| Weak protein-protein interactions are a commonly found in biological regulatory mechanisms, but current biophysical methods are limited in their ability to measure them. I developed a new method for quantitating weak interactions between proteins in which the interaction is "assisted" by a known DNA:DNA interaction. Oligonucleotides, which are conjugated to the proteins of interest, contain short complementary DNA sequences that provide additional binding energy for the protein:protein interaction. A stretch of unpaired bases links the protein to the hybridizing DNA sequence to allow formation of both the protein:protein and DNA:DNA interaction with minimal structural interference. I 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 (Kd = 0.37 mM) in the absence of arabinose and is weakened by the presence of arabinose. I also used the DNA-assistance method to investigate the interaction between two human proteins from the focal adhesion complex, PINCH and Nck2. I was unable to detect an interaction between these two proteins by the DNA-assistance method, indicating that the interaction between proteins under my experimental conditions, 300 mM NaCl pH 8, is weaker than 2 mM. |
