DE-loop mutations affect beta2 microglobulin stability, oligomerization, and the low-pH unfolded form

β2 microglobulin (β2m) is the light chain of class-I major histocompatibility complex (MHC-I). Its accumulation in the blood of patients affected by kidney failure leads to amyloid deposition around skeletal joints and bones, a severe condition known as Dialysis Related Amyloidosis (DRA). In an effort to dissect the structural determinants of β2m aggregation, several β2m mutants have been previously studied. Among these, three single-residue mutations in the loop connecting strands D and E (W60G, W60V, D59P) have been shown to affect β2m amyloidogenic properties, and are here considered. To investigate the biochemical and biophysical properties of wild-type (w.t.) β2m and the three mutants, we explored thermal unfolding by Trp fluorescence and circular dichroism (CD). The W60G mutant reveals a pronounced increase in conformational stability. Protein oligomerization and reduction kinetics were investigated by electrospray-ionization mass spectrometry (ESI-MS). All the mutations analyzed here reduce the protein propensity to form soluble oligomers, suggesting a role for the DE-loop in intermolecular interactions. A partially folded intermediate, which may be involved in protein aggregation induced by acids, accumulates for all the tested proteins at pH 2.5 under oxidizing conditions. Moreover, the kinetics of disulfide reduction reveals specific differences among the tested mutants. Thus, β2m DE-loop mutations display long-range effects, affecting stability and structural properties of the native protein and its low-pH intermediate. The evidence presented here hints to a crucial role played by the DE-loop in determining the overall properties of native and partially folded β2m. Published by Wiley-Blackwell. © 2010 The Protein Society.