The effects of an ideal {beta}-turn on {beta}-2 microglobulin fold stability

Beta-2 microglobulin (β2m) is the light chain of the MHC-I complex. β2m is an intrinsically amyloidogenic protein capable of forming amyloid fibrils in vitro and in vivo. β2m displays the typical immunoglobulin-like fold with a disulphide bridge (Cys25 - Cys80) cross-linking the two β-sheets. Engineering of the loop comprised between β-strands D and E has shown that mutations in this region affect protein structure, fold stability, folding kinetics and amyloid aggregation properties. Such overall effects have been related to the DE loop backbone structure, which presents a strained conformation in the w.t. protein, and a type I β-turn in the W60G mutant. Here, we report a biophysical and structural characterisation of the K58P-W60G β2m mutant, where a Pro residue has been introduced in the type I β-turn i+1 position. The K58P-W60G mutant shows improved chemical and temperature stability and faster folding relative to w.t. β2m. The crystal structure (1.25Å resolution) shows that the Cys25 - Cys80 disulphide bridge is unexpectedly severed, in agreement with ESI-MS spectra that indicate that a fraction of the purified protein lacks the internal disulphide bond. These observations suggest a stabilising role for Pro58, and stress a crucial role for the DE loop in determining β2m biophysical properties