STRUCTURE OF PRION PROTEIN AMYLOID FIBRILS AS DETERMINED BY HYDROGEN/DEUTERIUM EXCHANGE by XIAOJUN LU Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Witold K. Surewicz Department of Chemsitry CASE WESTERN RESERVE UNIVERSITY May, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. To My Family ii TABLE OF CONTENTS Page Table of Contents iii List of Tables vii List of Figures viii Acknowledgements xi List of Abbreviations xii Abstract xv Chapter 1: Prion Diseases 1 1.1 Amyloidosis 1 1.1.1 Amyloidoses 1 1.1.2 Amyloid 1 1.2 Prion diseases 3 1.3 Protein-only hypothesis 6 1.3.1 Historical overview of prion theory 6 1.3.2 “Protein-only” hypothesis 7 1.3.3 Evidence in support of the protein-only hypothesis 8 1.3.4 Prion strains and species barriers 10 1.4 PrPC 12 1.4.1 The structure of PrPC 12 iii 1.4.2 The physiological function of PrPC 13 1.4.3 Familial mutations associated with the PRNP gene 15 1.5 PrPSc 17 1.5.1 Current knowledge about PrPSc structure 17 1.5.2 In vitro conversion studies 19 1.5.3 Current structural models for PrPSc 21 1.6 Fungal prions 23 Chapter 2: Hydrogen-Deuterium Exchange 26 2.1 Background 26 2.1.1 Theory 26 2.1.2 Historical perspective 29 2.2 Techniques 33 2.2.1 Labeling 33 2.2.2 Rapid proteolysis 33 2.2.3 Fragment separation 34 2.2.4 MS measurement 35 2.2.5 Data analysis 35 2.3 Current application of HXMS to amyloid fibrils 36 2.3.1 Amyloid-β 36 2.3.2 α-Synuclein 37 iv 2.3.3 HET-s protein 37 Chapter 3: Structure of Wild-type HuPrP90-231 Amyloid Fibrils 39 3.1 Introduction 39 3.2 Materials and methods 40 3.2.1 Protein purification 40 3.2.2 Spontaneous conversion of PrP to amyloid fibrils 42 3.2.3 ThT assay 42 3.2.4 AFM 43 3.2.5 Annealing 43 3.2.6 Peptide mapping 43 3.2.7 Hydrogen/deuterium exchange 44 3.3 Results 48 3.3.1 Peptide mapping and coverage 48 3.3.2 H/D exchange data for the soluble monomer 50 3.3.3 H/D exchange on amyloid fibrils 53 3.3.4 Effect of fibril annealing on the pattern of H/D exchange 56 3.4 Discussion 60 Chapter 4: Structure of D178N HuPrP90-231 Amyloid Fibrils 67 4.1 Introduction 67 4.2 Materials and methods 67 v 4.3 Results 68 4.3.1 Optimization of digestion conditions 68 4.3.2 Peptide mapping and coverage 70 4.3.3 H/D exchange of amyloid fibrils 71 4.4 Discussion 74 Chapter 5: Structure of ShaPrP90-231 Amyloid Fibrils 78 5.1 Introduction 78 5.2 Materials and methods 80 5.2.1 Protein expression and purification 80 5.2.2 Preparation and characterization of amyloid fibrils 80 5.2.3 Hydrogen/deuterium exchange 81 5.2.4 Bimodal distribution 81 5.3 Results 82 5.3.1 Peptide mapping and coverage 82 5.3.2 H/D exchange data for the monomer 83 5.3.3 H/D exchange on spontaneously converted amyloid fibrils 83 5.3.4 H/D exchange on PrPPMCA 88 5.4 Discussion 93 Chapter 6: Future Directions 98 Bibliography 103 vi LIST OF TABLES Page Table 1.1. Human amyloid (or aggregate) proteins and their associated diseases 2 Table 1.2. The prion diseases 4 Table 3.1. Peptic fragments from wild-type huPrP90-231 that were used in HXMS experiments 49 Table 5.1. Peptic fragments from ShaPrP90-231 used in HXMS experiments 84 vii LIST OF FIGURES Page Figure 1.1. The solution structure of human PrP 14 Figure 1.2. Pathogenic mutations associated with the PRNP gene 16 Figure 1.3. Left handed β–helical model 22 Figure 1.4. Spiral model 24 Figure 2.1. Rate constant for isotopic exchange of hydrogen located on peptide amide linkages in polyalanine presented as a function of pH 28 Figure 2.2. The resulting MS spectra of the two kinetic limits, EX2 and EX1 30 Figure 3.1. MS/MS spectrum of peptides FTETDVKM (residues 198-205 of huPrP) 45 Figure 3.2. Overview of HXMS experimental procedure for amyloid fibrils 46 Figure 3.3. MS spectra of double-charged peptide 198–207 illustrating time-dependent deuterium uptake 51 Figure 3.4. Kinetic curves for deuterium incorporation by individual peptic fragments for huPrP90–231 monomer and amyloid fibrils 52 Figure 3.5. Characterization of amyloid fibrils of huPrP90–231 54 Figure 3.6. H/D exchange protection maps summarizing 25-h exchange data for huPrP90–231 fibrils before and after annealing 57 Figure 3.7. PK digestion of huPrP90–231 fibrils before and after annealing 59 viii Figure 4.1. Optimization of digestion conditions 69 Figure 4.2. Typical amyloid fibrils of D178N huPrP90–231 as seen by atomic force microscopy 72 Figure 4.3. H/D exchange protection maps summarizing 25-h exchange data for D178N huPrP90–231 fibrils 73 Figure 4.4. The simplest model of recombinant PrP amyloid consistent with EPR experimental data 77 Figure 5.1. Kinetic curves for deuterium incorporation by