:45 uur
door
Michelina Giuseppina Finiguerra
geboren te Foggia, Italië in 1967
Promotiecommissie
Promotores: Prof. dr. E. J. J. Groenen Prof. dr. M. Ubbink
Co-promotor: Dr. M. Huber
Overige leden: Prof. dr. J. Brouwer Prof. dr. G. W. Canters Prof. dr. J. M. van Ruitenbeek
Prof. dr. H.-J. Steinhoff (Universität Osnabrück)
A mia madre e mio padre
Cover image:
A detail of the spin-labeled Cyt f-Pc complex, created using the PyMOL Molecular Graphics System, Version 0.99rc6, Schrödinger, LLC
Printed by Ipskamp Drukkers, Enschede
ISBN: 978-94-6191-015-8
2011, Michelina Finiguerra
Abbreviations 6
Chapter I 7
Introduction
Chapter II 23
High-field (275 GHz) spin-label EPR for high-resolution polarity determination in proteins
Chapter III 37
Accurate long-range distance measurements in a doubly spin-labeled protein by a four- pulse, double electron-electron resonance method
Chapter IV 53
Paramagnetic NMR using spin-labeled proteins to study the structure of the complex between cytochrome f and plastocyanin
Concluding Remarks 89
Appendices 97
Summary 103
Samenvatting 106
List of publications 109
Biography 110
Acknowledgments 111
6
Abbreviations
Amp ampicillin
cam chloamphenicol
CSPs chemical shift perturbations
Cyt f soluble part of the transmembrane cytochrome f
CW continuous wave
DEER double electron-electron resonance
DDT dithiothreitol
EDTA N,N,N’,N’-ethylenediaminetetraacetic acid disodium salt
EPR electron paramagnetic resonance
FPLC fast protein liquid chromatography
FRET Förster resonance energy transfer
HSQC heteronuclear single-quantum coherence
IPTG isopropyl-β-D-thiogalactopyranoside
LB Luria – Bertani medium
MTS (1-acetyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-methanethiosulfonate
MTSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-methanethiosulfonate
NMR nuclear magnetic resonance
Pc Plastocyanin
PCSs pseudocontact shifts
PRE paramagnetic relaxation enhancement
rmsd root-mean-square deviation
RT room temperature
SDSL site-directed spin labelling
SL spin label
Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride
wt wild type
Zn-Pc Zinc Pc
τc rotational correlation time
15N-Pc 15N-labeled Pc
