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Studies of Amyloid Nanostructure

Sumner Makin

Clare College

University of Cambridge

This dissertation is submitted for the degree of Doctor of Philosophy.

Preface

This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text.

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Acknowledgements

Thanks to my supervisor Louise Serpell for her active encouragement, guidance and assistance throughout the project. Thusnelda Stromer for extensive discussions, her patience in testing Clearer and helpful feedback.

I am very grateful for the assistance provided by our collaborators. Professor Atkins for detailed discussions and access to facilities. Pawel Sikorski for his outstanding help with data collection, discussions and substantive suggestions for Clearer. Gayatri Chavali for her help with data collection, particularly the supply of a cryo-loop for alignment. Jan Johansson for the supply of the KFFEAAAKKFFE peptide. James Elliot for help with data analysis and Sam MacDonald for assistance with early data collection.

Professor David Lomas and his group were invaluable; Didier Belorgey, Robin Carrell, Damian Crowther, Mark Davies, Kerri Kinghorn, Meera Mallya, Elena Miranda, Richard Page, Russell Phillips, Lynda Sharp and Alison Warrington for the meetings, support and discussions.

Professor Randy Read and his group for providing a friendly environment, which was conducive to work.

Brenda Sumner and Nick, Seth, Tristan and Gully Makin, for their love, support and wonderfully critical reading.

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Abstract

Amyloid fibril self-assembly, from a disparate group of soluble precursor peptides, is central to the pathology of many diseases. Knowledge of the structure and formation of these fibrils is critical to the understanding required for the rational design of drugs capable of inhibiting fibrillogenesis and promoting disaggregation. Amyloidogenic potential is thought to be an almost universal property of protein. It is therefore desirable that the three-dimensional structure and architecture of amyloid be understood. The insolubility and texture of the amyloid fibrils frustrate the usual techniques of X-ray crystallography and solution nuclear magnetic resonance.

We have investigated the structure of amyloid fibrils formed from various peptides, including islet amyloid polypeptide (type II diabetes) and designed peptides. Computer programs have been written which enable structural analysis using data from X-ray fibre diffraction, electron microscopy and electron diffraction. Our twelve-residue, sequence-designed peptide forms fibrous nano-crystallites, which diffract to high resolution (> 0.1 nm). Our analyses favour a hydrogen-bonded -sheet as the fundamental crystalline entity within these fibrils and show how the fibril is held together. Fine structural details have been revealed, including salt-bridges and - bonding between adjacent phenylalanine residues. Consequently, the data from many different fibrils contributes to the understanding of the formation and structure of the generic amyloid fibril.

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Abbreviations

Å

Angstroms

AA

Amyloid A

AAAK

Peptide with sequence KFFEAAAKKFFE

A

Amyloid beta peptide

AFM

Atomic force microscopy

APP

Amyloid precursor protein

apoSAA

Apolipoprotein serum amyloid A

BSE

Bovine spongiform encephalopathy

CD

Circular dichroism

CJD

Creutzfeldt-Jakob disease

CR

Congo red

Cryo-EM

Cryo electron microscopy

DQCSA

Double quantum chemical shift anisotropy

DRAMA

Dipolar decoupling at the magic angle

DRAWS

Dipolar coupling in a windowless sequence

ED

Electron diffraction

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EM

Electron microscopy

EPR

Electron paramagnetic resonance

fpRFDR-CT

Finite-pulse radio-frequency driven recoupling constant time

FT

Fourier transform

FTIR

Fourier transform infrared spectroscopy

H1

First predicted -helical region, residues (109-122) of cellular prion

hCT

Human calcitonin

HDX

Hydrogen-deuterium exchange

HypF-N

N-terminal domain of the bacterial hydrogenase maturation factor HypF

IAPP

Islet amyloid polypeptide (amylin)

IgLC

Immunoglobulin light chain

JAI

Java advanced imaging library

MAS

Magic angle spinning

MS

Mass spectrometry

MQNMR

Multiple quantum nuclear magnetic resonance

NMR

Nuclear magnetic resonance

PDB

Protein data bank

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PrP

PrP

PrP

C

Sc

Prion protein

Prion protein, cellular form

Prion protein, scrapie form

REDOR

Rotational echo double resonance

RR

Rotational resonance

SANS

Small angle neutron scattering

SAXS

Small angle X-ray scattering

SDSL

Site directed spin labelling

SH3

Src-homology 3 domain

SSNMR

Solid state nuclear magnetic resonance

STEM

Scanning transmission electron microscopy

TEM

Transmission electron microscopy

TTR

Transthyretin

XD

X-ray diffraction

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Contents

Preface

 

2

Acknowledgements

3

Abstract

 

4

Abbreviations

5

Contents

8

Table of Figures

16

1

Introduction

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1.1

Biological Background

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1.1.1 Definition of Amyloid

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1.1.2 Amyloid in Disease

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1.1.3 Alzheimer’s Disease Amyloid

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1.1.4 Amyloid and Oligomer Toxicity

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1.1.5 Protein Folding and Misfolding

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1.1.6 Natural Amyloid-Like Products

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1.1.7 Bionanotechnology Applications

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1.2

Current Understanding of Amyloid Structure

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1.2.1

Introduction

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1.2.2

Problems Associated with Studying Amyloid Structure

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1.3

Techniques Used to Examine Amyloid Structure

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1.3.1 The Use of Synthetic Peptides for Studies of Amyloid Structure

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1.3.2 X-ray Diffraction

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1.3.3 Electron Microscopy

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1.3.4 Electron Diffraction

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1.4

Other Techniques Used to Examine Amyloid Structure

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1.4.1 Fourier Transform Infra Red Spectroscopy

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1.4.2 Atomic Force Microscopy

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1.4.3 Neutron Scattering

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1.4.4 Hydrogen-Deuterium Exchange

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1.4.5 Solid State Nuclear Magnetic Resonance

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1.4.6 Other Methods

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1.5

Electron Microscopy Theory

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1.5.1 Beam Specimen Interaction

50

1.5.2 Imaging

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1.6

Diffraction Theory

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1.6.1

Introduction

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1.6.2

Fourier Transform

53

 

1.6.3 Convolution Theorem

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1.6.4 Sample Texture

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1.6.5 Ewald Sphere

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1.6.6 Problems

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1.7

Experimental Methods for X-ray Fibre Diffraction

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1.7.1 Introduction

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1.7.2 Glass Capillary and Stretch Frame

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1.7.3 Magnetic Field

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1.7.4 Mat

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2

Application for the Structural Analysis of Amyloid

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2.1 Abstract

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2.2 Introduction

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2.3 Preparation

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2.3.1 Format Conversion

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2.3.2 Centring

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2.3.3 Background Removal

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2.3.4 Contrast Enhancement

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2.4

Peak Measurement

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2.4.1 Automated

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2.4.2 Manual

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2.5

Unit Cell Determination

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2.5.1 Search

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2.5.2 Spot Position Predictor

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2.6

Simulation of Amyloid Fibre Diffraction Patterns

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2.6.1 Sampling of Intensities in Reciprocal Space

77

2.6.2 Reflection Shape

80

2.6.3 Structure Factors

81

2.6.4 Other Factors

82

2.6.5 Optimisation

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2.6.6 Automation

84

2.6.7 Testing

84

2.7

Other Features

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2.7.1 Introduction

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2.7.2 Fourier Space Operations

86

2.7.3 Determining the Repeat Distance from Micrographs

86

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2.7.4

General Features

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2.7.5 PDB Display and Manipulation

89

2.7.6 Image Mathematics

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2.8

Discussion

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2.8.1 Comparison of Clearer with Other Programs

90

2.8.2 Application Use

91

2.8.3 General Improvements

92

2.8.4 Specific Improvements

93

2.9

Conclusion

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3

Characterisation of Islet Amyloid Polypeptide Fibrils

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3.1 Abstract

95

3.2 Introduction

95

3.3 Methods

98

3.3.1 Peptide and Incubation

98

3.3.2 Electron Microscopy

98

3.3.3 Cryo Electron Microscopy

98

3.3.4 Electron Diffraction

99

3.3.5 X-ray Diffraction

99

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3.4

Results

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3.4.1 Negative Stain Electron Microscopy

100

3.4.2 Platinum/Carbon Shadowed Electron Microscopy

100

3.4.3 Cryo Electron Microscopy

101

3.4.4 Electron Diffraction

103

3.4.5 X-ray Diffraction

105

3.4.6 Structural Interpretation

108

3.5

Discussion

109

3.5.1 General Fibril Morphology

109

3.5.2 Understanding of Structure Derived from Diffraction Data

110

3.5.3 Hypothetical Modelling

111

3.5.4 Conclusion

112

4

Molecular Basis for Fibril Formation and Stability

114

4.1 Abstract

114

4.2 Introduction

114

4.3 Methods

116

4.3.1 Peptide and Incubation

116

4.3.2 Electron Microscopy

116

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4.3.3

Electron Diffraction

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4.3.4 X-ray Diffraction

117

4.3.5 Structural Determination

117

4.4

Results

118

4.4.1 Electron Microscopy

118

4.4.2 X-ray Fibre Diffraction

118

4.4.3 Electron Diffraction

119

4.4.4 Indexing

120

4.4.5 Modelling

122

4.5

Discussion

129

4.5.1 The Role of Side-chains in Amyloid Formation and Structure

129

4.5.2 Conclusion

131

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Discussion

133

 

5.1

Models of Amyloid Structure

133

5.1.1 Introduction

133

5.1.2 Largely Native Structures

133

5.1.3 Water-Filled Nanotube

136

5.1.4 General -Helices

138

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5.1.5

Cross- Tubule

140

 

5.1.6

Conventional Cross- Structure

141

5.2

Conclusion

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Bibliography

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Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Figure 1.8

Figure 1.9

Figure 1.10

Figure 1.11

Figure 1.12

Figure 1.13

Figure 1.14

Figure 1.15

Table of Figures

The characteristic cross- pattern

Schematic showing cleavage of A from APP

Structural hierarchy in amyloid fibrils

Structure of transthyretin amyloid

Fibre diffraction patterns of A (11-25)

Electron microscope images of A (11-25) and A (1-40) fibrils

Electron diffraction pattern from IAPP amyloid fibrils

Atomic force micrograph of Sup35 fibrils

Summary of NMR distance information for A (10-35)

Diffraction geometry

Side view of the diffraction geometry showing circle traced out by a reflection

Side view of the diffraction geometry; the circle centred at the fibre axis does not intersect with the Ewald sphere

Side view of the diffraction geometry; the case in which no diffraction occurs

Unaligned A (1-40) fibrils and stretch frame aligned A (11- 25) fibrils

Stretch frame used to align fibrils

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21

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32

35

36

39

41

44

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56

57

58

58

60

61

Figure 1.16

Orientation of fibres after alignment

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Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 2.9

Figure 2.10

Figure 2.11

Figure 2.12

Figure 2.13

Figure 2.14

Figure 2.15

Procedure for processing general fibre diffractograms

Centring of the diffraction pattern

Image histogram and cumulative frequency distribution

Circularly symmetric background reduction

Comparison of electron diffraction pattern with contrast- enhanced version

Screenshot of the automated peak finder

Manually measuring the resolution of a peak

Indexing of reflections and determination of the unit cell

Diffraction simulation window for MacOS X

Process by which simulated diffractograms are calculated

Reflection shapes for a true fibre and the general case

Simulated and empirical diffraction patterns for A (11-25) fibrils

Simulated and empirical diffraction patterns for cellulose triacetate I

Graph relating visibility to length of box

Superposition of images

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67

70

70

72

73

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76

77

79

81

85

85

85

89

Figure 3.1

Negative stain electron micrograph of IAPP amyloid fibrils

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Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Platinum/carbon shadowed electron micrograph of mature IAPP amyloid fibrils

Cryo-electron micrographs of IAPP fibrils and Fourier transform

Electron diffraction patterns before and after dehydration

X-ray fibre diffraction patterns from IAPP fibrils

Negative stain electron micrographs of AAAK amyloid

X-ray fibre diffraction pattern from amyloid nanocrystals

Electron diffraction pattern from the fibrous nanocrystals

Comparison between simulated and observed diffraction patterns for incorrect model

Comparison between simulated and observed diffraction patterns for proposed model

Orientations of aromatic rings

Structure of amyloid nanocrystals

Pair of transthyretin molecules

Transthyretin amyloid fibrils

Perutz’s model of amyloid

Antiparallel -helix

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101

103

105

107

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119

120

125

126

127

128

135

136

138

140

Figure 5.5

Possible structure of A (1-40)

141

Figure 5.6

Models of insulin fibrils

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144

1

Introduction

1.1 Biological Background

Chapter 1. Introduction

1.1.1 Definition of Amyloid

Rokitansky is generally credited with the first description of amyloid in 1842 as a firm, greyish, “lardaceous-gelatinous” substance infiltrating the livers of patients with syphilis and tuberculosis (Rokitansky 1846). In 1854, Rudolph Virchow gave amyloid its name after noticing that it stained pale blue with iodine and sulphuric acid, similar to corpora amylacea in brain tissue. He believed that it was similar to cellulose and consequently amyloid means starch (amylase) like (Virchow 1854; Sipe and Cohen

2000). It is now known that this is due to associated proteoglycans and glycosaminoglycans and that the core of amyloid is protein (Snow et al. 1987; Kisilevsky and Snow 1988).

Amyloid fibrils are formed by the polymerisation of a normally soluble, non-toxic protein into extracellular, insoluble aggregates with a large molecular weight. Amyloid is defined by a series of empirical observations. Specific chemical staining forms part of the definition. After staining with Congo red (CR), plaques composed of the fibrils appear orange in tissue sections. An apple green birefringence pattern is seen when the stained sample is viewed between crossed polarisers, (Puchtler et al. 1961; Puchtler and Sweat 1965). The birefringence is due to an ordered structure in which dye binding sites are symmetry related (Carter and Chou 1998). Amyloid can be extracted using a water purification method (Pras et al. 1968). Binding to the histological benzothiazole dye thioflavine T results in a shift in fluorescence (LeVine 1993). Neither dye is completely specific but each empirical observation forms part of the definition. The aggregates are rich in -structure, with resistance to proteolysis and even strong chemical denaturants. Circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) both show a high percentage of -structure (Hilbich et al. 1991). X-ray diffraction reveals a characteristic pattern, similar to that observed in silk fibroin and described as cross- . The cross- pattern is comprised of two

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Chapter 1. Introduction

reflections, a sharp one at 4.7 Å parallel to the direction of the fibre and a more diffuse spot between 10 and 11 Å perpendicular to the fibre direction (Figure 1.1) (Eanes and Glenner 1968). Electron microscopy (EM) reveals fibrils that are unbranching, 70 to 120 Å in diameter and of indeterminate length (Cohen et al. 1982).

10-11 Å 4.7 Å Bundle of fibres X-ray beam Equatorial direction
10-11 Å
4.7 Å
Bundle of fibres
X-ray beam
Equatorial direction

Figure 1.1. The X-ray fibre diffractogram of amyloid has a characteristic cross- pattern, with the 4.7 Å reflection along the same direction as the fibrils.

1.1.2 Amyloid in Disease

The diseases collectively known as the amyloidoses are characterised by the deposition of extracellular amyloid fibrils. These are generally divided into five subdivisions; systemic, hereditary, central nervous system, ocular and other localized amyloidoses (Sipe and Cohen 2000). A substantial number of human and animal amyloidoses have been described to date (Table 1.1 and Table 1.2, respectively). Further examples continue to be found; amyloid deposits have been discovered in the cell-free fraction of bronchoalveolar lavage fluid in patients with pulmonary alveolar

proteinosis (Gustafsson et al. 1999). Whilst amyloid is defined to be extracellular, there are intracellular deposits thought to be otherwise of the same structural class.

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Chapter 1. Introduction

These include paired-helical filaments formed from tau in Alzheimer’s disease (Grundke-Iqbal et al. 1986), Lewy bodies formed from -synuclein in Parkinson’s disease (Spillantini et al. 1998) and polyglutamine huntingtin deposits in Huntington’s disease (Chen et al. 2002). Perhaps the correct nomenclature for such material is amyloid-like. Nevertheless, the literature does not generally use such a term (Perutz et al. 2002) and the distinction becomes impractical in the case of amyloid formed from synthetic peptide fragments or proteins not normally associated with disease.

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Chapter 1. Introduction

Table 1.1. Classification and nomenclature of the human amyloidoses, assembled from (Husby et al. 1994; Kelly 1996; Sunde et al. 1997; Xing and Higuchi 2002; Blumenthal 2004).

Amyloidogenic protein

Precursor to Amyloidogenic Protein

Syndrome

Immunoglobulin light chain (IgLC) or fragments of V L domain

Monoclonal immunoglobulin light chain (i.e. or )

AL amyloidosis: primary isolated or associated with multiple myeloma

AH

Immunoglobulin heavy chain ( )

AH amyloidosis: isolated

76-residue N-terminal fragment of amyloid A (AA)

Apolipoprotein serum amyloid A (apoSAA)

Secondary systemic amyloidosis: resulting from infections, chronic inflammation, tumours, familial Mediterranean fever, tumour necrosis factor receptor associated periodic syndrome, Muckle-Wells syndrome

Mutant transthyretin

Mutant transthyretin

Familial amyloid polyneuropathy I and II

Transthyretin (TTR) or its fragments

Native transthyretin

Senile systemic amyloidosis

2 -microglobulin

2 -microglobulin

Haemodialysis related amyloidosis: associated with chronic endstage renal failure

Approximately 90-residue apolipoprotein A-I N-terminal fragments

Apolipoprotein A-I variants

Familial amyloid polyneuropathy III

Apolipoprotein A-II

Apolipoprotein A-II

Familial amyloidosis

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Chapter 1. Introduction

Amyloidogenic protein

Precursor to Amyloidogenic Protein

Syndrome

71-residue gelsolin fragment

Gelsolin

Familial amyloid polyneuropathy IV

Lysozyme or its fragments

Lysozyme variants (not native)

Hereditary non-neuropathic systemic amyloidosis (Ostertag- type)

110-residue cystatin C fragment

Cystatin C

Hereditary cerebral haemorrhage

Amyloid peptide (A ) (39 to 43 residues)

Amyloid precursor protein (APP), native and variants

Alzheimer's disease, Down syndrome, hereditary or sporadic cerebral amyloidosis

Prion protein, scrapie form (PrP Sc ) (not cellular form, PrP C )

Prion precursors, native and variants

Transmissible spongiform encephalopathies, including kuru, Creutzfeldt-Jakob Disease (CJD) and variant Creutzfeldt-Jakob Disease

Calcitonin or its fragments

Procalcitonin

Medullary thyroid carcinoma associated amyloidosis

Atrial natriuretic factor

Atrial natriuretic factor

Isolated atrial amyloidosis

Islet amyloid polypeptide (IAPP)

Pro-islet amyloid polypeptide

Type 2 diabetes, insulinoma

Insulin

Insulin

Injection localised amyloidosis, insulinoma

Prolactin

Prolactin

Aging pituitary prolactinoma

ABri

BRI-L

Familial British dementia

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Chapter 1. Introduction

Amyloidogenic protein

Precursor to Amyloidogenic Protein

Syndrome

Keratoepithelin

Keratoepithelin

Keratoepithelin amyloidosis: lattice dystrophies of the cornea

Lactoferrin

Lactoferrin

Familial cornea amyloidosis

Lactadherin

Lactadherin

Aortic medial amyloidosis

Keratin

Keratin

Primary localised cutaneous amyloidosis

Fibrinogen -chain variant fragments

Fibrinogen -chain variants

Hereditary renal amyloidosis

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Chapter 1. Introduction

Table 1.2. Classification and nomenclature of animal amyloidoses (Xing and Higuchi 2002).

Amyloid protein

Precursor

Syndrome

PrP Sc

Prion precursors

Transmissible spongiform encephalopathies, including scrapie (sheep and goat) and similar bovine (BSE), feline, mink, elk and mule deer diseases.

AA

Apolipoprotein serum amyloid A

Reactive AA amyloidosis (mouse)

Apolipoprotein A-II

Apolipoprotein A-II

Mouse senile amyloidosis

The amyloidoses are often cited as examples of post evolutionary disease (Csermely

2001; Dobson 2002). Modern medical practices are one cause of such disease. 2 -

microglobulin accumulates during blood dialysis, which after several years, results in

skeletal deposition of amyloid (Gejyo et al. 1985; Gejyo et al. 1986). Cannibalism in

members of the Fore tribe in Papua New Guinea caused kuru. Bovine spongiform

encephalopathy appeared as a result of feeding cows with animal material,

demonstrating an infectious route for amyloidosis (Gajdusek 1988). Many

amyloidoses are diseases of old age and the incidence of such disease has grown as

life spans have increased over the last century. In addition to humans, a relationship

between aging and amyloidosis has been examined in mice, hamsters, cats, dogs,

cattle, ducks and horses (Blumenthal 2004).

1.1.3 Alzheimer’s Disease Amyloid

Alzheimer’s disease is the best-known amyloidosis. It is estimated to cost the US

economy some $100 billion annually (Ernst and Hay 1994). Alois Alzheimer, a

Bavarian neuropsychiatrist, first noted the disease. In November 1901, a patient

referred to as Auguste D. presented with an unknown brain disorder; the 1906 post-

mortem revealed lesions in the brain (Alzheimer 1907; Graeber and Mehraein 1999;

Gorman and Chakrabartty 2001). Some twenty years ago, the principal component of

these plaques was found to be amyloid formed from A (Glenner and Wong 1984;

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Chapter 1. Introduction

Masters et al. 1985). A is a 4-kDa peptide cleaved from amyloid precursor protein (APP) (Kang et al. 1987) by -secretase (beta-site APP cleaving enzyme) followed by -secretase as shown in Figure 1.2. Most APP is degraded in the secretory pathway before reaching the cell surface by -secretase. A is always present in healthy subjects (Seubert et al. 1992) and is a result of normal proteolytic processing (Shoji et al. 1992). The predominant A species in vivo has forty residues, whilst A (1-42) is the major component of senile plaques and may nucleate amyloidogenesis (Jarrett et al. 1993; Iwatsubo et al. 1994). All species of in vivo A are able to cofibrilise (Hasegawa et al. 1999). Alzheimer’s disease’s prominence among the amyloidoses has resulted in substantial effort being expended in examining fibrils formed from A (Serpell 2000).

