A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation.
Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.
Smith
David P
DP
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Jones
Susan
S
Serpell
Louise C
LC
Sunde
Margaret
M
Radford
Sheena E
SE
D000682:Amyloid / Q000378:metabolism*; D052160:Benzothiazoles; D002942:Circular Dichroism; D004396:Coloring Agents / Q000494:pharmacology; D003224:Congo Red / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011489:Protein Denaturation; D012965:Sodium Chloride / Q000494:pharmacology; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology; D014961:X-Ray Diffraction; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000378:metabolism
D000682:Amyloid; D052160:Benzothiazoles; D004396:Coloring Agents; D013844:Thiazoles; D001613:beta 2-Microglobulin; C009462:thioflavin T; D003224:Congo Red; D012965:Sodium Chloride; D014508:Urea