Sedimentation velocity analysis of amyloid oligomers and fibrils using fluorescence detection

doi:10.1016/j.ymeth.2010.10.004

Methods

Volume 54, Issue 1, May 2011, Pages 67-75

https://doi.org/10.1016/j.ymeth.2010.10.004 Get rights and content

Abstract

The assembly of proteins into large fibrillar aggregates, known as amyloid fibrils, is associated with a number of common and debilitating diseases. In some cases, proteins deposit extracellularly, while in others the aggregation is intracellular. A common feature of these diseases is the presence of aggregates of different sizes, including mature fibrils, small oligomeric precursors, and other less well understood structural forms such as amorphous aggregates. These various species possess distinct biochemical, biophysical, and pathological properties. Here, we detail a number of techniques that can be employed to examine amyloid fibrils and oligomers using a fluorescence-detection system (FDS) coupled with the analytical ultracentrifuge. Sedimentation velocity analysis using fluorescence detection is a particularly useful method for resolving the complex heterogeneity present in amyloid systems and can be used to characterize aggregation in exceptional detail. Furthermore, the fluorescence detection module provides a number of particularly attractive features for the analysis of aggregating proteins. It expands the practical range of concentrations of aggregating proteins under study, which is useful for greater insight into the aggregation process. It also enables the assessment of aggregation behavior in complex biological solutions, such as cell lysates, and the assessment of processes that regulate in-cell or extracellular aggregation kinetics. Four methods of fluorescent detection that are compatible with the current generation of FDS instrumentation are described: (1) Detection of soluble amyloid fibrils using a covalently bound fluorophore. (2) Detection of amyloid fibrils using an extrinsic dye that emits fluorescence when bound to fibrils. (3) Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates. (4) Detection of green fluorescence protein (GFP) constructs and their interactions within mammalian cell lysates.

Introduction

The self-assembly of specific proteins into amyloid fibrils occurs via the transient formation of a range of soluble oligomers and protofibrillar intermediates. These aggregates may be deposited extracellularly, as in Alzheimer’s disease [1]; or intracellularly, in related conditions such as Huntington’s [2]. Both mature fibrils and oligomeric intermediates have been implicated as pathogens in amyloid-linked diseases [1], [3], [4]. In addition, the bulk accumulation of amyloid fibrils within large insoluble deposits leads to organ dysfunction as manifest in the systemic amyloidoses [5]. The extracellular deposition of amyloid fibrils in vivo is accompanied by the accumulation of non-fibrillar components such as serum amyloid P (SAP), apolipoprotein (apo) E, proteoglycans and lipids [6]. Inside cells, components such as chaperones and proteasomes accumulate with the aggregates [2]. These components influence the interactions of amyloid fibrils and have the potential to exert regulatory effects on both proteolytic and innate immune surveillance mechanisms [5]. Resolution and separation of the variously-sized aggregates and complexes involved in amyloid diseases provides a key to understanding the mechanism of amyloidogenesis and developing approaches to control the process.

The analytical ultracentrifuge has a long history in studies of protein self-assembly. Sedimentation velocity and equilibrium techniques are widely used for characterizing protein complexes, protein–protein interactions (recent reviews [7], [8], [9]), and more specifically, for the analysis of amyloid oligomers and fibrils [10]. The development of a commercial fluorescence-detection system (FDS) for the analytical ultracentrifuge has significantly extended the sensitivity and specificity of these techniques [11], [12]. This detection system allows the sedimentation behavior of fluorescently labeled amyloid oligomers and fibrils to be explored over a much broader range of concentrations. Fluorescence detection using extrinsic fluorescence probes and ligands provides a new approach to characterizing the sedimentation and binding properties of amyloid fibrils. Of particular interest is the development of green fluorescence protein (GFP) constructs as in vivo fluorescence tags, permitting the use of sedimentation velocity studies and fluorescence detection for the analysis of protein aggregation and fibril formation in cell lysates and ex vivo [13]. The present work describes these new methods and provides examples of their use for the analysis of amyloid oligomers and fibrils.

Section snippets

Description of methods

All sedimentation experiments described here were performed using a Beckman XL-A analytical ultracentrifuge equipped with an AVIV Biomedical fluorescence-detection system (FDS). The rotor and cells employed were the same as those typically used for absorbance detection experiments (An-Ti60 rotor, cells with double-sector charcoal-epon centerpieces, and either quartz or sapphire windows). A Delrin 5-sector calibration cell manually filled with fluorescein was used in place of the counterbalance

Detection of soluble amyloid fibrils using a covalently bound fluorophore

Covalent modification of proteins by specific fluorophores provides a general method to monitor the distributions of proteins using the FDS in sedimentation velocity and equilibrium experiments. This approach, referred to as normal use tracer sedimentation (NUTS) [12], permits studies down to the picomolar concentration range and is therefore suited to studies of the high affinity interactions observed with amyloid fibril self-assembly. An example of this approach is provided by studies of the

Detection of an extrinsic dye that fluoresces when bound to amyloid fibrils

An alternative to the direct covalent fluorescence labeling of proteins for FDS experiments is to choose a fluorophore that binds specifically to the species of interest; an approach that has been referred to as biological on-line tracer sedimentation (BOLTS) [12]. 4-(Dicyanovinyl)-julolidine (DCVJ, Fig. 2) is a fluorescent probe used to monitor the formation of amyloid fibrils in vitro [22]. DCVJ belongs to a class of fluorescent molecules known as ‘molecular rotors’, and binds to amyloid

Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates

Many of the proteins known to form amyloid fibrils in vivo are lipid binding proteins, suggesting that fluorescently-labeled lipids can be used in sedimentation velocity experiments with fluorescence detection to study lipid-bound amyloid intermediates. Fibril formation by apoC-II is activated by sub-micellar concentrations of the short chain phospholipid, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) [16], [17], [24]. This activation is mediated by a lipid induced, tetrameric apoC-II

Detection of green fluorescent protein constructs and their interactions within mammalian cell lysates

A particularly useful feature of the FDS is the capacity to study how proteins aggregate in the context of their natural environment, such as in cells or in the extracellular milieu. Early studies using the FDS showed that GFP could be detected specifically in solutions containing high concentrations of a “background” protein (bovine serum albumin), which demonstrated the capacity for its application in complex solutions [11]. FDS was also shown to be useful for the study of fluorescent

Conclusions

The availability of fluorescence detection for the analytical ultracentrifuge has broadened the possible applications of sedimentation analysis of biological macromolecules. The system offers advantages in the accessible range of analyte concentrations, in the precise and specific detection of molecular interactions in oligomeric systems, and in the ability to specifically detect molecules within complex mixtures. In recent years, the FDS has been utilized to study the biophysical properties of

Acknowledgments

This work was funded in part by grants from the Australian Research Council (DP0877800, DP0984565).

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Sedimentation velocity analysis of amyloid oligomers and fibrils using fluorescence detection

Abstract

Introduction

Section snippets

Description of methods

Detection of soluble amyloid fibrils using a covalently bound fluorophore

Detection of an extrinsic dye that fluoresces when bound to amyloid fibrils

Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates

Detection of green fluorescent protein constructs and their interactions within mammalian cell lysates

Conclusions

Acknowledgments

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