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) forma tion of interchain disulfide bonds; and (iii) formation of interchain isope ptide cross-links. Oxidative refolding and thermal unfolding of ribonucleas e A, lysozyme, and protein disulfide isomerase led to the formation of cros s-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophor esis, Chemical modification of free amino groups in these proteins or unfol ding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, pre formed interchain disulfide bonds were pivotal for promoting subsequent int erchain isopeptide crosslinks; no dimers/oligomers were detected when the r efolding and unfolding solution contained the reducing agent dithiothreitol , Similarly; the Cys326Ser point mutation in protein disulfide isomerase ab rogated its ability to cross-link into homodimers, Heterogeneous proteins b ecome cross-linked following the formation of heteromolecular interchain di sulfide bonds during thermal unfolding of a mixture of of ribonuclease A an d lysozyme, The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-link s and interchain isopeptide cross-links, No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of therm al denaturation temperature.