Abstract：
: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.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
O'Nuallain B,Thakur AK,Williams AD,Bhattacharyya AM,Chen S,Thiagarajan G,Wetzel Rdoi
10.1016/S0076-6879(06)13003-7subject
Has Abstractpub_date
2006-01-01 00:00:00pages
34-74eissn
0076-6879issn
1557-7988pii
S0076-6879(06)13003-7journal_volume
413pub_type
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