APP Extracellular Space Membrane Cytosol NH2 COOH 671 714 -Secretase -Secretase -Secretase 40 4 243
APP
Extracellular Space
Membrane
Cytosol
NH2
COOH
671
714
-Secretase
-Secretase
-Secretase
40
4
243
A
12345678901234567890123456789012345678901234567890
1
10
20
30
40
NH2
VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKK COOH
NL
GQ
VI
X
G
F
Swedish double
Flemish
Dutch
Florida
London
Australia
mutation
mutation
mutation
mutation
mutations
mutation

Figure 1.2. Schematic showing cleavage of A from APP; the A sequence is in bold type (Storey and Cappai 1999; Burkoth et al. 2000). A dashed box indicates the transmembrane domain. Vertical lines show the sites of secretase cleavage. Hydrophobic regions of A are in blue.

1.1.4 Amyloid and Oligomer Toxicity

The amyloid hypothesis suggests that the formation and accumulation of amyloid is central to disease pathology. Amyloid toxicity has been demonstrated on many occasions (Kelly 1996; 1998b). Alzheimer's amyloid interacts with endothelial cells to

produce superoxide free radicals (Thomas et al. 1996; Hardy and Selkoe 2002) and is responsible for the destruction of neurons (Lansbury 1999). Prion aggregates are responsible for disease transmission in the transmissible spongiform encephalopathies

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Chapter 1. Introduction

(Prusiner 1998). In other cases, it is the sheer quantity of amyloid; kilograms of this material disrupt organs such as the liver or spleen (Tan and Pepys 1994; Pepys 1996).

The oligomer hypothesis takes the view that it is the oligomeric precursors to amyloid which are primarily responsible for toxicity (Kirkitadze et al. 2002; Stefani and Dobson 2003). Fibrils are clearly immobile which raises the question of how they can effect change in nearby cells. One piece of evidence, apparently contrary to the amyloid hypothesis, is that the degree of cognitive impairment shows poor correlation with the quantity of amyloid deposited in the brain in Alzheimer’s disease (Morris et al. 1996). Furthermore, studies using amyloid fibrils not associated with the amyloidoses show that they can have a similar effect on cells as those made of proteins linked to disease (Stefani and Dobson 2003). These include aggregates formed from the src-homology 3 (SH3) domain of the phosphatidyl inositol-3-kinase (Frederikse 2000) and the N-terminal domain of the bacterial hydrogenase maturation factor HypF (HypF-N) (Chiti et al. 1999). The studies observed a series of well- defined, pre-fibrillar species, which substantially impaired cultured cell viability. Mature amyloid was not found to have any substantial effect on the cultures. This implies that cytotoxicity is due to the supramolecular structure of pre-fibrillar aggregates and not the amino acid sequence (Bucciantini et al. 2002). Alzheimer’s A oligomers have been studied in detail (Lambert et al. 1998; Hartley et al. 1999; Lin et al. 1999; Walsh et al. 1999; Bhatia et al. 2000; Monji et al. 2000; Nilsberth et al. 2001; Walsh et al. 2002). Similar studies concerning familial amyloidotic polyneuropathy (Sousa et al. 2001) and Parkinson’s disease (Conway et al. 2000b; Goldberg and Lansbury 2000) have been conducted. These studies suggest that oligomers may interact with cell membranes, leading to oxidative stress and increases in free Ca 2+ causing apoptosis or necrotic cell death.

It should not be concluded from the above studies that amyloid is unimportant. The amyloid cascade hypothesis takes account of both oligomers and amyloid (Hardy and Selkoe 2002). Neither should it be neglected that amyloid fibrils are large inactive reservoirs of toxic species (Walsh et al. 2002). Contrarily, mature fibrils are a potential sink for toxic agent. Any doubt as to harm caused by amyloid is removed in

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Chapter 1. Introduction

those diseases in which amyloid forms the greater part of the diseased organ (Pepys

1996).

Once a link between amyloid and amyloidosis pathology has been established, the fibril structure is more than intellectual curiosity. Understanding of the structure of amyloid is necessary for the rational design of drugs to prevent aggregation and promote disaggregation. One way in which such an agent can be devised is by using peptide mimetics (Lashuel et al. 2000). Examples of agents have been conceived in many cases (Kolstoe and Wood 2004), including IAPP (Kapurniotu et al. 2002; Rijkers et al. 2002; Scrocchi et al. 2002) and A (Tjernberg et al. 1996; Talaga 2001).

1.1.5 Protein Folding and Misfolding

Knowledge of amyloid structure also contributes to the understanding of protein folding and misfolding. Amyloidogenic potential may be an almost universal property of protein (Stefani and Dobson 2003). This important alternative protein-folding state addresses fundamental aspects of biophysics. Indeed, a change in our understanding of protein folding, previously established for soluble globular proteins, may be

required. Amyloid precursor proteins appear to be extremely diverse. There is considerable variation in size and sequence (Fandrich and Dobson 2002) with molecular masses varying from less than a single kilodalton to tens of kilodaltons. Precursors do not share a common tertiary structure; they may even be unstructured or -helical. In spite of this, amyloid fibrils have a similar external morphology and internal structure. The molecular basis of amyloid toxicity also seems to display common features (Stefani and Dobson 2003).

Proteins not associated with disease have been shown to form fibrils in vitro. These include SH3 (Guijarro et al. 1998), acylphosphatase (Chiti et al. 1999), cold shock protein A (Alexandrescu and Rathgeb-Szabo 1999) and cold shock protein B (Gross et al. 1999). Conditions for amyloid fibril formation include a low pH, lack of specific ligands, high temperature and moderate concentrations of salts or co-solvents. Although amyloid is very stable, there might be a significant activation energy barrier to amyloidogenesis (Kusumoto et al. 1998). Mutations associated with hereditary

29

Chapter 1. Introduction

amyloidoses often destabilise the native state of the protein, thus reducing this barrier (Kelly 1998a; Dobson 1999; Radford and Dobson 1999). Natural proteins in their native states deploy several strategies to avoid intermolecular association of -strands (Richardson and Richardson 2002). Strategies include covering with helical or non- repetitive structure, having no edges ( -barrel), a sharp bulge in the -sheet or prolines and charged residues. Statistical analysis of globular protein sequences shows that nature disfavours both sequences of alternating polar and non-polar residues (Broome and Hecht 2000) and clusters of several consecutive hydrophobic residues (Schwartz et al. 2001). Most aggregation disease proteins are either secreted or membrane bound, or a fragment of such a protein. Although the aggregated form may have the lowest Gibbs energy (Gazit 2002a), the removal of the source of unaggregated protein results in disaggregation (Pepys et al. 2002). It is therefore important to know how these proteins adopt highly organised multi-molecular structures that are not specifically encoded for in the sequence (Uversky 2002). This is important in protein handling and production, where general protein aggregation is a serious problem (Clark 1998).

1.1.6 Natural Amyloid-Like Products

Amyloid-like material is also a natural product (Kelly and Balch 2003), the earliest example being the silk of the egg-stalk of the lacewing Chrysopa (Parker and Rudall 1957; Geddes et al. 1968). The cross- structure is also found in mammalian ocular lenses (Frederikse 2000) and it is hypothesised that fibrillar deposits of serum amyloid

A induce lysis of bacterial cells, thus protecting the host in chronic amyloid diseases (Hirakura et al. 2002). Curli are highly aggregated amyloid-like fibres produced by Escherichia coli and Salmonella (Chapman et al. 2002) and cross- spider silk fibres also have the characteristic features of amyloid (Li et al. 2001).

1.1.7 Bionanotechnology Applications

Finally, the emerging field of bionanotechnology offers industrial application for

amyloid (Zhang 2003; Waterhouse and Gerrard 2004). This requires a fundamental understanding of the macromolecules, which are the elemental components from which nanostructures are constructed. Nano-structural components require the

30

Chapter 1. Introduction

properties of structural compatibility and chemical complementarity. Amyloid precursors satisfy this requirement, so investigation is needed to understand the structure, assembly and dynamic behaviour of amyloid fibres (Mihara and Takahashi 2001; Zhang et al. 2002). By way of demonstration, a conducting nanowire was constructed by using Sup35 amyloid as a natural scaffold to align gold particles (Scheibel et al. 2003). Similarly, silver nanowires were also assembled; ionic silver was reduced inside amyloid nanotubes formed from diphenylalanine (Reches and Gazit 2003). A great many potential applications have been envisaged including using fibrils in catalysis and electronics, as a plastic, a support for the growth of cells or a therapy for treating humans and animals (Dobson and MacPhee 2004).

1.2 Current Understanding of Amyloid Structure

1.2.1 Introduction The characteristic cross- diffraction pattern gives insight into the basic structure of amyloid. Peptides are in a -strand conformation, with a distance of 6.9 Å between adjacent pairs of residues along the direction of the chains (Figure 1.3). -strands, perpendicular to the direction of the fibre, are spaced 4.7 Å apart and laterally associated to form a -sheet ribbon. These -sheets run along the direction of the fibre and are stacked approximately 10 to 11 Å apart. The laminated -sheets comprise the protofilaments, which form the fibril (Figure 1.3). The ribbons may be twisted and the protofilaments themselves intertwined (Lashuel et al. 2000). Protofilaments should be distinguished from the pre-fibrillar intermediates known as protofibrils (Harper et al. 1997b; 1999; Walsh et al. 1999). Substantive evidence for protofilaments is provided by electron microscopy. Helical reconstruction showed twisted structure for SH3 and insulin fibrils (Jimenez et al. 1999; Jimenez et al. 2002). Similarly, intracellular paired-helical filaments (Crowther 1991) have been shown to have a protofilament substructure. Cross-sections of fibres analysed using single particle methods allowed the number of protofilaments to be determined (Serpell et al. 2000b). In the case of IAPP, the splitting of fibrils can be explained using protofilaments (Goldsbury et al. 1997), an electrolucent core may also be detected (Cohen et al. 1982; Serpell et al.

2000b).

31

Chapter 1. Introduction

Single

-strand

-sheets

Protofilament

Two to six protofilaments

Fibril

10-11 Å 4.7 Å
10-11 Å
4.7 Å

6.9 Å

Two to six protofilaments Fibril 10-11 Å 4.7 Å 6.9 Å 25-30 Å 70-120 Å Figure
Two to six protofilaments Fibril 10-11 Å 4.7 Å 6.9 Å 25-30 Å 70-120 Å Figure
Two to six protofilaments Fibril 10-11 Å 4.7 Å 6.9 Å 25-30 Å 70-120 Å Figure
Two to six protofilaments Fibril 10-11 Å 4.7 Å 6.9 Å 25-30 Å 70-120 Å Figure

25-30 Å

70-120 Å

Figure 1.3. The structural hierarchy in amyloid. Fibrils are composed of protofilaments. Protofilaments are built from laminated -sheet ribbons.

Amyloid fibrils formed from the same precursor peptide may have different structures

at all levels of detail. Under the same experimental conditions, differing numbers and

organisations of protofilaments are observed, whilst the structure within the

protofilament appears to remain the same (Fraser et al. 1991b; Bauer et al. 1995;

Goldsbury et al. 1997; Jimenez et al. 1999; Bouchard et al. 2000; Jimenez et al.

2002). Several varieties of fibril may be present in a single electron micrograph. This

polymorphism was demonstrated in many types of in vitro amyloid, including A ,

calcitonin, IAPP, SH3 and insulin fibrils. Cryo-electron microscopy of insulin fibrils

combining helical reconstruction with single particle analysis revealed diverse low-

resolution three-dimensional fibrils containing two, four and six protofilaments.

