Amyloid and the Cross-Beta Architecture

Summary

Amyloid fibrils are formed by numerous proteins, and the resulting fibrils share a "cross β-sheet" structure. Here we describe how amyloid fibril samples may be prepared for X-ray fiber diffraction and how the patterns may be analysed.

Abstract

Amyloid fibrils are made up of a misfolded, self-assembled protein organised into a well-defined, repetitive structure. They are well known for their important role in protein misfolding diseases, including Alzheimer's disease and type 2 diabetes, in which the insoluble amyloid fibrils are deposited in the tissues. Furthermore, they have been found to play an important functional role in many organisms, providing strength, scaffolding, and protection. The first structural models of amyloid were informed by X-ray fiber diffraction data collection from bundles of aligned amyloid fibrils. This resulted in early models of the generic cross-β structure which has been superseded by more detailed diffraction analysis from highly oriented and semi-crystalline samples, resulting in details of the organization of peptides into a repetitive architecture. Here, we focused on describing methods of determining the underlying architecture of these fibrils, and we have described the common features of amyloid fibrils from diverse pathogenic, functional, and synthetic sources. We describe the preparation of the samples, data collection, and subsequent data analysis to produce a model structure that can be compared to the experimental data.

Introduction

Amyloid fibrils were first identified within a group of diseases known as Amyloidoses, in which proteinaceous fibrils accumulate in the extracellular spaces of tissues¹. Each disease is characterized by a particular precursor protein which undergoes a conformational change to form highly ordered, stable protein fibrils^2,3. The more recent definition of amyloid fibrils now incorporates fibrils that accumulate within the cellular environment. More recently, a number of functional systems have been identified that utilize the amyloid structure to provide stability, adhesion, or even inform....

Protocol

1. Preparation of fibrillar samples for X-ray fiber diffraction

1. Dissolve protein or peptide of interest in optimal conditions for fibril formation for the selected amyloidogenic peptide and assess fibril formation using electron microscopy or atomic force microscopy. Details of how to prepare samples and conduct imaging are available elsewhere¹³. Ensure that the fibrils are long, straight, and unbranched as shown in Figure 1. For example, the fibrils shown in Figure 1 were imaged using transmission electron microscopy (TEM). Place a droplet containing fibrils on an electron....

Results

CcbMet example²⁴: Figure 1 shows electron micrographs showing a high density of amyloid fibrils on a grid suitable for preparation of an X-ray fiber sample. Figure 2 describes the different textures that can be generated from fibrous samples fiber bundle (Figure 2A), disk (Figure 2B), mat/film (Figure 2C), and shows t.......

Discussion

X-ray fiber diffraction can be used to evaluate whether a sample is forming an amyloid structure and to gain further insight into the molecular organization of the precursor protein or peptide within the fibers. Here, we have described the methods by which fibrous samples may be prepared for X-ray fiber diffraction, data may be obtained, and then analyzed. Critically, the quality of the data obtained will depend on both the order within the individual fibers. Therefore, initial sample preparation is of major importance, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.7 mm diameter X-ray  quartzglass capillaries	GLAS, W. Muller, D-13503, Berlin, Germany	Q-07-001-80
Glass capillaries 1,5 mm borosilicate	Harvard appatus Ltd, (Edenbridge, Kent, UK)	BS4 30-0074	example, available at other manufacturers
Protein crystallography X-ray diffractometer	Various		Various manufacturers