Solution NMR-based characterization of the structure of the outer mitochondrial membrane protein Tom40 and a novel method for NMR resonance assignment of large intrinsically
disordered proteins
Dissertation
for the award of the degree “Doctor rerum naturalium”(Dr. rer. Nat) Division of Mathematics and Natural Sciences
of the Georg-August-Universität Göttingen
Submitted by Xuejun Yao from Shanghai, China
Göttingen, 2013
Members of the Examination Board
Referee: Prof. Dr. Markus Zweckstetter, NMR based Structural Biology, Max Planck Institute for Biophysical Chemistry
(Name, Department/Group, Institution)
2nd Referee: Prof. Dr. Kai Tittmann, Department of Bioanalytics, Georg-August-Universität Göttingen (Name, Department/Group, Institution)
(if applicable) 3rd referee: ...
(Name, Department/Group, Institution)
Further members of the Examination Board
Prof. Dr. Holger Stark, 3D Electron Cryo-Microscopy, Max Planck Institute for Biophysical Chemistry (Name, Department/Group, Institution)
Prof. Bert de Groot, Computational biomolecular dynamics, Max Planck Institute for Biophysical Chemistry
(Name, Department/Group, Institution)
Dr. Adam Lange, NMR based Structural Biology, Max Planck Institute for Biophysical Chemistry (Name, Department/Group, Institution)
Dr. Lars Kuhn, NMR spectroscopy, European Neuroscience Institute (Name, Department/Group, Institution)
Date of oral examination: 23rd Oct. 2013
Affidavit
I hereby declare that this thesis has been written independently and with no other sources and aids than quoted.
………
Xuejun Yao
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Abstract
This dissertation is composed of two parts. In the first part, the characterization of the structure of liposome-embedded Tom40 using a novel hydrogen/deuterium (H/D) exchange method is described. In the second part, a new high-dimensional NMR experiment for the automatic backbone resonance assignment of intrinsically disordered proteins is described.
The Tom40 protein is the central component of the translocase of the outer mitochondrial membrane (TOM) complex. Tom40 forms the conductive channel for translocation of proteins into mitochondria and has been proposed to fold into a β-barrel. As part of my PhD thesis, I developed an H/D exchange protocol to characterize the structure of integral membrane proteins embedded into liposomes. The H/D exchange protocol consists of two steps of H/D exchange followed by NMR spectroscopic measurement of the denatured monomer. Application of the method to liposome-embedded Neurospora crassa Tom40 (ncTom40) showed that the N- and C- terminal tails are disordered. In addition, slow solvent exchange provided experimental support for several β-strands in the predicted barrel region and an α-helix N-terminal to the barrel.
Evidence was also provided for the presence of conformational instability in the first three N- terminal β-strands, which might be involved in Tom40 oligomerization and interaction with other TOM subunits. In addition, NMR-based titration analysis of a fragment comprising the N- terminal disordered part of ncTom40 with presequence revealed multiple presequence binding sites on Tom40 that may facilitate Tom40 binding to unfolded precursor proteins transiently and subsequent transfer of the preproteins into the translocation pore. Our interaction study of Tom40 and DHPC micelle further identified several hydrophobic clusters on Tom40, which could be the initial lipid binding sites during Tom40 folding into the membrane.
In the second part of my work, I developed a high-dimensional NMR experiment named 6D HACACONCAH APSY to facilitate the assignment of intrinsically disordered proteins (IDPs). The sequence-specific assignment of IDPs is challenging mainly due to severe chemical shift degeneracy of IDPs. Moreover, conventional resonance assignment based on amide proton detection is complicated due to fast exchange of amide proton signals in HN-detected NMR spectra. Automated Projection Spectroscopy (APSY) enables the measurement of very high- dimensional NMR spectra by simultaneous chemical shift evolution of several nuclei, whereas NMR experiments based on alpha proton (HA) detection is able to avoid the line broadening due
2 days, highly reducing the overall analysis time and tedious manual assignment procedures. Our method is ready to be used as a quick and reliable assignment protocol for IDPs even with large molecular weight.
3
Acknowledgements
The work in this thesis was accomplished in the department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, under supervision of Prof.
Dr. Markus Zweckstetter.
I express my profound gratitude to Prof. Dr. Markus Zweckstetter for providing this great opportunity to work on these challenging projects, for his constant support, stimulating suggestions and advices throughout my thesis.
I would like to thank Prof. Dr. Christian Griesinger, director of the department, for providing state-of-art equipments and excellent scientific atmosphere.
I deeply thank to Dr. Stefan Becker and Yvonne Laukat for their immense effort in preparation of numerous excellent protein samples for NMR measurement.
I extend my sincere thanks to Dr. Ulrich Dürr for introducing the project to me and providing help with NMR experiments and data analysis at the beginning of project. Without his pioneering work, this project couldn’t be accomplished.