Atomic force microscopy also revealed similar diversity in amyloid derived from A

(Goldsbury et al. 2001) and 2 -microglobulin (Kad et al. 2001; Kad et al. 2003).

Cryo-electron microscopy of ex vivo material from patients with hereditary non-

neuropathic, systemic amyloidosis demonstrated that spontaneous polymorphism is

also present in disease fibrils (Jimenez et al. 2001).

In the case that experimental conditions are changed, then both the arrangement of

protofilaments and their internal structure may differ. Polymorphism may be

32

Chapter 1. Introduction

attributed to staining conditions (Cohen et al. 1982), since ion concentration has been shown to affect fibril conformation (Fraser et al. 1991b). Solid-state nuclear magnetic resonance of fibrils formed from A (11-25) and human calcitonin at various pHs both showed structural diversity at the atomic level, whilst maintaining a cross- structure (Naito et al. 2004; Petkova et al. 2004).

1.2.2 Problems Associated with Studying Amyloid Structure

Structural studies of amyloid fibrils are impeded by a series of problems relating to the characteristics of amyloid fibrils. They are neither three-dimensionally crystalline nor soluble (Lansbury 1992; Kelly 1997), which prevents the use of conventional

techniques such as single-crystal X-ray crystallography and solution nuclear magnetic resonance. Although amyloidogenesis resembles crystallisation (Jarrett and Lansbury 1993), single crystal formation has proved as difficult as for insoluble fibrous silk proteins (Lotz et al. 1982). Disorder within the sample is a serious issue since it reduces the amount of available information. Polymorphic structures also present problems when attempting to compare data from X-ray diffraction and electron microscopy (Diaz-Avalos et al. 2003a).

1.3 Techniques Used to Examine Amyloid Structure

1.3.1 The Use of Synthetic Peptides for Studies of Amyloid Structure

Structural examination requires as much order within the sample as possible. Ex vivo material is rarely used, since synthetic samples can be grown in the conditions to maximise orientational order. Additionally, synthetic peptides are more readily available and purification is not required. Full-length peptides form amyloid that appears to be less ordered than truncated versions. A (11-25) amyloid fibrils have a similar morphology and pH dependence as full-length A (Fraser et al. 1991b) and may form the core of fibrils formed from full-length A . Similar results have been shown for other A fragments including A (11-28), A (13-28), A (15-28) and A (29-42) (Castano et al. 1986; Gorevic et al. 1987; Halverson et al. 1990; Barrow and Zagorski 1991; Fraser et al. 1991a; Barrow et al. 1992; Burdick et al. 1992; Fraser et al. 1992b).

33

Chapter 1. Introduction

Other peptides, including human non-disease related peptides, such as SH3 and those that are sequence designed are employed as amyloid precursors. These can be used to investigate the generic nature of amyloidogenesis and the importance of side chain interactions. Amyloid formed from proteins with four to six residues has all the characteristics of fibrils formed from 100-residue peptides (de la Paz et al. 2002; Reches et al. 2002; Tjernberg et al. 2002). The advantages of such an approach are a far higher quality of experimental data, the fibrils being more amenable to alignment and that it also allows the removal of disordered regions.

1.3.2 X-ray Diffraction

Much of the published information on protein structure is based on X-ray diffraction data (XD). Detailed information at high resolution (approximately 1 to 3 Å) can be obtained from the analysis of X-ray scattering patterns. Although single-crystal X-ray crystallography is not possible, X-ray fibre diffraction has proved to be a very powerful technique. The primary evidence for amyloid fibrils having a common core structure composed of laminated, continuous -sheets running along the fibre arises

from X-ray fibre diffraction (Eanes and Glenner 1968; Blake and Serpell 1996; Sunde et al. 1997).

The cross- pattern’s characteristic features were first described in diffraction from the egg stalk of the lacewing Chrysopa (Geddes et al. 1968). The proposed structure was formed from extended -strands folded back on themselves, forming a -sheet

25 Å wide. The -ribbons described were flat and stacked face-to-face (Pauling and

Corey 1951). The same pattern was observed for amyloid formed from the Bence Jones protein (Eanes and Glenner 1968).

Indexing of meridional diffraction signals suggested the presence of helical repeats. Transthyretin amyloid was found to have a helical pitch of 115.5 Å, corresponding to

24 -strands and a twist of 15 degrees per strand (Figure 1.4). A twisted -pleated

sheet has been shown to have a lower free energy than an untwisted sheet (Chothia 1973). On this basis, a model of the core structure of the generic amyloid fibril was built (Blake and Serpell 1996; Sunde et al. 1997).

34

Chapter 1. Introduction

Chapter 1. Introduction Figure 1.4. The structure of transthyretin amyloid, based on X-ray diffraction data (Serpell

Figure 1.4. The structure of transthyretin amyloid, based on X-ray diffraction data (Serpell et al.

1997).

Examination of peaks on the equator of X-ray diffraction patterns allows the number and arrangement of protofilaments to be interpreted. A (1-40) fibrils were modelled using three to five tubular protofilaments, each built from a pair of -sheets (Malinchik et al. 1998). Small angle X-ray scattering (SAXS) studies on fibrils formed from a wide variety of A fragments arrived at similar models involving hollow cylinders (Lu et al. 2003) or bundles thereof (Inouye et al. 1993). In one case it was not possible to distinguish between an 86 Å diameter tubule or walls of cross- sheets 71 Å apart (Kirschner et al. 1987). Difficulties may also arise in the event of polymorphism being present in the sample.

Much of the early work on amyloid used fibrils formed from A fragments and mutants thereof (Kirschner et al. 1987; Halverson et al. 1990; Fraser et al. 1992b; Inouye et al. 1993; Fraser et al. 1994; Inouye and Kirschner 1996). A detailed structure of A (11-25) fibrils was built based on X-ray fibre diffraction data (Sikorski et al. 2003). Diffraction patterns were recorded from the magnetically aligned specimen using three mutually orthogonal beam directions, one of which was parallel to the magnetic field. All three patterns were different, suggesting that the fibrils are composed of crystallites with a preferred orientation. The A (11-25) peptide was in an extended -strand conformation; these -strands were then stacked to form an antiparallel -sheet. The pseudo-unit cell was monoclinic ( = 122°) with dimensions a = 9.42 Å, b = 25 Å and c = 6.9 Å. There was therefore a slip along the chain direction of 6.9 Å, which is twice the length of an amino acid unit in an extended conformation.

Other studies have postulated similar cross- structures from X-ray data in the cases of amyloid fibrils formed from short prion protein (PrP) fragments (Nguyen et al.

35

Chapter 1. Introduction

1995); the first predicted -helical region, residues (109-122) of PrP C (H1) (Inouye and Kirschner 1996); eleven-residue N-termini of the apoSAA family (Kirschner et al. 1998) and IAPP (Makin and Serpell 2004b).

b b a c a c
b
b
a c
a
c

Figure 1.5. Fibre diffraction patterns from A (11-25) fibrils (Sikorski et al. 2003). Images a and b are perpendicular to the direction of preferred orientation, while image c is parallel to the fibre axis.

1.3.3 Electron Microscopy

Transmission electron microscopy (TEM) is a form of microscopy in which the properties of the specimen are measured in terms of the specimen’s interaction with a beam of electrons. This beam is passed though lenses composed of electric and magnetic fields allowing very high-resolution data to be obtained in a manner analogous to the operation of a light microscope. Electron microscopy does not require a crystalline sample and the result need not be implicitly averaged as with neutron and X-ray diffraction experiments. Unfortunately, the contrast of micrographs of many biological samples is poor and increasing the flux of the electron beam to compensate results in damage to the specimen. Cryo electron microscopy (cryo-EM) reduces the radiation damage by cooling the specimen using liquid nitrogen or helium. Alternatively, the specimen may be stained with a material such as uranyl

36

Chapter 1. Introduction

acetate; however the image will be of the stain rather than the specimen itself; hence little information about the interior of the fibril is revealed. Shadow electron microscopy involves imaging heavy metal atoms that had previously been deposited at an angle, delivering improved definition of vertical features. EM is a visual technique, which allows the examination of a sample to confirm the presence of ordered fibrils (Figure 1.6).

Much of the information about the number of protofilaments involved in the fibril substructure is derived from EM (Kirschner et al. 1987; Fraser et al. 1991a; Fraser et al. 1991b; Serpell et al. 1995; Goldsbury et al. 1997; Jimenez et al. 1999; Serpell et al. 2000b; Jimenez et al. 2001; Jimenez et al. 2002). Groups of filamentous structures wound around a common axis are sometimes observed, whilst cross-sections show between three and six units composing the fibril. Single particle analysis combined with helical reconstruction of micrographs has revealed low-resolution three- dimensional structures of insulin fibrils with two, four or six protofilaments (Jimenez et al. 2002). As discussed earlier, polymorphism may arise spontaneously or as a result of the differing conditions for fibril formation.

Three-dimensional reconstruction of cryo-electron microscope images resulted in

low-resolution (25 Å) structures of protofilaments for amyloid formed from SH3 (Jimenez et al. 1999) and insulin (Jimenez et al. 2002). Ex vivo Asp67His lysozyme and Leu60Arg apolipoprotein were also studied but structural variability and disorder prevented reconstruction (Jimenez et al. 2001). A great diversity of helical fibrils was observed. Single particle averaging and analysis was combined with helical reconstruction, which dealt with the problem of variable pitch (Boettcher et al. 1996; Jimenez 2000). The resulting structures showed how the protofilaments were arranged around an electrolucent core. These structures implied possible arrangements of strands within the protofilaments. The model suggested that protofilaments had the same overall twist as the fibril itself. SH3 fibrils were composed of four protofilaments with cross-section dimensions of 20 by 40 Å and a crossover repeat of ~600 Å. Insulin fibrils had a larger cross-section of 30 by 40 Å with crossover repeats of between 355 and 900 Å depending on the number of protofilaments in the fibril.

37

Chapter 1. Introduction

Models of lysozyme fibrils, with a periodicity of ~3000 Å, were also constructed; a six-protofilament model most closely matched the experimental results.

Images of fibrils composed of A (11-25) compared to A (1-42) showed that fibrils formed from the shorter peptide were more consistently homogenous, straight and

uniform with higher contrast and better-defined edges (Serpell and Smith 2000). Tjernberg found that making substitutions and deletions in the A (14-23) peptide had

a significant effect on the morphology of the amyloid formed, however all A

peptides longer than eleven residues formed fibrils of similar morphology (Tjernberg et al. 1999). Structural elucidation of the protofilament has then proceeded by either three-dimensional reconstruction (Jimenez et al. 1999) or direct visualisation (Serpell and Smith 2000). Fourier transforms of regions of electron micrographs showed a similar cross- pattern to X-ray diffractograms and in the case of A (11-25) fibrils, striations 4.7 Å apart were visible. These striations perpendicular to the fibre axis

were clear after the application of translational averaging. They have been interpreted

as being stacks of -strands. It was not known whether the fibril was twisted so the

fibre was cut into boxes containing a specified number of striations and boxes with the same number of striations averaged along the fibre. Boxes containing six or twelve striations gave qualitatively better results than those with four, eight or ten. The core of the fibril also appeared to be electrolucent, which was consistent with images of the cross-sections of cores that have been processed using single-particle averaging techniques (Kirschner et al. 1987; Fraser et al. 1991a; Fraser et al. 1991b; Serpell et al. 1995; Serpell et al. 2000b). Electron micrographs of A (10-35) fibrils showed a 1200 Å helical twist, allowing a model to be built (Lakdawala et al. 2002).