individual peptic fragments for ShaPrP90–231 monomer , spontaneously converted amyloid fibrils and the PMCA product 85 Figure 5.2. Typical spontaneously converted amyloid fibrils of ShaPrP90–231 as seen by atomic force microscopy 87 Figure 5.3. MS spectra of doubly-charged peptic fragment 145-154 illustrating the bimodal envelopes of isotope peaks 89 Figure 5.4. Deuterium incorporation of all peptic fragments for ShaPrP90–231 fibrils after 2 h and 25 h of exchange 91 Figure 5.5. The fractions of highly protected species for different peptic fragments in the PMCA product formed by ShaPrP90-231 92 Figure 5.6. H/D exchange protection maps summarizing 25-h exchange data for spontaneously converted amyloid fibrils and highly protected ix PrPPMCA species formed by ShaPrP90-231 94 Figure 5.7. In vitro conversion of recombinant Sha 90-231 to PK-resistant form by PMCA seeded with purified PrPSc from 263K-infected hamster brain 97 Figure 6.1. Peptide mapping for wild-type huPrP90-231 digested by protease type XVIII at enzyme/protein ratio (wt/wt) of 4 100 x ACKNOWLEDGEMENTS First and most importantly, I would like to thank my advisor Dr. Witold Surewicz. Without his continued support and guidance, I couldn’t have finished this work. I would also like to acknowledge my committee members including Dr. Irene Lee, Dr. Mary Barkley, Dr. James Burgess and Dr. Patrick Wintrode for their critical review of my doctoral work. I want to express many thanks to my colleagues: to Dr. Krystyna Surewicz, for her continued encouragement and help; to Dr. Jonathan Cannon and Dr. Nathan Cobb, for proofreading this thesis; to Dr. Jae-Il Kim, for preparing all PMCA samples for me; to all former members of the Surewicz lab, particularly to Drs. David Vanik, Bishwajit Kundu, Constantin Adrian Apetri and Eric Jones for all time we spent together. I would also like to thank Dr. Patrick Wintrode and his student Yuko Tsutsui, for their help on my projects; to my co-advisor Dr.Michael Zagorski, for introducing me to amyloid field. xi LIST OF ABBREVIATION Aβ Amyloid-β AFM Atomic force microscopy BSE Bovine spongiform encephalopathy CID Collision induced dissociation CJD Creutzfeldt–Jakob disease CWD Chronic wasting disease D178N Aspartic acid to asparagine substitution at position 178 EPR Electron paramagnetic resonance ESI Electrospray ionization FAB Fast atom-bombardment FFI Fatal familial insomnia FT-ICR Fourier transform ion cyclotron resonance GPI Glycosylphosphatidylinositol GSS Gerstmann–Sträussler–Scheinker syndrome GdnHCl Guanidine hydrochloride HPLC High-performance liquid chromatography Hu Human HX Hydrogen/deuterium exchange IPTG Isopropyl-β-D-thiogalactopyranoside xii LB Luria Bertani MALDI Matrix-assisted laser desorption ionization mo Mouse MS Mass spectrometry MS/MS Tandem mass spectrometry NMR Nuclear magnetic resonance PDB Protein Data Bank PK Proteinase K PMCA Protein misfolding cyclic amplification PMSF Phenylmethylsulphonyl fluoride PrP Prion protein PrP27-30 The 27-30 kDa fragment resulted from treatment of PrPSc with PK PrPC Prion protein, cellular conformer PrPPMCA The PMCA product formed by recombinant ShaPrP90-231 PrPSc Prion protein, scrapie-associated conformer PrPSp Spontaneously formed ShaPrP90-231 amyloid fibrils SDSL Site-directed spin labeling SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Sha Syrian hamster TCEP Tris (2-carboxyethyl) phosphine hydrochloride xiii TFA Trifluoroacetic acid ThT Thioflavine T TME Transmissible mink encephalopathy TSE Transmissible spongiform encephalopathy vCJD Variant Creutzfeldt–Jakob disease xiv Structure of Prion Protein Amyloid Fibrils as Determined by Hydrogen/Deuterium Exchange Abstract by Xiaojun Lu Propagation of transmissible spongiform encephalopathies is associated with the conversion of normal prion protein, PrPC, into a misfolded, oligomeric form, PrPSc. While the high-resolution structure of PrPC is well characterized, the structural properties of PrPSc remain elusive. Here we used mass spectroscopic analysis of hydrogen/deuterium backbone amide exchange (HXMS) to examine the structure of amyloid fibrils formed by the recombinant human prion protein corresponding to residues 90-231 (PrP90-231), a misfolded form recently reported to be infectious in transgenic mice overexpressing PrPC. Analysis of H/D exchange data allowed us to map the systematically hydrogen-bonded β-sheet core of PrP amyloid to the C-terminal region (starting at residue ~169) that in the native structure of the PrP monomer corresponds to α-helix 2, a major part of α-helix 3, and the loop between these two helices.