Oxidative Refolding of Amyloidogenic Variants of Human Lysozyme

Normal and Aberrant Biological Self-Assembly: Insights from Studies of Human Lysozyme and Its Amyloidogenic Variants

“…Although, the I56T and D67H variants refold in a manner qualitatively similar to that of wild-type lysozyme, an interesting and initially unexpected finding is that they fold faster by a factor of 2 and 3, respectively. 11 The origin of this observation, however, can be attributed primarily to the fact that the lower stabilities of the native-like intermediates of the variants compared with those of the wild-type protein facilitate the conformational rearrangements associated with the final folding step.…”

Familial Amyloidosis Caused by Lysozyme

“…The final and slowest step involves a conformational rearrangement requiring at least local unfolding of the latter species in order to allow the remaining free thiol groups to form the fourth disulfide linkage and hence to generate the fully oxidized native protein. Although the I56T and D67H variants refold in a manner qualitatively similar to that of wild-type lysozyme, they fold faster by a factor of 2 and 3, respectively [25]. This finding can be attributed primarily to the fact that the lower stabilities of the native-like intermediates of the variants compared with those of the wild-type protein facilitates the conformational rearrangements associated with the final folding step.…”

“…Folding of the wild-type lysozyme in vitro from both the reduced and oxidized states is characterized by the existence of multiple pathways, involving a series of metastable intermediates [10,11,[22][23][24][25]. The I56T and D67H variants refold in a manner qualitatively similar to that the wild-type protein, although the rate at which they do so differs.…”

“…Unfolded lysozyme under reducing conditions (i.e., with all of its eight cysteine residues in the thiol form) corresponds to ensembles of unfolded conformers, and in the initial stages of folding these species collapse rapidly via a large number of parallel pathways to form a multitude of relatively unstructured intermediates having one or two disulfide bonds [25]. The majority of these species then fold to form a nativelike three-disulfide intermediate lacking the 77-95 bond.…”

Normal and Aberrant Biological Self‐Assembly: Insights from Studies of Human Lysozyme and Its Amyloidogenic Variants

“…The refolding of the wild-type, I56T, and D67H lysozymes from their reduced states in guanidinium chloride (where the protein is highly denatured and with all eight cysteine residues in the free thiol form) has therefore been studied in vitro under oxidative conditions designed to mimic, at least in general terms, protein folding in the endoplasmic reticulum. 11 Under these conditions, the reduced proteins all correspond to ensembles of unfolded conformers, and in the initial stages of folding these species collapse rapidly via a large number of parallel pathways to form a multitude of relatively unstructured intermediates having one or two disulfide bonds. The majority of these species then fold to form a native-like three-disulfide intermediate lacking the (77-95) bond.…”