The role of conformational flexibility in β2-microglobulin amyloid fibril formation at neutral pH.
BACKGROUND
Amyloid formation is implicated in a number of human diseases. β(2)-Microglobulin (β(2)m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and β(2)m amyloid formation at physiological pH has been investigated.


METHODS
HDX-ESI-MS was used to study the conformational dynamics of β(2)m. Protein engineering, or the addition of Cu(2+) ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of β(2)m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further.


RESULTS
ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type β(2)m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation.


CONCLUSIONS
The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of β(2)m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from β(2)m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly.
Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
John P
JP
Hodkinson
Sheena E
SE
Radford
Alison E
AE
Ashcroft
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D058767:Protein Unfolding; D012521:Sarcosine / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014199:Trehalose / Q000737:chemistry; D014270:Trifluoroethanol / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000378:metabolism
D000682:Amyloid; D001613:beta 2-Microglobulin; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol; D014199:Trehalose; D002118:Calcium; D012521:Sarcosine