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Mapping the Core of the B2-Microglobulin Amyloid Fibril by H/D Exchange

 

DMSO-dissolution and NMR assignment

H/D exchange combined with 2D NMR is a powerful technique to probe the structured regions in proteins at single-residue resolution. However, direct application of this technique to characterize amyloid fibrils is difficult because solution NMR is mostly limited to small proteins. We noticed that among various protein denaturants and organic solvents, dimethylsulfoxide (DMSO) is exceptionally effective for dissolving amyloid fibrils of ß2-m into the monomeric form, which is amenable to NMR analysis. Moreover, DMSO, (CH3)2S=O, has no exchangeable protons. In the presence of more than 90% (v/v) DMSO, the base-catalyzed H/D exchange is substantially decreased. These two properties make it possible to monitor the H/D exchange kinetics of the ß2-m amyloid fibrils by the following procedures;

[1] H/D exchange: The exchange reaction is carried out with amyloid fibrils at pD 2.5 and 25°C, conditions under which the ß2-m amyloid fibrils are stable.
[2] Quenching: After a specified period, the reaction is quenched by decreasing the temperature to 0°C, followed by desalting and lyophilization.
[3] Dissolution and NMR analysis: The lyophilized sample is dissolved with DMSO at pD 5.0, the minimal pH of exchange rate in DMSO.
[4] NMR analysis: The 1H-15N HSQC spectrum is measured to estimate the extent of exchange.
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