Abstract
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.
Highlights
Ordered assembly of monomeric human 2-microglobulin (2m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis
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
We have demonstrated the power of Electrospray ionization mass spectrometry (ESI-MS) to resolve complex mixtures of protein conformers that rely on differences in the net positive charge between native and nonnative conformations
Summary
Samples were diluted 1:10 (v/v) into the appropriate low salt buffer and allowed to equilibrate in a 10-mm quartz cuvette (Hellma UK Ltd., Southend on Sea, Essex, UK) contained in a heated Peltier unit at 37 °C for 6 min prior to data acquisition Under these conditions, the fluorescence signal was constant over the acquisition time. Comparison of Acid Denaturation Monitored by Fluorescence and ESI-MS—To compare the pH denaturation profiles of 2m measured by ESI-MS and fluorescence, the fluorescence emission of the protein at 327 nm ( excitation 280 nm) was measured as a function of pH and compared with data computed directly from the known relative populations of native, partially folded, and acid-unfolded 2m determined by ESI-MS This process assumes that each conformer has a different fluorescence intensity at pH 6.0 and that each signal varies linearly with pH. All images were taken using a CM10 electron microscope (FEI, Eindhoven, Holland) operating at 100 keV
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