PROBING THE MOLECULAR MECHANISM OF ACTION OF CO-REPRESSOR IN THE ESCHERICHIA-COLI METHIONINE REPRESSOR-OPERATOR COMPLEX USING SURFACE-PLASMON RESONANCE (SPR)

We have studied quantitatively the effect of the compressor S-adenosylmethionine (SAM), on the interaction between the E.coli methionine repressor, MetJ, and an idealised operator fragment, by recording measurements of surface plasmon resonance using a BIAcore(TM) instrument. We have recorded kinetic binding data in the presence of SAM, which carries a net positive charge, and two corepressor analogues, adenosylornithine (AO) and aza-SAM, which differ in the location of the atom carrying the positive charge. Our data support the hypothesis that the effect of the compressor is electrostatic in origin. The difference in electrostatic interaction energy between the SAM- and AO-repressor-operator complexes of approximate to 3.5 kJ/mol calculated from the known three-dimensional structure is within the range of our experimentally determined values of 2.8-4.3 kJ/mol. These results illustrate the potential of SPR measurements for studying protein-nucleic acid interactions.