Crosslinking of the cAMP receptor protein of Escherichia coli by o-phenylenedimaleimide as a probe of conformation

Abstract

Reaction of the c[cyclic]AMP receptor protein (CRP) of E. coli with the bifunctional reagent o-phenylenedimaleimide (oPDM) results in the cross-linking of the 2 subunits of a CRP protomer. In the presence of cAMP the rate of cross-linking increases. CRP modified with oPDM retains [3H]cAMP binding activity but loses [3H]d(I-C)n binding activity. Proteolysis of cross-linked CRP gives distinct sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns depending upon whether cAMP was present during the reaction with oPDM. CRP cross-linked in the absence of cAMP retains the same relative resistance to proteolysis as unmodified CRP. The presence of 0.1 mM cAMP during proteolysis results in the production of 2 fragments, 1 of .apprx. 13000 daltons and a 2nd of .apprx. 20000 daltons. CRP cross-linked with oPDM in the presence of cAMP (then dialyzed to remove cAMP) remains sensitive to .alpha.-chymotrypsin digestion even in the absence of added cAMP producing only the 13,000 fragment. The nature of the oPDM cross-link may be a consequence of the conformational state of CRP.