Scanning transmission electron microscopy (STEM) uses a smaller probe than TEM, which scans across the image. It allows the mass per unit length of fibrils to be determined by comparison with a standard, such as the tobacco mosaic virus. This information can be combined with data from other techniques, especially nuclear magnetic resonance, to build a model. A protofilament composed of a pair of -sheets was proposed for A (1-40) fibrils (Antzutkin et al. 2000; Antzutkin 2004; Petkova et al. 2004) and similar structures postulated for A (10-35) and A (1-42) fibrils

38

Chapter 1. Introduction

(Antzutkin et al. 2002). STEM complemented protease digestion, cryo-EM and mass spectroscopy in the analysis of Ure2p, to build a model based on a cross- core (Baxa et al. 2003). In the study of a designed seventeen-residue peptide, STEM formed part of a battery of techniques in conjunction with X-ray diffraction, TEM and nuclear magnetic resonance (Kammerer et al. 2004). It can also be used simply as another form of microscopy (Goldsbury et al. 2000a; Goldsbury et al. 2000b).

(Goldsbury et al. 2000a; Goldsbury et al. 2000b). Figure 1.6. Electron microscope images showing the

Figure 1.6. Electron microscope images showing the morphology of A (11-25) and A (1-40) fibrils (Sikorski et al. 2003).

1.3.4 Electron Diffraction

Electron diffraction (ED) patterns are theoretically very similar to X-ray diffractograms (Dyson 2004). A sample is illuminated with a beam of electrons and the resulting diffraction pattern recorded using film or another detector. A sharp, strong 4.7 Å reflection is normally very clear but little else can generally be seen. Most examples of this type of pattern are from amyloid-like fibrils formed from - synuclein (Serpell et al. 2000a), paired-helical filaments (Berriman et al. 2003) and polyglutamine fibrils (Perutz et al. 1994). In general fibre diffraction, high-resolution reflections are spread over a greater volume in reciprocal space than those at lower resolution, resulting in a lower relative intensity. If an electron beam is used, then the relative intensity of high resolution reflections are further reduced owing to changes in the structure factors. The structure factors for an electron beam are calculated from the X-ray structure factors using the Mott formula, which involves division by the distance from the origin in reciprocal space squared. Thus, the reflection intensity is

39

Chapter 1. Introduction

divided by a total factor proportional to the radius to the fourth power and hence some reflections are much stronger than others. The incident electron beam also obscures low angle data; it is very strong compared with the intensity of the reflections and saturates the film, thereby preventing their recovery.

Electrons are charged and have a far stronger interaction with matter in comparison to X-rays. Electron diffraction can therefore take advantage of a much lower beam width than X-ray diffraction; hence a far smaller sample can be examined. This enabled individual amyloid nanocrystals from a seven-residue peptide to be selected and the diffraction patterns from single crystals recorded (Diaz-Avalos et al. 2003a; b). The image area being diffracted from can be viewed in real space. Consequently, the relative orientations of fibres and their diffraction patterns can be determined. Another result of the strength of interaction is radiation damage; the microscope operator may observe the diffraction pattern fading as the irradiated region is permanently damaged. Long exposures increase the signal to noise ratio and reveal weak signals, whereas short exposures ensure that the specimen remains intact.

Quantitative analysis of intensities is further impeded by the requirement for calibration of the exposed film to establish the relationship between film transparency and incident flux density. Other electron diffraction studies have used amyloid fibrils formed from IAPP (Chapter 3) (Makin and Serpell 2004b), a twelve-residue sequence designed peptide with sequence KFFEAAAKKFFE (see Chapter 4), scrapie prion (Wille et al. 2002), Sup35 (King and Diaz-Avalos 2004) and the WW domain FBP28 (Ferguson et al. 2003).

40

Chapter 1. Introduction

4.7 Å
4.7 Å

Figure 1.7. Electron diffraction pattern from IAPP amyloid fibrils (Chapter 3).

1.4 Other Techniques Used to Examine Amyloid Structure

1.4.1 Fourier Transform Infra Red Spectroscopy

A Michelson interferometer, with a movable mirror, has an interference pattern,

which is the Fourier transform of the spectrum of the source. In Fourier Transform Infrared Spectroscopy (FTIR), this interference pattern is Fourier inverted to allow the

measurement of the absorbance spectrum of a sample (Jackson and Mantsch 1995). Normal mode analysis of simple molecules allows the theoretical spectrum to be calculated in these cases. The reverse of this process is not possible and different structures may have indistinguishable absorption spectra.

Especially in conjunction with isotopic labelling (Anderson et al. 1996), FTIR is able

to distinguish between parallel and antiparallel arrangements and obtain detailed

information about the orientation of amide carbonyls. The amide I band, which is the carbonyl stretching vibration of the amide group (1700 to 1600 cm -1 ), is sensitive to -structure. Evidence for antiparallel sheets involves splitting of the amide band into low and high frequency bands due to strong inter-strand dipole coupling. Studies of amyloid of various types observed a strong amide I band near 1630 cm -1 and a weaker band near 1690 cm -1 (Hilbich et al. 1991; Fraser et al. 1992b; Conway et al. 2000a). This spectrum was close to that simulated for Pauling’s cross- structure (Krimm and Bandekar 1986). Percentages of secondary structure were calculated by curve fitting

41

Chapter 1. Introduction

to the experimental spectrum. Unfortunately, another study showed that that the position of bands should not be over-interpreted (Wilder et al. 1992), particularly since amyloid might be regarded as being a separate class of structures. Therefore, the presence of a low frequency amide band is a necessary but not sufficient condition for the assignment of antiparallel -structure (Lansbury 1992). Measurements on parallel -sheet protein showed that these amide I bands were not unique to antiparallel structures (Khurana and Fink 2000). Booth used FTIR to show that there was some residual helical and disordered fold in lysozyme fibrils (Booth et al. 1997); this more flexible structure may surround the rigid core. FTIR provides the only support for the -sheets in full-length A fibrils being in an antiparallel conformation (Halverson et al. 1990; Hilbich et al. 1991). This was contradicted by solid-state nuclear magnetic resonance studies, which were claimed to be more reliable, since the nuclear magnetic resonance data was not based on purely empirical models (Yamada et al. 1998) or normal mode calculations (Bandekar and Krimm 1988) that were based on substantially simplified model systems (Antzutkin et al. 2000).

1.4.2 Atomic Force Microscopy

Atomic force microscopy (AFM) offers direct, high-resolution visualisation without the requirement for averaging (Stine et al. 1996). The height of a solid probe over a specimen surface is measured, with individual molecules examined in air or solution. Physiological conditions are possible without the requirement for fixing or staining. Time dependent aspects of the molecular interactions can be observed including the molecular conformation and aggregation state.

In vitro assembly of amyloid, including fibrils and protofibrils, can be studied using time lapse AFM. Thus, information about the directionality, growth rate and morphology of individual protofibrils is obtained (Goldsbury et al. 1999). Atomic resolution is not possible with biological samples, due to the end radii of available tips, limiting this resolution to flat periodic structures, such as graphite. Fibrils imaged using AFM appear larger than those in electron micrographs. Additionally, the tip- sample interaction may also distort or destroy many soft biological samples. Tapping

42

Chapter 1. Introduction

rather than dragging the tip over the sample reduces but does not eliminate this effect (Chamberlain et al. 2000).

Fibrils may be pre-assembled in a test tube, then absorbed on to a mica surface or assembled on the surface. Different distributions of fibrils are formed in each case. The cause is probably fibril immobilisation due to the constraining effect of the mica. It is not known which better represents physiological conditions, since in vivo, there are many surfaces, including cell membranes. Substantial work has been done on A (Harper et al. 1997a; Harper et al. 1997b; 1999) and amyloid formed from other peptides, including IgLC (Ionescu-Zanetti et al. 1999), IAPP (Goldsbury et al. 1997) and 2 -microglobulin (Kad et al. 2001; Kad et al. 2003). The images showed that growth was normally bi-directional (Blackley et al. 1999; Blackley et al. 2000) but often blocked at one end. Protofibrils themselves were observed to be growing from one end of a fibril (Goldsbury et al. 1999). Structural diversity and twisting protofilament substructure were often observed (Kad et al. 2001).

43

Chapter 1. Introduction

1 m
1 m

Figure 1.8. Atomic force micrograph of Sup35 fibrils (Stromer and Serpell, unpublished).

1.4.3 Neutron Scattering

Neutron diffraction (Bacon 1975) is similar to both electron diffraction and X-ray diffraction. A beam of neutrons is fired at the sample and the scattering pattern observed. Unlike electrons, neutrons have no charge and therefore do not interact with the charges on the nuclei or electron clouds. Instead, neutrons have a nuclear magnetic moment, resulting in an interaction with the nuclei of atoms making up the specimen. This interaction makes it easier to detect light atoms, such as hydrogen, in the presence of heavier ones, which would normally be expected to dominate the interaction. Additionally, atoms with similar atomic numbers such as deuterium and hydrogen, can be distinguished, allowing isotopic substitution for labelling purposes. Radiation damage is largely eliminated, owing to the weak nature of the interaction. Unfortunately, the technique is limited by the available neutron sources, which are nuclear reactors and high-energy spallation sources.

44

Chapter 1. Introduction

Studies of micelle-like oligomers, involved in A fibril formation, used curve fitting of small angle neutron scattering (SANS) data to study a solution cooled to slow fibrillogenesis (Yong et al. 2002). Spherocylindrical structures were modelled with sizes consistent with the diffractogram. Likewise, A (10-21) fibril formation was monitored in the presence of zinc ions (Morgan et al. 2002a). X-ray and neutron scattering studies showed that a crystallised human serum amyloid P component was a pentameric species (Wood et al. 1988). Nuclear magnetic resonance and electron microscopy studies of mature A (10-35) fibrils were complemented by small angle neutron scattering data (Burkoth et al. 2000).

1.4.4 Hydrogen-Deuterium Exchange

Hydrogens in a protein structure normally undergo rapid exchange with those in the solvent. If the solvent contains deuterium, then this is exchanged into the protein, thus increasing the protein’s mass. Determination of the quantity of incorporated deuterium is normally accomplished using two-dimensional nuclear magnetic resonance, mass spectrometry (MS) or FITR. The rate of exchange is dependent on

the protein structure and solvent accessibility. Therefore, by measuring protein exchange, inferences about the protein dynamics can be made. Hydrogens bonded to carbons do not, for the purposes of these experiments, exchange and those bonded to heteroatoms on the side-chains generally exchange too rapidly to be detected. Backbone amide hydrogen exchange rates can be measured, so information can be gathered along the length of the backbone. All amino acids, except prolines, have amide hydrogens. Proton exchange is much slower if the hydrogen is involved in a hydrogen bond, as is the case in a -sheet or -helix. In contrast, solvent accessible hydrogens will exchange very rapidly. Therefore, hydrogen-deuterium exchange (HDX) does not provide a high-resolution three-dimensional structure but probes structure at the resolution of a single residue (Li and Woodward 1999; Englander 2000). A problem is the loss of exchange information via back exchange during fibril solubilisation.

