Investigation of D76N β₂-Microglobulin Using Protein Footprinting and Structural Mass Spectrometry

Abstract

NMR studies and X-ray crystallography have shown that the structures of the 99-residue amyloidogenic protein β2-microglobulin (β2m) and its more aggregation-prone variant, D76N, are indistinguishable, and hence, the reason for the striking difference in their aggregation propensities remains elusive. Here, we have employed two protein footprinting methods, hydrogen–deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), in conjunction with ion mobility-mass spectrometry, to probe the differences in conformational dynamics of the two proteins. Using HDX-MS, a clear difference in HDX protection is observed between these two proteins in the E-F loop (residues 70–77) which contains the D76N substitution, with a significantly higher deuterium uptake being observed in the variant protein. Conversely, following FPOP-MS only minimal differences in the level of oxidation between the two proteins are observed in the E–F loop region, suggesting only modest side-chain movements in that area. Together the HDX-MS and FPOP-MS data suggest that a tangible perturbation to the hydrogen-bonding network in the E–F loop has taken place in the D76N variant and furthermore illustrate the benefit of using multiple complementary footprinting methods to address subtle, but possibly biologically important, differences between highly similar proteins.

Pathogenic D76N Variant of β2-Microglobulin: Synergy of Diverse Effects in Both the Native and Amyloid States

“…Native structures were found to be similar by X-ray crystallography and NMR [ 20 , 22 , 24 , 53 ]. Besides its significantly decreased stability, the amyloidogenic propensity of native D76N β2m was attributed to alterations in conformational dynamics and unfolding-refolding kinetics [ 22 , 23 , 24 , 28 , 57 , 58 , 59 ]. By finding that it is not the overall net charge or pI of the protein, nor the overall charge distribution on the β2m surface, De Rosa et al speculated that “the decreased stability and remarkable aggregation trends of the D76N β2m mutant must be the result of specific yet uncharacterized properties, strictly linked to the structural location of the protein 76 site” [ 24 ].…”

“…Recently, several studies have investigated the structure and amyloid forming propensity of the D76N β2m variant [ 23 , 24 , 25 , 26 , 27 ], but it is still not clear how this single point mutation may affect the amyloidogenic properties and cause a disease different from that caused by the WT protein. A recent investigation of β2m fibrils by ssNMR suggests rearrangement of intramolecular interactions in D76N β2m, which might be responsible for the elevated amyloidogenic propensity of the protein [ 28 ].…”