Probing the molecular mechanism of action of co-repressor in the E. coli methionine repressor-operator complex using surface plasmon resonance (SPR).
We have studied quantitatively the effect of the corepressor, 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 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 corepressor is electrostatic in origin. The difference in electrostatic interaction energy between the SAM- and AO-repressor-operator complexes of approximately 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.
Parsons
I D
ID
Department of Genetics, University of Leeds, UK.
Persson
B
B
Mekhalfia
A
A
Blackburn
G M
GM
Stockley
P G
PG
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009876:Operon; D011994:Recombinant Proteins; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000494:pharmacology*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C092076:aza-S-adenosylmethionine; C060486:methionine repressor protein, Bacteria; C489220:methionine repressor protein, E coli; D012436:S-Adenosylmethionine; D000241:Adenosine; C006235:sinefungin