Structural folding dynamics of an archetypal conformational disease using Nuclear Magne PDF
Preview Structural folding dynamics of an archetypal conformational disease using Nuclear Magne
Structural folding dynamics of an archetypal conformational disease using Nuclear Magnetic Resonance Spectroscopy Ph.D. Thesis Géraldine Rosalie Levy Institute of Structural and Molecular Biology (ISMB) University College London Submitted September 2013 Declaration I, Géraldine Rosalie Levy, declare that all the work presented in this thesis is the result of my work only. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. The work herein was carried out while I was a post-‐graduate student at University College London, Institute of Structural and Molecular biology, under the supervision of Dr John Christodoulou and Dr Bibekbrata Gooptu. 1 Acknowledgements 2 Abstract Members of the serpin (serine protease inhibitor) superfamily of proteins regulate key physiological processes through their ability to undergo major conformational transitions. In conformational diseases, native protein conformers convert to pathological species that polymerise. Structural characterization of these key transitions is challenging. Mechanistic intermediates are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. I use Nuclear Magnetic Resonance (NMR), and Circular Dichroism (CD) spectroscopy, to investigate the interrelated processes of serpin folding, misfolding and polymerisation in solution using the 45kDa prototypic serpin α -antitrypsin, the 1 recent assignment of the backbone resonances of α -antitrypsin by our group, allows 1 us to ask more sophisticated questions by a range of NMR techniques to study its structure and dynamics. In this study, I analysed early unfolding behaviour of α - 1 antitrypsin across a urea titration within what is apparently the largest two-states system yet characterised. In order to assess the dynamics of the native state, I have used hydrogen/deuterium exchange nuclear magnetic resonance spectroscopy (HDXNMR) to characterise motions on the slow (ms) timescale. I have conducted a detailed analysis of residue-specific changes in protection from exchange across a pH titration using SOFAST-HMQC. This is complemented by a detailed a preliminary analysis of fast motions (ps-ns) using NMR relaxation experiments. Moreover, a forme fruste deficiency variant of α -antitrypsin (Lys154Asn) that forms polymers 1 recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo was characterized in this study. Lys154Asn α -antitrypsin populates an 1 intermediate ensemble along the polymerisation pathway at physiological temperatures. Together, this study shows how the use of powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions, provides novel insights into pathological conformational behaviour. 3 List of abbreviations Serpin: Serine Protease Inhibitor FENIB: Familial Encephalopathy with Neuronal Inclusion Body NMR: Nuclear Magnetic Resonance spectroscopy SDS: Sodium Dodecyl Sulfate PAGE: Polyacrylamide Gel Electrophoresis LB: Luria Broth E. coli: Escherichia coli Cryo-‐EM: Cryo electron-‐microscopy CD: Circular Dichroism RPM: Revolutions Per Minute OD: Optical Density PEG: Poly Ethylene Glycol TUG: Transverse Urea Gel βME: β Mercapto-‐Ethanol EDTA: EthyleneDiamineTetracetic Acid IPTG: Isopropyl β-‐D-‐1-‐thiogalactopyranoside ER: Endoplasmic Reticulum HSQC: Heteronuclear Single Quantum Coherence TROSY: Transverse Relaxation-‐Optimized Spectroscopy RDC: Residual Dipolar Coupling HMQC: Heteronclear Multiple quantum coherence SOFAST: Selective Optimized Flip Angle Short Transient DSS: 4,4-‐dimethyl-‐4-‐silapentane-‐1-‐sulfonic acid MW: Molecular Weight RNA: Ribonucleic Acid 4 mRNA: Messenger Ribonucleir Acid tRNA: Transfer Ribonucleir Acid DNA: Deoxyribonucleic acid cDNA: Complementary DNA v/v: volume: volume ratio w/v: weight: volume ratio kDa: kilo Daltons Δν : Linewidth 1H τ : Rotational correlation time C RCL: Reactive Centre Loop MS: Mass Spectrometry IM/MS: Ion Mobility Spectrometry -‐ Mass Spectrometry experiments ToF: Time of Flight m/z: mass:charge ratio CID: Collision Induced Dissociation HDXNMR: Hydrogen Deuterium Exchange Nuclear Magnetic Resonance Spectroscopy HDXMS: Hydrogen Deuterium Exhange Mass Spectrometry FID: Free Induction Decay NOE: Nuclear Overhauser Effect FT: Fourier Transformation PDB: Protein Data Base ω : Precession rate 0 γ: Gyromagnetic ratio F Apparent fraction unfolded app: WT: Wild Type ∆G: Gibbs Free energy ∆H: Difference in enthalpy T: temperature 5 ∆S: Difference in entropy k : Rate of exchange ex k : Intrinsic rate int k : Rate of opening op k : Rate of closing cl ∆G : Free