Exchange protection in mature 2 -microglobulin fibrils has been observed in a variety of conditions, over large regions of the chain, including those in the native loops

45

Chapter 1. Introduction

(McParland et al. 2000; Hoshino et al. 2002; Yamaguchi et al. 2004). This suggested a wide -sheet or an ideal -helix. This was also the case for cold shock protein A fibrils, in which all amide protons were found to be protected from exchange (Alexandrescu 2001). A study using A (1-40) amyloid fibrils found that at least 50 % of residues did not exchange even after a thousand hours of exposure (Kheterpal et al. 2000). These exchange times are not generally observed in globular proteins. Whilst much of the protein exists in a highly protected, rigid core, a substantial proportion is not involved in the protective -sheet structure. In the case of A (25-35) fibrils, residues 28-35 formed this core (Ippel et al. 2002). Later studies on A (1-40) fibrils showed the N-terminus to be substantially better protected (> 50 %) than the C- terminal segment (35 %) (Wang et al. 2003). Similarly, fibrils formed from lysozyme had some residual helical and disordered fold present (Booth et al. 1997). Such structures contradict the view that strong hydrogen bonding is present along the whole length of the peptide.

1.4.5 Solid State Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) is a well-established technique used to describe the high-resolution structure of molecules. Nuclear spins are aligned by a large magnetic field of the order of one Tesla. These spins are then perturbed by sequences of radio frequency waves and the resulting electromagnetic output detected by means of induction coils. Solution NMR is not possible due to amyloid being extremely insoluble. Solid state NMR (SSNMR) refers to the use of NMR on condensed matter that is neither liquid nor in solution (reviewed (McDowell and Schaefer 1996)). Unfortunately, the spectral resolution of solid state NMR is orders of magnitude lower than solution NMR. This line broadening, of the order of 10 kHz, is due to the angularly dependent anisotropic interactions and dipole-dipole interactions between nuclei in the solid. In liquids, these interactions are averaged out since the molecules rotate rapidly and randomly. Chemical shift data can be obtained using magic angle spinning (MAS). This can substantially reduce the line-width by spinning the solid sample about an axis at 54.5˚ (the magic angle) to the external magnetic field (Stejskal and Memory 1994). Relaxation measurements can be made by selectively reintroducing weak dipolar recoupling.

46

Chapter 1. Introduction

If the angular speed is matched to a multiple of the chemical shift difference between appropriate spins, then rotational resonance (RR) leads to recoupling. RR therefore requires a substantial isotropic chemical shift difference in the homonuclear spin pair. Techniques such as rotational echo double resonance (REDOR) (Gullion and Schaefer 1989) and dipolar decoupling at the magic angle (DRAMA) (Tycko and Dabbagh 1990) involve dephasing pairs of nuclei of the same isotope with no isotropic shift difference. Dipolar recoupling in a windowless sequence (DRAWS) allows detection of weak, dipolar-coupling interactions between spin ½ nuclei with substantial chemical-shift anisotropies (Mehta et al. 1996; Gregory et al. 1997; Gregory et al. 1998). Constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) is also an example of a dipolar recoupling technique; it minimises distortions in the experimental data due to pulse sequence imperfections (Ishii et al. 2001a).

In the case of amyloid, 13 C labels are usually employed. Pulse sequences use the direct dipolar interactions to allow the determination of distances between strategically placed spin pairs (Figure 1.9). The dipolar interaction is inversely proportional to the cube of the distance between the nuclei in the spin pair. It also depends on the magnetic moments of the nuclei and the cosine of the angle between the internuclear vector and the external magnetic field. More recently, multiple quantum NMR (MQNMR) (Yen and Pines 1983) has been employed. Signals from groups of coupled nuclear spins are detected; showing that labelled residues occur in groups of a particular size. Measurement of internuclear distances using these techniques is possible at up to 6 Å in favourable cases, with a standard error of between 0.1 Å and 0.2 Å (Tycko 2000). Additionally, torsion angles and absolute orientations of bonds and functional groups with respect to the external magnetic field can be determined. Double quantum chemical shift anisotropy (DQCSA) and 2D MAS exchange techniques both measure the relative orientations of a pair of 13 C tensors. These tensors depend on the orientations of the carbonyl groups and therefore the torsion angles. Line widths indicate the degree of structural order.

47

Chapter 1. Introduction 5.7 4.9 5.1 5.2 5.7 4.9 5.1 5.1 5.1 5.6 5.5 5.8
Chapter 1. Introduction
5.7
4.9
5.1
5.2 5.7
4.9
5.1 5.1
5.1 5.6 5.5
5.8

1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Figure 1.9. Summary of NMR distance information for A (10-35); this fits the model for a - strands being parallel and in register. No evidence for a -turn is observed. (Data taken from (Burkoth et al. 2000; Lynn and Meredith 2000).)

Most papers concerning SSNMR have reported on amyloid composed of truncated or full-length A . The exceptions were two early papers on two amyloidogenic fragments, IAPP (20-29) (Griffiths et al. 1995) and hamster prion H1 (109-122) (Heller et al. 1996). Both used rotational resonance to conclude that the structure was composed of -strands. Many studies concentrated on short truncated A peptides and found antiparallel -strands in A (34-42), A (26-40), A (26-43) and A (16-22) (Jarrett et al. 1994; Lansbury et al. 1995; Balbach et al. 2000). A cis linkage between residues 37 and 38 was proposed (Spencer et al. 1991) but later concluded to be a trans linkage (Costa et al. 1997). Parallel and in-register -strands were found in a series of DRAWS experiments on A (10-35) (Benzinger et al. 1998; Gregory et al. 1998; Benzinger et al. 2000; Burkoth et al. 2000). Similarly, experiments using a wide variety of NMR techniques on full-length A revealed in-register parallel - sheets (Antzutkin et al. 2000; Balbach et al. 2002; Petkova et al. 2002; Antzutkin et al. 2003). Line widths suggested a degree of structural disorder at the N-terminus. Some non- structure was found at residues 25, 26 and 29 (Antzutkin et al. 2003). This may be necessary, since a fully extended -strand is too wide to fit into the diameter of fibres observed in micrographs and therefore there must be a turn somewhere. The authors postulated the existence of a turn between -sheets, though this is not common in general protein structure.

Limitations on the distances measurable using SSNMR mean that it is often combined with other techniques. Using the constraints established by SSNMR data, some

48

Chapter 1. Introduction

members of the ensemble of possible structures can be eliminated. STEM has been used to obtain mass per unit length data so that a model may be constructed (Antzutkin et al. 2000; Petkova et al. 2002). They did not however completely refute the possibility of a -helical structure. Whilst FTIR measurements generally indicated antiparallel organisations of -strands (Jarrett et al. 1994; Lansbury et al. 1995), they were not consistent with later, more reliable NMR studies (see FTIR section). The SSNMR investigation into A (11-25) gave a pair of pH dependent structures, different to that expected from X-ray studies under different conditions (Sikorski et al. 2003; Petkova et al. 2004). This suggested that the -strand packing is environmentally dependent and the result may be explained by slippage along the chain axis (Makin and Serpell 2004a).

1.4.6 Other Methods

Site directed spin labelling (SDSL) involves the use of electron paramagnetic resonance (EPR) to determine structure at the level of the backbone fold in native-like conditions. EPR uses the same principle as NMR but applied to unpaired electrons rather than nuclei. Most stable molecules have closed shells without unpaired spins. Unpaired spins are introduced by means of site specific labelling of engineered cysteine residues with a sulphydryl-specific paramagnetic nitroxide compound. The EPR spectrum depends on the structural microenvironment of the spin label including its secondary structure, proximity to other spin labels and solvent accessibility. A study of full-length A fibrils found parallel in-register -strands (Torok et al. 2002), whilst a mostly native structure in a head-to-head, tail-to-tail arrangement was proposed for amyloid formed from transthyretin (Serag et al. 2002).

Protease digestion leaves the cross- core of the fibril. The technique was employed to assign the -turn position to be between residues 26 and 29 in A (10-43) fibrils (Hilbich et al. 1991). In the case of Ure2p it is disputed as to whether the fibrils are amyloid at all. Although they have the characteristic birefringence pattern and micrograph appearance, which in conjunction with mass spectrometry and protease digestion suggest a cross- structure (Baxa et al. 2003), other studies have used these

49

Chapter 1. Introduction

techniques to propose a structure is that largely native and therefore predominantly - helical (Bousset et al. 2004).

Selective mutation experiments have proved effective, including demonstrating the effect of charge-charge interactions to stabilise -sheet conformation (Fraser et al. 1994). Other studies have used amyloid formed from Sup35 mutated with cysteine residues (Scheibel et al. 2001), rat IAPP with point mutations from human IAPP (Green et al. 2003), A (Serpell 2000), 2 -microglobulin (Smith et al. 2003) and IgLC (Hurle et al. 1994; Stevens et al. 1995). Finally, circular dichroism spectroscopy has been applied to A (10-43) and -synuclein fibrils (Hilbich et al. 1991; Conway et al. 2000a) to describe the fibrils’ -sheet content.

1.5 Electron Microscopy Theory

1.5.1 Beam Specimen Interaction

If electron microscopy is used to describe the structure of amyloid, it is necessary to understand how the structure influences the image on the micrograph. As imaging takes place using an electron beam, the interaction between the electron beam and the specimen should be considered. The electrons in the column travel at speeds comparable to the speed of light, hence relativistic quantum mechanics is required. The requisite mathematical machinery requires the use of the Dirac equation. Fortunately, the effect of spin can be neglected, as it represents only about 1 % of the total interaction (Fujiwara 1961); meaning that a suitable first order approximation is the Schrödinger equation, with relativistic corrections to the wavelength and electron mass,

2 h − ∇ 2 ψ + V ψ = Eψ p 2 m f
2
h
2 ψ +
V ψ
=
p
2 m
f
eV
p
m
=
m
+
f
e
2
c

50

λ =

h

eV A c
eV
A
c

2

+ 2 eV m

p

e

Chapter 1. Introduction

where m e is the rest mass of the electron, is the electron wave function, E is the total

energy of the electron, e is the charge on the electron, h is Planck’s constant, V A is the

accelerating voltage and V p is the potential. Passing through a specimen changes the

potential in the Schrödinger equation and thus the wavelength of the electrons,

introducing a phase shift φ(x,y). φ ( x , y ) = 2 2 π
introducing a phase shift φ(x,y).
φ
(
x
,
y
)
=
2
2
π
eV
φ
A
=
+
2 eV m
A
e
h
c
 

1

1

dz

 

π

 

λ

vacuum

 

λ

specimen

(

x

,

y

,

z

)

 

e V

(

A

+

V

s

(

x

,

y

,

z

))

2

 

2

(

e V

A

 

V

s

(

 

))

 
 

 

c

 

+

+

x

,

y

,

z

m

e

2

dz

Approximating this shows that passing through the specimen advances the phase by

(

φ

x

,

y

)

=

2 π em eV e A 1 + 2 h m c e
2
π
em
eV
e
A
1
+
2
h
m c
e

V

s

(

x

,

y

,

)

z dz

where V s (x, y, z) is the potential at a point (x, y, z) in the specimen, and the line

integral is parallel to the electron beam (Saxton 1980). This equation shows that by

finding the phase of the electron beam leaving the specimen, the projection of the

potential can be found. Once the projection is known, the potential at any point can be

calculated using the methods of three-dimensional reconstruction. The potential is

related to the charge density s and permittivity of free space 0 by the Poisson

equation.

2

V

s

= −

51

ρ s

ε

0

Chapter 1. Introduction

This is in contrast to X-ray diffraction, in which the scattering factors depend almost entirely on the electron charge density; whereas electron scattering amplitudes are also dependent on the nuclear charge.

