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Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro
N M Kad1, N H Thomson, D P Smith
1School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
Abstract:
Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.
Insights
Growth conditions significantly impact beta(2)-microglobulin (beta(2)M) amyloid fibril morphology. Lowering ionic strength during in vitro formation yields longer, straighter fibrils, challenging previous assumptions about amyloid assembly intermediates.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Beta(2)-microglobulin (beta(2)M) amyloidosis is a complication of long-term dialysis.
- In vitro studies have produced short, curved beta(2)M fibrils under specific conditions.
- Patient-derived and seeded fibrils exhibit different morphologies.
Purpose of the Study:
- Investigate the influence of growth conditions on beta(2)M fibril morphology.
- Examine the impact of a specific beta(2)M mutation (N17D) on fibril formation.
- Characterize the structural diversity of in vitro-formed beta(2)M fibrils.
Main Methods:
- Recombinant wild-type and N17D mutant beta(2)M protein expression and purification.
- In vitro amyloid fibril formation under varying pH and ionic strength conditions.
- Electron microscopy (EM) and atomic force microscopy (AFM) for morphological analysis.
- Kinetic experiments to probe fibril assembly pathways.
Main Results:
- Deamidation of Asn17 to Asp (N17D) destabilizes beta(2)M at neutral pH but still requires acidification for amyloid formation.
- Low pH and low ionic strength promote the formation of long, straight recombinant beta(2)M fibrils.
- Three distinct fibril morphologies were identified by EM and AFM, differing in periodicity and protofibril number.
- Kinetic data suggest previously observed immature fibrils are not necessarily intermediates in mature amyloid assembly.
Conclusions:
- Fibril morphology is highly sensitive to environmental factors like ionic strength and pH.
- The N17D mutation affects protein stability but not the fundamental requirement for acidic conditions in amyloidogenesis.
- Beta(2)M amyloid fibrils exhibit significant structural heterogeneity.
- The study provides new insights into the assembly process and structural variations of beta(2)M amyloid fibrils.