Occurrence of S‐(1,2‐dicarboxyethyl)‐cysteine at position 77 in mutant human lysozyme secreted by Saccharomyces cerevisiae

Abstract

A mutant human lysozyme PI1', in which Val110 was replaced with Pro, was secreted by Succharomyces cerevisiae; modification of the cysteine residue at position 77 was found in a purified mutant protein (Pl'O-B) upon primary structure analysis. A peptide fragment containing 15 amino acid residues from Thr70 to Leu84 was obtained by proteolytic digestion of the protein and subsequently isolated by reverse-phase HPLC. This fragment was analyzed by high-resolution fast-atom-bombardment (FAB) mass spectrometry, which showed that 1,2-dicarboxyethyl group was attached to the thiol group of Cys77. This modification was confirmed by comparing it with a sample of chemically synthesized S-(l,2-dicarboxyethyl)-~-cysteine.It was found that the modification caused a disruption of the disulfide bond Cys77-Cys95 in the mutant molecule. These observations, plus structural considerations, suggest that Cys77 and Cys95 either remain uncrosslinked or the disulfide bond Cys77 -Cys95, once formed, is opened during the final step in the folding of human lysozyme in vivo.

Synthesis of Aromatic Monothiols and Aromatic Dithiols to Increase the Folding Rate and Yield of Disulfide Containing Proteins

“…Additionally, in another experiment at pH 7.4, des [76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94] was allowed to refold in the presence or absence of PDI, buffer containing 1 M urea, and 2 mM/0.4 mM GSH/GSSG. They found that the refolding rate of des [76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94] to native protein was ~8 times faster in the presence of PDI than in the absence of PDI. Furthermore, the data for mutant human lysozyme also suggested that the thiol group of Cys76 in des [76][77][78][79][80][81][82][83][84]…”

“…Therefore, des [76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94] appears to act as a kinetic trap.…”

“…86 Furthermore, they observed two other three-disulfide intermediates, des [64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80] and des . Des [64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80] accumulated to lower levels (10-15%) and oxidized more quickly than des [76][77][78][79][80][81][82][83][84][85][86][87][88][89][90]…”

“…Additionally, a short double stranded antiparallel β-sheet and one of the four disulfide bonds [76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94] link the two domains. 83 Two disulfide bonds and are present within the α-domain, and the remaining disulfide bond [64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80] is located within the β-domain (Figure 11).…”

“…The four disulfide bonds occur between residues Cys6-Cys127, Cys30-Cys115, Cys64-Cys80, and Cys76-Cys94 and provide stability to the native structure of lysozyme. 42 The disulfide bonds in native lysozyme are denoted as , , [64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80], and [76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94]. Lysozyme is made up of two domains, one consisting of four α-helices and a C-terminal 3 10 helix (called the α-domain), and one consisting of a triple-stranded antiparallel β-sheet, a 3 10 helix, and a…”