1.5.2

Let

ψ

Imaging

o be the object wave function, which is the wave function of the electrons

leaving the specimen and

the electrons in the image plane.

ψ

i the image wave function, that is, the wave function of

The Fourier transforms of the image and object wave functions are related by the

wave function transfer function W (k) (Teague 1983).

Ψ

i

(k)

= W

(k)

W

(

k

)

= exp

(

i

πλ

D

k

2

Ψ

o

+ i

(k)

π

2

3

C

S

λ

k

4

)

is the spherical

aberration. Here D > 0 is over focus. (There are also a number of envelope functions, which are not shown here.) If the specimen is such that the phase of the object wave function is small and the amplitude unity, then the weak phase object approximation (Frank 1996) can be employed. This gives the result that the measured intensity is twice the convolution of the phase of the object wave function with the inverse Fourier transform of the imaginary part of the wave function transfer function,

where λ is the electron wavelength, D is the defocus and

C

S

FT

[

ψ

i

2 ]

[

= 2FT arg

(

ψ

o )]

sin

( πλ

D

k

2

+ π

2

λ 3 C

S

k

4 )

.

Within this approximation, the phase of the object wave function can be found simply by dividing by

2sin

( πλ

D

k

2

+ π

2

3

C

S

λ

k

4 )

,

known as the contrast transfer function, in Fourier space.

52

1.6 Diffraction Theory

Chapter 1. Introduction

1.6.1 Introduction

Much of what is known about the three-dimensional structure of protein is from diffraction studies. X-ray diffraction theory and fibre diffraction in particular are

covered in detail in many excellent books (Vainshtein 1966; Blundell and Johnson 1976; Fraser et al. 1976; Holmes and Blow 1980; Vibert and Squire 1987). This brief summary illustrates how this theory relates to the included work.

The problem is the scattering of a beam of electrons from a sample. Formally, the Huygens-Kirchhoff Integral should be solved. In the far field approximation, the phase varies linearly across the aperture and Fraunhofer diffraction occurs. The three- dimensional diffraction pattern is the three-dimensional Fourier transform of the sample structure. The space in which the three-dimensional diffraction pattern exists is known as reciprocal space.

1.6.2 Fourier Transform

Fourier transforms are written as capitals, hence the Fourier transform (FT) of a

function f(x) is written

[

FT f

( )]

x

=

F

(

k

)

=

−∞ −∞ −∞

f

(

x

)

e

2

πi

k.x

d

3

x

Discrete Fourier transforms are used for computations in practice.

1.6.3 Convolution Theorem

The Fourier transform of the sample structure can be worked out by repeated application of the convolution theorem. This theorem states that multiplication in real space is equivalent to convolution in reciprocal space.

FT[f (x)]FT[g(x)]= FT[f (x)* g(x)]

53

Chapter 1. Introduction

The sample is composed of a series of crystallites at varying angles. These crystallites

can be thought of a being a small part of an infinitely large crystal. This crystal is

constructed from a lattice of parallelepiped shaped building blocks (unit cells). Points

on the lattice r lmn are defined by linear combinations of integer multiples of three

lattice vectors {a,b,c}.

r

lmn

=

la

+

mb

+

nc

These unit cells are themselves composed of identical symmetry related objects

known as the crystallographic asymmetric units. The space group defines the number

and orientation of asymmetric units. Mirror and inversion symmetries are not allowed

since proteins are chiral and only one type of enantiomer is allowed.

A real space sample can be represented by a series of multiplications and

convolutions. The Fourier transform of the unit cell is the molecular transform. The

Fourier transform of the lattice is another lattice. This reciprocal lattice has reciprocal

lattice vectors {

*

a

,b

*

,c

*

}

calculated using

*

a

=

b

×

c

a .

(b

× c)

,

b

*

=

c

×

a

a (b

.

×

c)

,

*

c

=

a

×

b

a (b

.

× c)

Points on the reciprocal lattice

r

* , with Miller indices [h k l] are defined thus

r

*

hkl

=

ha

*

+

kb

*

+

lc

*

Structure factors F hkl are the values of the molecular transform at r hkl * .

The crystallites have a finite size, which is equivalent to multiplying an infinite crystal

by a cut-off function, such as a top hat function. Hence, by applying the convolution

theorem, the infinitely narrow spots are convolved with the Fourier transform of the

cut-off function. So the result is a lattice of spots of finite size. Next, the orientational

disorder is applied by convolving the lattice of spots with an angular function,

representing the distribution of crystallites. In the case of a true fibre, the crystallites

54

Chapter 1. Introduction

are randomly distributed about the fibre axis. As a result, the spots are convolved with circles centred on the fibre axis and in planes perpendicular to this axis; the diffraction produced is equivalent to a rotation photograph. Hence fibre diffraction patterns have layers of reflections perpendicular to the fibre axis, known as layer lines.

1.6.4 Sample Texture

Texture is a description of the distribution of fibril orientations (Kocks et al. 1998). It

determines the shape of the reflection in reciprocal space and hence affects both the shape and intensity of the signals on the diffractogram. In general, the texture can be complicated (Detavernier et al. 2003), however amyloid fibrils are relatively simple. In the case of a true fibre, there is a rotational degree of freedom about the fibre axis; this results in a diffraction pattern with infinite cyclic symmetry about that axis. The diffraction pattern’s symmetry is like that of a rotating single crystal experiment; however there is also disorder in the orientations of individual fibrils about the mean. Sample texture depends on the method of alignment, the precursor peptide and the experimental conditions.

1.6.5 Ewald Sphere

The diffractograms observed by the detector are two-dimensional. Only points lying on the surface of a sphere in reciprocal space satisfy the geometrical requirements for diffraction to occur. Given the position of a lattice point, the corresponding position of the reflection on the detector can be calculated (Figure 1.10).

55

Chapter 1. Introduction

y Detector Ewald sphere x z X-rays
y
Detector
Ewald sphere
x
z
X-rays

Figure 1.10. The diffraction geometry, a sample lattice point (x lattice , y lattice , z lattice ) in red and its circle are shown, diffraction occurs where this circle intersects with the sphere. The resulting signal on the detector plate has the coordinates (x imageplate , y imageplate ).

To achieve this, the position of the reflection on the Ewald sphere must first be determined. The constraint provided by the Ewald sphere is

x

sphere

2

+ y

2

sphere

(

+ z

sphere

) 2 2 +1 λ = 1 λ
)
2
2
+1 λ
= 1 λ

In the case that the fibre axis is parallel to the y-axis then there are three possibilities. If the circle intercepts with the Ewald sphere (Figure 1.11), then the condition is

x

sphere

2

+ z

sphere

2

= x

lattice

2

+ z

56

lattice

2

and

y

sphere

= y

lattice

Chapter 1. Introduction

These three equations can be solved to give:

x sphere

= ±

z

sphere

= −

(

1 2 λ x

lattice

2

+ y

lattice

2

+ z

lattice

2

)

x

x

lattice

2

+ z

lattice

2

1

4

2

λ

(

x

lattice

2

+ y

lattice

2

+ z

lattice

2

) 2

y ( ) x ,y imageplate imageplate ( ) x ,y ,z ( x ,y
y
(
)
x
,y
imageplate
imageplate
(
)
x
,y
,z
(
x
,y
,z
)
sphere
sphere
sphere
lattice
lattice
lattice
2
2 + z
x lattice
lattice
y lattice
x
z

Figure 1.11. Side view of the diffraction geometry shows the circle traced out by the reflection (red) intersecting with the Ewald sphere at (x sphere , y sphere , z sphere ) (green).

Alternatively, the circle may not intercept with the Ewald sphere, in which case the angular disorder perpendicular to this circle is important in giving the circle a finite thickness on the surface of a sphere (shown in red, Figure 1.12) and the equations are as follows:

y

sphere

 

2

+ z

z

2

sphere

sphere

= x

= −

2

lattice

1

2

(

λ x

+ y

lattice

2

lattice

2

+ y

+ z

lattice

2

lattice

2

+ z

and

2

lattice

x

)

sphere

= 0

 
( x lattice 2 + y 2 lattice + z lattice 2 ) − 1

(

x

lattice

2

+ y

2

lattice

+ z

lattice

2

)

1

4

2

λ

(

x

lattice

2

+ y

2

lattice

+ z

lattice

2

) 2

y

sphere

=

57

Chapter 1. Introduction

y ( ) x ,y imageplate imageplate ( ) x ,y ,z ( x ,y
y
(
)
x
,y
imageplate
imageplate
(
)
x
,y
,z
(
x
,y
,z
)
sphere
sphere
sphere
lattice
lattice
lattice
x
z
2
2 + y
2 + z
x lattice
lattice
lattice

Figure 1.12. The circle centred at the fibre axis (y-axis) does not intersect with the Ewald sphere. Nevertheless diffraction may still occur since the diffraction circle has a finite thickness and is represented by a two-dimensional circular surface on the surface of a sphere (red).

2 2 There is no intercept if 2 + y 2 + z > 4
2
2
There is no intercept if
2 + y
2 + z
> 4 λ
(Figure 1.13).
x lattice
lattice
lattice
y
(
x
,y
,z
)
lattice
lattice
lattice
x
z
2
2
2 + y
2 + z
x lattice
lattice
lattice
λ

Figure 1.13. The case in which no diffraction occurs. The radius of the sphere containing the lattice point is greater than the diameter of the Ewald sphere.

58

Chapter 1. Introduction

Once the position on the Ewald sphere is known, the position of the spot on the image plate can be calculated

x

imageplate

=

x

sphere

D

λ

z

sphere

λ

+ 1

,

y

imageplate

=

y

sphere

D

λ

z

sphere

λ

+ 1

1.6.6 Problems

Diffraction detectors only measure the intensity of the wave function, not its phase. In single crystal X-ray diffraction, the integrated intensity of reflections is measured and after the application of intensity correction factors, the amplitude of the structure factors can be calculated.

In order to calculate the structure of the sample from its structure factors, both the amplitude and phase of the structure factors are required. The absence of the phase information is known as the phase problem. Crystallographers are able to solve this problem using a variety of methods including molecular replacement, multiple isomorphous replacement and anomalous dispersion. Neither is there a problem in electron microscopy, in which phases can be experimentally determined under the weak phase object approximation. Fibre diffraction structures are generally determined by modelling a structure and then comparing the simulated diffractogram with the observed pattern. The most prominent exceptions are the use of isomorphous replacement to determine the structure of the tobacco mosaic virus TMV (Stubbs and Diamond 1975) and molecular replacement in the case of the ribgrass mosaic virus (Wang et al. 1997).

Overlapping reflections can also be a problem, further reducing the amount of information available. Reflections may overlap due to angular disorder or the diffuse nature of the signals. The choice of the Lorentz intensity correction factor also depends substantially on the sample texture (Vainshtein 1966). These make calculation of the intensity of individual reflections difficult or impossible.

59

Chapter 1. Introduction

1.7 Experimental Methods for X-ray Fibre Diffraction

1.7.1 Introduction

The quantity and quality of diffraction information is very dependent on the amount of order in the sample (Lorenz and Holmes 1993). The general problem was first considered in the case of tobacco mosaic virus fibrils (Bernal and Fankuchen 1941). In the absence of alignment, the pattern is a series of concentric rings; if there is some alignment, the reflections form arcs (Sunde et al. 1997) (Figure 1.14). Investigators therefore wish to align fibrils with respect to one another, which may achieve a high degree of orientation and a diffraction pattern with far more information (Sikorski et al. 2003). The correct technique and maximum degree of orientation depends strongly on the type of fibre.

degree of orientation depends strongly on the type of fibre. Figure 1.14. Comparison of unaligned A

Figure 1.14. Comparison of unaligned A (1-40) amyloid fibrils (left) and stretch frame aligned A (11-25) fibrils (right). Arrows indicate the location of the 4.7 Å reflections.

