FIG. 2.

In vivo chemical cross-linking between C24 and the second cysteine of di-Cys BglF proteins. (A) The three homo-bifunctional cysteine cross-linkers used. (B) Di-Cys BglF derivatives were specifically labeled with [³⁵S]methionine as described in Materials and Methods. Cells were pelleted, washed, resuspended with PBS, and incubated with BMH, p-PDM, or o-PDM dissolved in DMF or with only DMF. The reaction was quenched by addition of NEM and DTT, as described in Materials and Methods. Proteins were denatured in electrophoresis sample buffer and analyzed on 7.5% SDS-polyacrylamide gels, followed by autoradiography. Results are presented only for BglF variants that generated cross-links. The arrows indicate the positions of the cross-linked products of BglF.