Interchain disulfide bonds promote protein cross-linking during protein folding.
We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.
Gao
Y
Y
Department of Bioimmunotherapy, The University of Texas M.D. Anderson Cancer Center, Holcombe Boulevard, Houston, TX 77030, USA.
Mehta
K
K
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D009113:Muramidase / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic; D009113:Muramidase; D019704:Protein Disulfide-Isomerases