Co-populated conformational ensembles of beta2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry.
Ordered assembly of monomeric human beta(2)-microglobulin (beta(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 degrees C), wild-type beta(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, beta(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of beta(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of beta(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.
Borysik
Antoni J H
AJ
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Radford
Sheena E
SE
Ashcroft
Alison E
AE
D019943:Amino Acid Substitution; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D016011:Normal Distribution; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014364:Tryptophan / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; D014364:Tryptophan