1.7.2 Glass Capillary and Stretch Frame

Long fibres are likely to be very viscous and thus a stretch frame will yield the best results. The frame can be used to mount a pair of glass capillaries. These capillaries

are sealed flat using melted wax and a droplet rests between the two (Figure 1.15). The fibrils orient parallel to the capillaries as the drop dries over several hours (Damas et al. 1995; Sunde et al. 1997) (Figure 1.16). A threaded arrangement allows

60

Chapter 1. Introduction

the capillaries to be separated gradually during drying, leading to some stretching of the fibre sample, although the sample may break even without stretching.

Laterally aggregated samples can also be encouraged to align by drying them down to form
Laterally aggregated samples can also be encouraged to align by drying them down to
form a disk. This can be obtained by drawing 2 to 3 cm of solution into a siliconized
capillary of 0.7 mm diameter and then allowing the solution to dry.
Sample
Stretch frame
Capillary
Wax
Plasticine

Figure 1.15. A stretch frame can be used to align fibrils. Inset shows sample suspended between the waxed ends of two glass capillaries. The capillaries are attached to the stretch frame by means of Plasticine.

1.7.3 Magnetic Field

Most filamentous macromolecular assemblies are oriented by magnetic fields (Glucksman et al. 1986). Magnetic alignment is more appropriate for samples composed of small crystallites, which are grown in a magnetic field. Alignment of

these samples can be achieved using a 2.4 Tesla permanent magnet (Hummingbird

61

Chapter 1. Introduction

Instruments, Arlington, MA). The fibrils are oriented parallel to the direction of the magnetic flux density vector (Figure 1.16). Bond resonance in certain side-chains causes anisotropy in the diamagnetic susceptibility, resulting in a force on the fibrils and thus a preferred orientation (Worcester 1978; Pauling 1979; Glucksman et al. 1986). The sample is prepared in the same manner as for a disk, with the sample placed between the poles of the magnet and dried over several weeks. Kirschner first used this to study A and its fragments (Inouye et al. 1993). Magnetic alignment has also proved very effective for the alignment of other amyloid fibrils (Inouye and Kirschner 1997; Malinchik et al. 1998; Sikorski et al. 2003).

1.7.4 Mat

Alternatively, a thin flat film can be formed with the fibres aligned parallel to the

plane of the film (Figure 1.16). A larger volume of solution is required and the film must be mounted such that the X-ray beam can be passed both in the plane of film and perpendicular to that plane.

The mat can be produced by depositing solution on a glass slide, drying and detaching the residue from the substrate. Whilst this method has been successfully applied in the case of poly-amino acids (Fandrich and Dobson 2002), removing the mat from the surface can be difficult if the mat is brittle, perhaps owing to short fibres. Parafilm and Teflon can also be used as a substrate and the mat has been used for aligning polymers, AA (Turnell et al. 1986) and polyglutamine fibrils (Perutz et al. 2002). To avoid problems with lifting off the mat, a cryo-loop (Hampton) normally used to freeze single crystals can be employed. The loop is immersed in the fibril solution and then lifted out to dry, resulting in a film across the plane of the loop (Makin and Serpell 2005).

62

Chapter 1. Introduction

Magnetic Field
Magnetic Field

Figure 1.16. Illustration of the orientation of fibres after alignment by stretch frame, cryo-loop and magnetic field (from top to bottom). Block arrows indicate the X-ray beam directions.

63

Chapter 2. Application for the Structural Analysis of Amyloid

2 Application for the Structural Analysis of Amyloid

2.1 Abstract

Amyloid has a characteristic cross- structure. Structural studies of amyloid necessarily reflect its characteristics. The examination of amyloid structure is impeded by problems peculiar to amyloid. Existing applications, which are not specific to amyloid diffraction, have difficulty with these issues and are unable to exploit amyloidal features. We have therefore developed a suite of programs specifically for the study of amyloid fibre diffraction patterns. It is also suitable for many general fibre diffraction problems.

Our Java based application (Clearer), employs a series of libraries to process experimental data, particularly X-ray and electron diffractograms. Components within the suite aid and automate crucial elements in the sequence of analysis. Background subtraction and contrast enhancement allow weak signals to be observed and then peak profiling gives their resolutions. The indexing process is semi-automated, so the user can obtain the unit cell. Finally, simulation of diffraction patterns ensures that structures modelled using an external program can be tested and compared with the experimental diffractogram. This allows the structure of amyloid to be examined in detail.

2.2 Introduction

The purpose of the application was to facilitate the process of examining amyloid structure. Both portability and usability were intrinsic to the design; these made Java (Sun Microsystems) the obvious choice of language. Rapid application development and the Java Advanced Imaging library (JAI) enabled better use of development resources. The aim was to be as user friendly as possible. Accordingly, the system was built with a modern graphical user interface and without batch files, lists of parameters or idiosyncratic user-interface metaphors. Sensible defaults and clear language were also essential to help new users and enable rapid working.

64

Chapter 2. Application for the Structural Analysis of Amyloid

Each component was developed in response to a specific practical requirement. Development proceeded as a response to issues arising from the analysis of the peptides described in Chapter 3 and Chapter 4. Feedback from users was crucial, both in terms of demand for features and human-computer interaction issues.

We have benefited greatly from the availability of ancillary software. The underlying image processing engine relies on the Java Advanced Imaging library, in which many of its methods are available as C routines for speed. One-dimensional graphs are displayed by JFreeChart. The molecular visualisation component allows protein data bank (PDB) files to be viewed; this was based on an existing application (Jmol), implemented with the assistance of Jmol developers.

The sequence of analysis for fibre diffraction patterns generally involves several stages (Figure 2.1). First, the image is prepared for processing, including removing the background. Secondly, the required data is extracted from the image. Finally, this data is processed to reveal information about the structure of the sample.

Amyloid samples suffer from low crystallinity, small crystal size and lack of good packing. A carefully chosen processing strategy is required to make best use of the available data. Each stage is analysed in turn.

65

Chapter 2. Application for the Structural Analysis of Amyloid

Format Conversion

for the Structural Analysis of Amyloid Format Conversion Image Processing Image Analysis Indexing Structure Modelling

Image Processing

Analysis of Amyloid Format Conversion Image Processing Image Analysis Indexing Structure Modelling Figure 2.1.

Image Analysis

of Amyloid Format Conversion Image Processing Image Analysis Indexing Structure Modelling Figure 2.1. Flowchart showing

Indexing

Format Conversion Image Processing Image Analysis Indexing Structure Modelling Figure 2.1. Flowchart showing the

Structure Modelling

Figure 2.1. Flowchart showing the procedure for processing general fibre diffractograms. Based on (Stubbs 1999).

2.3

Preparation

2.3.1 Format Conversion

A very wide variety of image formats are output by detectors and used by diffraction processing programs. The range of formats in general use is even greater. Writing input filters is time consuming and does not result in new structures. The CCP13 website lists some 22 types, before accounting for byte order and other nuances. We therefore chose to limit the input to those available from the underlying library including RAW, BMP, GIF, JPEG, PNG, PNM and TIFF. Marcvt (Marresearch, Norderstedt), Fit2D (A. Hammersley, ESRF), Denzo (Otwinowski and Minor 1997) and XCONV (CCP13) all offer the ability to convert the output of detectors into TIFF files, which are compatible with most image processing applications.

2.3.2 Centring

The centre of the pixel array collected from the detector rarely matches the centre of the beam. It is therefore necessary to realign the image so that systematic error does not affect the following analysis. Figure 2.2 shows the component used for this

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purpose. On the right, traces along the x and y axes in the positive and negative directions will line up once the image is properly centred. Pressing the calculate button crudely calculates the optimum x and y shifts by comparing peak positions on each of the traces. Alternatively, blue concentric rings are overlaid so the process can be completed by eye.

rings are overlaid so the process can be completed by eye. Figure 2.2. The user can

Figure 2.2. The user can centre the diffraction pattern to prevent the introduction of systematic error into the analysis.

2.3.3 Background Removal

The background is unwanted low frequency data, which is added to the diffraction pattern. Reasons for its existence include detector-specific noise (fog) and white radiation – the incident beam not being completely monochromatic. Additionally X- ray scattering from air, the sample holder, amorphous material in the specimen such as solvent and disordered polymer and components of the camera all contribute. The background may show a high level of variation across the field and is a source of error

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in intensity measurements. In the case of electron diffraction, the background is particularly strong and weak spots may be obscured.

There are three types of methods to determine the background, experimental measurement, simulation and estimation from the diffractogram. The diffraction experiment can be repeated without the sample; this method does not account for amorphous scattering from the sample and is time consuming, as it must be repeated many times. Computational simulation is unreliable since a great many parameters must be estimated to be input into the model, which may not correctly simulate the amorphous scattering. Finally, the background can be estimated from the spaces between reflections in the diffractogram. This estimation is difficult if layer lines overlap, which is often a problem at larger radii. We implemented several methods of estimation.

The simplest form of background estimation is to calculate the local mean, using a box filter. Each pixel is replaced by the average of the pixels in a box centred on its position. Since the background is low resolution, a large box is used and the calculation may require a large number of calculations. It may be better to do the calculation in Fourier space. A box filter is a convolution operation, so it is equivalent to multiplication by a sinc function in reciprocal space. Alternatively, the box may be replaced by a Gaussian, so the effects of the sharp box edges are avoided. This method can also be used for background removal in electron micrographs.

In single crystal X-ray crystallography, the individual reflections are small; hence an inclined plane is a suitable function for the local background (Rossmann 1979). Early attempts employed a similar strategy, using a series of inclined planes or splines. Unfortunately, amyloid’s angular disorder results in large reflections, many of which may overlap. Therefore, a single, global function, interpolated from the gaps between signals, may be more appropriate. One approximation is that the background varies linearly with the polar angle, with the parameters determined using a set of experimental and computationally derived values (Fraser et al. 1976). Alternatively,

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the lower orders of two-dimensional cylinder function expansion can be calculated using selected points between layer lines (Millane and Arnott 1985).

If the background is circular, other methods are appropriate. A polynomial fit to the circularly averaged image is surprisingly effective. The beam stop must be excluded and non-quadratic polynomials are prone to over fitting. We have developed a statistical approach in which the image is divided into concentric circular annuli, each one pixel thick. If an annulus contains a signal, its histogram (intensity frequency distribution) will be the sum of two approximately normal distributions (Figure 2.3). These are a tall, narrow Gaussian at low intensities from the background and a low, wide distribution at higher intensities due to the reflection. The aim of the program is to determine the mean of the background distribution. If the background distribution has a roughly constant standard deviation, then it is only necessary to determine the mean plus a constant offset. The algorithm finds the background intensity by considering the cumulative frequency distribution and using a user supplied value for the minimum percentage of the image that is background. A value of 50 % was found to work well in most cases (Figure 2.4). Care should be taken not to introduce artefacts by underestimating the percentage of any annulus occupied by the signal, since this will result in overestimation of the background intensity at some resolutions.

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