J. Biochem, 2001, Vol. 129, No. 1 179-183
© 2001 Japanese Biochemical Society
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Interchain Disulfide Bonds Promote Protein Cross-Linking during Protein Folding1
Department of Bioimmunotherapy, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard Houston, TX 77030, USA
2To whom correspondence should be addressed. Tel: +1-713-792-2649, Fax: +1-713-745-4167, E-mail: kmehta{at}mailmdanderson.org
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 crosslinks; 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.
1This work was supported by a grant from the World Health Organization.