β2-Microglobulin (β2-m), a light chain of the major histocompatibility complex type I, is also found as a major component of amyloid fibrils formed in dialysis-related amyloidosis. Denaturation of β2-m is considered to initiate the formation of fibrils. To clarify the mechanism of fibril formation, it is important to characterize the intermediate conformational states at the atomic level. Here, we investigated the refolding of β2-m from the acid-unfolded state by heteronuclear magnetic resonance and circular dichroism spectroscopies. At low temperature, β2-m refolded slowly, accumulating a rate-limiting intermediate with non-native chemical shift dispersions for several residues, but with compactness and secondary structures similar to those of the native protein. β2-m has a cis proline residue at Pro32, located on the turn connecting the βB and βC strands. The slow refolding phase disappeared upon mutation of Pro32 to Val, indicating that Pro32 is responsible for the accumulation of the intermediate. The distribution of the perturbed residues in the intermediate suggests that the non-native prolyl peptide bond of Pro32 affects large areas of the molecule. A cis proline residue is common to various immunoglobulin domains involved in amyloidosis, implying that a non-native prolyl peptide bond that might occur under physiological conditions is related to the amyloidogenicity of these immunoglobulin domains.
- Dialysis-related amyloidosis
- Prolyl cis-trans isomerism
- Protein folding and misfolding