Interchain Disulfide Bonds Promote Protein Cross-Linking during Protein Folding1

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 disul-fide 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. Hetero-geneous proteins become cross-linked following the formation of heteromolecular inter-chain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfoldin