| Abstract: | The fluorescent probe N-(iodoacetyl)amino]-ethyl]-5-naphthylamine-1-sulfonate (I-AEDANS) reacts selectively with Cys-140 of the lac repressor. The reasons for this selectivity were investigated. The ability of 8-anilino-1-naphthalenesulfonate and 5,5'-bis(8-anilino-1-naphthalene-sulfonate) to bind noncovalently to the interface between the core and headpiece regions of the repressor suggested that I-AEDANS might also bind to this interface and then react intramolecularly with Cys-140 nearby. Two observations strongly support this model. (1) The selectivity for Cys-140 was lost when the headpiece regions were removed from the repressor. The rate of reaction with Cys-140 relative to Cys-107 in the repressor was 13.5 +/- 1.4, from trypsin digestions of labeled repressor. This ratio decreased to 2.1 +/- 1.0 for the core protein. (2) Iodoacetamide, which lacks the naphthylaminesulfonate portion of I-AEDANS, showed little selectivity for Cys-140 in either the repressor or the core. Nonreactive analogues of I-AEDANS did not alter the reaction of I-AEDANS with the repressor, presumably because they bound too weakly. Decreasing the ionic strength from 0.61 M to 56 mM decreased the selectivity of I-AEDANS for Cys-140 in the repressor, suggesting that I-AEDANS is not bound to the repressor by ionic interactions. Decreasing the pH from 8.5 to 7.5 increased the selectivity for Cys-140 only slightly. Fluorescent probes attached to Cys-140 appear to be ideally located to report motions of the headpieces , relative to the core, that attend DNA binding. |
