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

Beta-2 microglobulin (\u3b22m) is the light chain of Class I major histocompatibility complex (MHC-I) complex. \u3b22m is an intrinsically amyloidogenic protein capable of forming amyloid fibrils in vitro and in vivo. \u3b22m displays the typical immunoglobulin-like fold with a disulphide bridge (Cys25-Cys80) cross-linking the two \u3b2-sheets. Engineering of the loop comprised between \u3b2-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 wild-type (wt) protein, and a type I \u3b2-turn in the W60G mutant. Here, we report a biophysical and structural characterization of the K58P-W60G \u3b22m mutant, where a Pro residue has been introduced in the type I \u3b2-turn i + 1 position. The K58P-W60G mutant shows improved chemical and temperature stability and faster folding relative to wt \u3b22m. The crystal structure (1.25 resolution) shows that the Cys25-Cys80 disulphide bridge is unexpectedly severed, in agreement with electrospray ionization-mass spectrometry (ESI-MS) spectra that indicate that a fraction of the purified protein lacks the internal disulphide bond. These observations suggest a stabilizing role for Pro58, and stress a crucial role for the DE loop in determining \u3b22m biophysical properties