Rapid Generation of Amyloid from Native Proteins In vitro

Summary

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Abstract

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Introduction

Proteins are the most abundant biological macromolecules present in all types of cells. They occur in a great variety of sizes, structures, and post-translational modifications, and fulfill an enormous range of important biological functions when in their native forms. More than two dozens of aberrant polypeptides have been implicated in numerous human pathological conditions, such as Alzheimer's disease, Parkinson disease,  and Type 2 Diabetes^1-4. The terminal misfolded proteins accumulate as amyloid fibrils the insoluble stable aggregates that occur extracellularly or intracellularly.

Despite the implication of sp....

Protocol

1. Prepare MES Buffer

1. Use MES buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid and 0.9 M NaCl) to prepare both protein-only amyloid as well as stabilized soluble protein oligomers. Weigh 1.95 g of MES and 5.27 g of NaCl. Add H₂O and adjust pH to 4.7. Make up the final volume to 100 ml. Store at 4º C for up to 6 months.

2. Prepare Protein-only Amyloid Directly from Native Proteins

1. Weigh protein of choice (any native protein or peptide) and reconstitute to 10 mg/ml in MES buffer.
2. Incubate at 65 ºC in a waterbath for 4 hr. By the end of the period, white precipitate is visible in s....

Results

The ability to convert native proteins directly to amyloid relies on the conformational change incurred at high temperature in the MES buffer. Precipitation in the solution is a good indication of protein aggregation and possibly amyloid formation. On the other hand, to allow soluble protein oligomer to stabilize, EDC is used to crosslink the proteins to solidify the oligomeric conformation induced in MES buffer. As a result, the stabilized protein oligomers are multimeric proteins (Figure 1), distinct f.......

Discussion

The method described here offers a rapid and flexible means to prepare amyloid fibrils in vitro from virtually any protein or peptide of choice. Several different types of amyloid can be reliably prepared - protein-only fibrils or hybrid aggregates containing DNA, RNA,  or glycosaminoglycans. The procedures involved are simple, straight forward, and do not require advanced technical training. The amyloid prepared by this method is quite stable and can be safely stored at 4 ºC or frozen at -80 ºC f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-(N-morpholino)ethanesulfonic acid (MES)	Sigma-Aldrich	M8250
NaCl	Fisher	BP358-10
1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC)	Thermo/Pierce	22980
DNA from salmon sperm	Sigma	D1626
RNA from torula yeast	Sigma	R6625
Heparin from porcine intestinal mucosa	Sigma	H3149
Tris base	Fisher	BP152-1
Equipment:
Water bath	Fisher Science	Isotemp 210
Slide-A-Lyzer dialysis cassette	Thermo/Pierce	66380