I am very grateful to Dr. Zrinka Gattin and Dr. Adam Lange for supplying solid state NMR data.
I am thankful to Shengqi Xiang for giving a lot of suggestions and tips on my work. I also want to thank Raghavendran Lakshmi Narayanan for the help with MARS software. My thanks go to Rakhi Bajaj and Dr. Piotr Wysoczanski for their time and effort reading and commenting on this manuscript.
I also want to thank to Dr. Saskia Villinger, Philip Lottmann for their kind help in spectrometer maintenance. Especially Saskia, she has always been patient to share with me a lot of her experience on administration of NMR spectrometer.
I thank Dr. Dirk Bockelmann and Jürgen Arve for their supports on IT systems and our secretaries Mrs. Breiner and Mrs. Silberer for helping me dealing with paper works.
My sincere thanks go to my current and former colleagues in this department for their support and friendship: Guowei, Han, Elias, Rakhi, Hari, Sheng Qi, Guohua, Aldo, Luis, Piotr, Martin, Saskia, Hessam, Stefan, Jean-Philippe, Davood, Edward, Philip, Francesca and other people from our department.
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Table of Contents
Abbreviations ··· VIII
Part I Solution NMR-based characterization of the structure of the outer mitochondrial membrane protein Tom40
1 Introduction ··· 10
1.1 Import of mitochondrial proteins··· 10
1.1.1 Presequence translocation pathway ···························································································11
1.1.2 β-strand pathway ························································································································13
1.2 Tom40, the central proteins forming the conductive pore of the TOM complex ··· 16
1.2.1 Translocase of outer membrane (TOM) complex ·······································································16
1.2.2 The structure of Tom40 ··············································································································18
1.2.3 Structural prediction of Tom40 based on VDAC ········································································20
1.3 Solution NMR characterization of membrane protein structures ··· 21
1.3.1 Membrane media for solution NMR studies of IMPs ·································································21
1.3.2 Solution NMR methods for membrane proteins ··········································································23
1.4 Aims and Outline ··· 24
2 Materials and Methods ··· 26
2.1 Material ··· 26
2.1.1 Chemical reagents ······················································································································26
2.1.2 Equipments ·································································································································26
2.1.3 Scientific software ······················································································································27
2.2 Sample preparation ··· 27
2.2.1 Production of ncTom40 ··············································································································28
2.2.2 Production of the N-terminal (1-59) peptide of ncTom40 ·························································29
2.2.3 Synthesis of presequence peptide ·······························································································29
2.3 Backbone resonance assignments ··· 29
2.3.1 Assignment of ncTom40 in GdnSCN ··························································································29
2.3.2 Assignment of ncTom40 in urea ·································································································31
2.3.3 Assignment of ncTom40 N-terminal peptide(1-59) in the presence and absence of TFE ···········32
2.4 Secondary chemical shift analysis ··· 33
2.5 Best HSQC(Heteronuclear single quantum coherence) ··· 33
3.1.4 Structural characterization of Tom40 by hydrogen/deuterium exchange ···································43
3.1.4.1 Amino acid selective 15N- labeling to relieve the spectral overlap ···43
3.1.4.2 Optimization of experimental conditions ······44
3.1.4.3 Residue-specific solvent protection ···46
3.1.4.4 Structural elements identified by H/D exchange ···50
3.2 Recognition of Presequence by Tom40 ··· 53
3.2.1 Secondary structure propensity of ncTom40 N-terminal peptide ···············································53
3.2.2 Interaction of Tom40 with rALDH presequence·········································································54
3.2.3 TFE induces helix-helix interaction between N-terminal Tom40 and presequence····················56
3.3 Lipid-dependent folding of ncTom40 ··· 57
3.3.1 Secondary structure propensity of ncTom40 in urea ··································································58
3.3.2 Interaction of ncTom40 with micelle in urea ··············································································59
4 Discussion ······························································································································· 61
4.1 Hydrogen/Deuterium exchange characterizes transmembrane proteins structure ··· 61
4.2 Structure of Tom40 ··· 62
4.3 Weakly stable β-strands in Tom40 ··· 64
4.4 Multiple interaction sites between Tom40 N-terminus and presequence ··· 64
4.5 Initial contact sites on Tom40 for membrane insertion ··· 65
Part II A novel method for NMR resonance assignment of large intrinsically disordered proteins 5 Introduction ····························································································································· 68
5.1 Intrinsically disordered proteins (IDPs) ··· 68
5.2 Solution NMR methods for structural characterization of IDPs ··· 69
5.2.1 Secondary chemical shift ···········································································································70
5.2.2 3JHNHα scalar couplings ···········································································································70
5.2.3 Residual Dipolar Coupling ········································································································70
5.2.4 Paramagnetic Relaxation Enhancement ····················································································71
5.3 NMR resonance assignment of IDPs ··· 72
5.3.1 HN-detected triple resonance NMR spectroscopy for resonance assignment ····························72
