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Solution State NMR Investigations on the Structure and Function of Proteins: Application to Riboflavin Synthase

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Solution State NMR Investigations on the Structure and Function of Proteins: Application to Riboflavin Synthase Institut für Organische Chemie und Biochemie der Technischen Universität München Solution State NMR Investigations on the Structure and Function of Proteins: Application to Riboflavin Synthase Vincent Truffault Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. St. J. Glaser Prüfer der Dissertation: 1. Univ.-Prof. Dr. H. Kessler 2. Univ.-Prof. Dr. W. Hiller Die Dissertation wurde am 20.12.2001 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 28.01.2002 angenommen. Ce travail est dédié à mes parents, qui m’ont donné tous les moyens de développer mes aptitudes, et qui m’ont fait confiance lors de mon départ à l’étranger. A Corinne, Eric, Anaïs et Maxime, qui m’ont soutenu et encouragé dans toutes les étapes difficiles. Die vorliegende Arbeit wurde am Institut für Organische Chemie und Biochemie der Technischen Universität München in Garching unter Anleitung von Prof. Dr. Horst Kessler in der Zeit von Oktober 1998 bis Dezember 2001 durchgeführt. Meinem akademischen Lehrer Prof. Dr. Horst Kessler möchte ich an dieser Stelle besonderes danken für das mir entgegengebrachte Vertrauen, sein Interesse an meiner Arbeit, seine uneingeschränkte Unterstützung in allen Belangen, die freie und ungezwungene Arbeitsatmosphäre und die hervorragenden Arbeitsbedingungen. Mein weiterer Dank gilt: -Prof. Dr. Dr. A. Bacher und seiner Gruppe für die fruchtbare Zusammenarbeit am RiSy-Projekt; dabei möchte ich vor allem Dr. Sabine Eberhardt und Dr. Holger Lüttgen für die unermüdliche Herstellung der zahlreichen RiSy-Proben und für die Kooperation bei den Bindungsstudien danken, -Dr. Murray Coles, Dr. Tammo Diercks und Kerstin Abelmann für die großartige Zusammenarbeit am RiSy-Projekt, die die Basis für den Abschluß dieser Arbeit war, -Dr. Gerd Gemmecker für die vielen lehrreichen Gespräche und Diskussionen sowie bei allen sonstigen spektroskopischen Problemen, -meinen Praktikant Andreas Enthart, Nicolas Giraud, und Alexander Genest für Ihre selbständige und konstruktive Arbeit, -nochmals Gustav Gemmecker, Michael John, Markus Heller und Melina Haupt für das intensive Korrekturlesen dieser Arbeit, -Dr. Rainer Haeßner, Monika Goede und Beate Diaw für Rat und Tat bei den diversen Tücken des Computer- und Spektrometeralltags, -Dr. Jens Liermann für die Hilfe bei Computerproblemen, -den Sekretärinnen Frau Machule und Frau Bruckmaier für ihre professionelle Arbeit, -allen weiteren Mitgliedern des Arbeitkreises für die zahlreiche Diskussionen und die angenehme Atmosphäre im Arbeitskreis, -ma famille pour leur aide et soutient constant pendant mes études et la thèse. Abbreviations Index Abbreviations.................................................................................................................IV 1 Introduction and Aim of this Work.....................................................................1 2 Protein Structure Determination by NMR-Spectroscopy ..............................4 2.1 Preamble....................................................................................................................4 2.2 Structure Determination of [U-15N, 13C] Labeled Proteins...............................12 2.2.1 Introduction.....................................................................................................12 2.2.2 Assignment of the backbone chemical shifts .............................................13 2.2.3 Assignment of the side-chains chemical shifts...........................................17 2.2.4 Secondary structure determination ..............................................................20 2.2.5 Tertiary structure determination...................................................................22 2.2.6 Quaternary structure determination.............................................................25 Chemical shift mapping............................................................................26 Hydrogen exchange rates (surface mapping) .........................................28 Heteronuclear NOE (steady state NOE) .................................................29 Isotope filter experiments .........................................................................29 Filter experiments based on a difference technique ..............................31 Isotope filters based on purging schemes ...............................................32 3 Structure Determination of the N-Terminal Domain of Riboflavin Synthase ....................................................................................................................................36 3.1 Biochemical Background......................................................................................36 3.2 Materials and Methods ..........................................................................................41 3.2.1 Sample preparation ........................................................................................41 3.2.2 NMR Spectroscopy and structure calculations ..........................................41 3.2.3 Database details..............................................................................................42 3.3 Chemical Shift Assignments.................................................................................43 3.3.1 Oligomerization state of RiSy-N..................................................................43 3.3.2 Resonance assignment...................................................................................48 3.4 Secondary Structure...............................................................................................51 3.5 Tertiary Structure ...................................................................................................54 3.5.1 Structural data.................................................................................................54 I Abbreviations 3.5.2 Structure and structure calculation...............................................................55 3.5.3 Structure comparison.....................................................................................56 3.6 Quaternary Structure..............................................................................................61 3.6.1 Structural data.................................................................................................61 Cross-labelled sample and mass spectroscopy.......................................61 Water exchange experiment .....................................................................65 Heteronuclear NOE ...................................................................................67 Ligand titration...........................................................................................67 Isotope filter experiment ...........................................................................69 3.6.2 Structure and structure calculation...............................................................72 The RiSy-N dimer......................................................................................75 Riboflavin binding.....................................................................................77 3.6.3 Relevance of the structure.............................................................................80 RiSy-N and the functional trimer.............................................................80 RiSy-N and the X-ray structure ...............................................................81 4 Reaction Mechanism.............................................................................................86 4.1 General knowledge at the beginning of this work.............................................86 4.2 Riboflavin synthase a Diels-Alderase?................................................................90 5 Summary..................................................................................................................97 6 Appendix..................................................................................................................99 A Spectrometer specifications..........................................................................99 B Data appendix of RiSy-N........................................................................... 100 B 1: RiSy-N spectral regions .......................................................................... 100 B 2: RiSy-N sample conditions ...................................................................... 101 Minimal medium and inoculum cultures ...................................................... 101 Fermentation..................................................................................................... 102 B 3: NMR experiments that were carried out for the structure determination of RiSy-N ......................................................................................................... 103 Experiments for backbone assignment.......................................................... 103 Experiments for side-chains assignment ....................................................... 103 Experiments for NOE assignment ................................................................. 103 Experiments for determination of binding and dimerisation sites............. 104 II Abbreviations Experiments for determination of the oligomerization state ...................... 104 Diffusion experiments ....................................................................................
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