Kinetics and thermodynamics of amyloid assembly using a high-performance liquid chromatography-based sedimentation assay.
Nonnative protein aggregation has been classically treated as an amorphous process occurring by colloidal coagulation kinetics and proceeding to an essentially irreversible endpoint often ascribed to a chaotic tangle of unfolded chains. However, some nonnative aggregates, particularly amyloid fibrils, exhibit ordered structures that appear to assemble according to ordered mechanisms. Some of these fibrils, as illustrated here with the Alzheimer's plaque peptide amyloid beta, assemble to an endpoint that is a dynamic equilibrium between monomers and fibrils exhibiting a characteristic equilibrium constant with an associated free energy of formation. Some fibrils, as illustrated here with the polyglutamine repeat sequences associated with Huntington's disease, assemble via highly regular mechanisms exhibiting nucleated growth polymerization kinetics. Here, we describe a series of linked methods for quantitative analysis of such aggregation kinetics and thermodynamics, focusing on a robust high-performance liquid chromatography (HPLC)-based sedimentation assay. An integrated group of protocols is provided for peptide disaggregation, setting up the HPLC sedimentation assay, the preparation of fibril seed stocks and determination of the average functional molecular weight of the fibrils, elongation and nucleation kinetics analysis, and the determination of the critical concentration describing the thermodynamic endpoint of fibril elongation.
O'Nuallain
Brian
B
University of Tennessee Medical Center, Graduate School of Medicine, Knoxville, TN, USA.
Thakur
Ashwani K
AK
Williams
Angela D
AD
Bhattacharyya
Anusri M
AM
Chen
Songming
S
Thiagarajan
Geetha
G
Wetzel
Ronald
R
D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D016229:Amyloid beta-Peptides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D020005:Propanols / Q000737:chemistry; D020836:Protein Structure, Quaternary; D013696:Temperature; D013816:Thermodynamics; D014269:Trifluoroacetic Acid / Q000737:chemistry; D006845:Hydrocarbons, Fluorinated
D000682:Amyloid; D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D010455:Peptides; D020005:Propanols; D014269:Trifluoroacetic Acid; C078592:amyloid beta-protein (1-40); C097188:polyglutamine; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated