Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS.
We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding.
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Ricardo
R
Martí-Arbona
Munehiro
M
Teshima
Penelope S
PS
Anderson
Kristy L
KL
Nowak-Lovato
Elizabeth
E
Hong-Geller
Clifford J
CJ
Unkefer
Pat J
PJ
Unkefer
D000225:Adenine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D003839:Deoxyadenosines / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D008024:Ligands; D008715:Methionine / Q000096:biosynthesis* / Q000235:genetics; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013872:Thionucleosides / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D003839:Deoxyadenosines; D008024:Ligands; D012097:Repressor Proteins; D013872:Thionucleosides; C060486:methionine repressor protein, Bacteria; C008500:5'-methylthioadenosine; D012436:S-Adenosylmethionine; D008715:Methionine; D000225:Adenine