energy between the close app GdmCl: Guanidine Hydrochloride K: Equilibrium constant F: Folded U: Unfolded I: Intermediate M*: Molten Globule-‐like CFTR: Cystic Fibrosis Transmembrane Conductance Regulator ERAD: ER-‐associated degradation EOR: ER Overload Response S: Stressed R: Relaxed B Externally applied magnetic field 0: R Transverse relaxation rate 2: τ Rotational correlation time c: pET: pTermat Expression vector system PCR: Polymerase chain reaction AmPS: Ammonium persulphate IM-‐MS: Ion Mobility -‐ Mass Spectrometry DTT: Dithiothreitol U.V.: Ultra Violet DSC: Differential Scanning Calorimetry mAb: Monoclonal antibody P: Protection factor 6 IEF: Iso-‐Electric Focusing SI: Stoichiometry of Inhibition K : Association rate constant app PAI-‐1: Plasminogen Activator Inhibitor-‐1 CCS: Collision Cross Section INEPT: Insensitive Nuclei Enhanced by Polarization Transfer 7 Table of Contents Declaration 1 Acknowledgements 2 Abstract 3 List of abbreviations 4 Chapter 1 – INTRODUCTION 12 Protein folding 13 Physical forces and principle underlying protein folding 13 1.1.1.1. Thermodynamics 14 1.1.1.2. Kinetics 16 1.1.1.3. Models of protein folding 16 1.1.1.4. Polypeptide chain behaviour and energy landscapes 18 1.1.1.5. Tools for the study of protein folding. 20 1.1.1.6. Relationship of polypeptide folding within cells and in isolation 20 1.1.1.7. Protein misfolding and conformational disease 21 1.2. Serpins and serpinopathies 23 1.2.1. α -‐antitrypsin deficiency 24 1 1.2.2. Metastability and inhibitory mechanism 25 1.2.3 Alternative serpin conformations 27 1.2.4. Studies of serpin folding 29 1.2.5. Serpin polymerisation 31 1.2.5.1. Polymerisation induced by loop cleavage or peptide insertion 32 1.2.5.2. Three models of polymerisation proposed for disease mechanism and intermediate formation 34 1.3. NMR spectroscopy in studying protein folding 38 1.3.1. General principles of NMR 38 1.3.2. Study of protein structure and dynamics using NMR spectroscopy 41 1.3.3. NMR studies of high molecular weight proteins 42 1.3.4. Introduction to NMR of α-‐antitrypsin 46 1 1.3.5. Multiple timescales allow structural and dynamical behaviour to be characterized 48 Chapter 2 – MATERIALS AND METHODS 54 2.1 Materials 55 2.1.1 Regents 55 2.1.2 Instruments 55 2.1.2.1. Cell purification and characterization 55 2.1.2.2. Circular Dichroism 56 2.1.2.3. Nuclear Magnetic Resonance (NMR) Spectroscopy 56 2.1.2.4. Mass Spectrometry (MS) 56 2.2 Methods 57 2.2.1 Protein recombinant expression in Escherichia coli (E. coli) and purification 57 8 2.2.1.1. Plasmid constructs 57 2.2.1.2 Transformation of BL-‐21-‐Gold E. coli with plasmid pQE-‐31 containing α-‐antitrypsin 1 cDNA 59 2.2.1.3 Unlabelled recombinant α-‐antitrypsin protein production 59 1 2.2.1.4 Uniform incorporation of 15N-‐ and 13C-‐15N-‐2H-‐ isotopic labeling for NMR 60 2.2.2 Purification of α-‐antitrypsin 60 1 2.2.3 Biophysical and biochemical characterization 62 2.2.3.1 Activity Assay 62 2.2.3.2 Far U.V. Circular Dichroism spectroscopy 63 2.2.3.3 Native-‐PAGE 63 2.2.3.4 Transverse Urea Gradient-‐PAGE 64 2.2.3.5 1H NMR 65 2.2.3.6 Native Mass Spectrometry (MS) 65 2.2.4 Hydrogen Deuterium exchange (HDXNMR) 66 2.2.4.1 Sample preparation 66 2.2.4.2 1H-‐15N SOFAST HMQC NMR for HDXNMR experiments 66 2.2.4.3. Spectral processing and NMR frequency referencing 67 2.2.4.4. Data processing and analysis 68 2.2.5. NMR studies of α-‐antitrypsin in urea 70 1 2.2.5.1. Sample preparation 70 2.2.5.2 1H-‐15N -‐TROSY HSQC 71 2.2.6. NMR relaxation of α-‐antitrypsin 71 1 2.2.6.1 Sample preparation 71 Chapter 3 – NMR STUDIES OF α-‐ANTITRYPSIN SOLUTION BEHAVIOUR IN UREA 74 1 3.1. Introduction 75 3.1.1. Metastability/Z-‐mutant and formation of intermediate ensemble. 75 3.1.2. Equilibrium folding intermediate 76 3.1.3. Probing the folding/unfolding pathway of α-‐antitrypsin in guanidine 78 1 3.1.4. Probing the folding/unfolding pathway of α-‐antitrypsin in urea 80 1 3.1.5. Effect of stabilizing ‘cavity-‐filling’ mutations on the native state α-‐antitrypsin 81 1 3.2 Results 82 3.2.1. NMR studies of α-‐antitrypsin unfolding structural behaviour 82 1 3.2.2. Circular Dichroism studies of the plasma derived and recombinant α-‐antitrypsin 92 1 3.2.3. Transiently populated α-‐antitrypsin intermediates form off-‐pathway polymers with 1 defined secondary structure in urea 95 3.3. Discussion 97 Chapter 4 – STRUCTURAL DYNAMICS OF THE NATIVE α-‐ANTITRYPSIN STUDIED BY 1 HYDROGEN DEUTERIUM EXCHANGE NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 101 4.1. Introduction 102 4.1.1. General principle of HDXNMR 102 4.1.2. HDXMS studies of α-‐antitrypsin 105 1 9
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