A DNA-assisted binding assay for weak protein-protein interactions.
We describe a new method used for quantitating weak interactions between proteins in which the weak interaction is "assisted" by a known DNA-DNA interaction. Oligonucleotides, which are conjugated to proteins of interest, contain short complementary DNA sequences that provide additional binding energy for protein-protein interactions. A stretch of unpaired bases links the protein to the hybridizing DNA sequence to allow formation of both protein-protein and DNA-DNA interactions with minimal structural interference. We validated the DNA-assisted binding method using heterodimerizing coiled-coil proteins. The method was then used to measure the predicted weak interaction between two domains of the Escherichia coli L-arabinose operon regulatory protein AraC. The interaction between domains has the expected magnitude (K(d)=0.37 mM) in the absence of arabinose. Upon addition of arabinose, we detected a weaker and unexpected interaction, which may necessitate modification of the proposed mechanism of AraC. The DNA-assisted binding method may also prove useful in the study of other weak protein-protein interactions.
Frato
Katherine E
KE
Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA. kfrato@jhu.edu
Schleif
Robert F
RF
D051777:AraC Transcription Factor / Q000235:genetics / Q000378:metabolism; D044704:DNA, A-Form / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000378:metabolism*
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D044704:DNA, A-Form; D029968:Escherichia coli Proteins; D009698:Nucleoproteins; D011506:Proteins