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A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation
David P Smith1, Susan Jones, Louise C Serpell
1School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Abstract:
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.
Insights
Beta-2-microglobulin (beta(2)m) amyloid formation depends on protein stability and specific denatured states. Mutagenesis reveals distinct fibril morphologies correlate with precursor states, offering insights into amyloidogenesis.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Beta-2-microglobulin (beta(2)m) forms amyloid fibrils in vitro under acidic conditions.
- Distinct fibril morphologies (short/curved vs. long/straight) observed at different pH values.
- Both fibril types exhibit amyloid characteristics like Congo red binding and cross-beta structure.
Purpose of the Study:
- Investigate the role of partially folded states in beta(2)m fibril formation.
- Examine the effect of protein stability on amyloidogenesis.
- Correlate specific denatured states with distinct fibril morphologies.
Main Methods:
- Detailed mutagenesis study of beta(2)m, creating 13 point variants.
- Analysis of protein structure, stability, and fibril formation propensity across a pH range.
- Characterization of fibril morphology and precursor states.
Main Results:
- Destabilization of the native beta(2)m state is crucial for amyloid fibril generation.
- Population of specific denatured states is a prerequisite for amyloid formation.
- Different fibril morphologies correlate with the relative populations of distinct precursor states.
Conclusions:
- Amyloid formation from beta(2)m is dependent on accessing specific denatured states, not just general destabilization.
- The study elucidates the relationship between protein precursor states and the resulting fibril morphology.
- Findings provide a deeper understanding of the mechanisms underlying beta(2)m amyloidogenesis.