4] Linkage of protein assembly to protein-DNA binding

Abstract

Abstract In summary, we list a series of steps to serve as a general protocol to determine whether protein assembly is linked to DNA binding. Although no general procedure will be suitable for every system, judicious application of a combination of these steps when experimentally practical should enable one to identify and study systems in which protein assembly is linked to nucleic acid binding. 1. 1. Examine the assembly states of the free protein as well as the protein complexed with nucleic acid by direct, independent methods. If possible, these should be done under solution conditions and at protein and DNA concentrations that cover the range of conditions that will be used in DNA-binding titrations. However, for systems in which DNA-binding affinity is high, it may not be practical to perform these assembly state studies at the low concentrations of proteins used in the protein-DNA titrations. In these cases, the usefulness of these direct studies is limited as one cannot conclude that an assembly state that is stable at micromolar concentrations will also be stable at nanomolar concentrations. 2. 2. Titrations should be performed both by titrating DNA into protein to obtain plots of r vs DT and by titrating protein into DNA to obtain plots of θ vs PT. Determine if these are well described by a simple 1:1 Langmuir isotherm using the same equilibrium constant. If not, then an assembly-linked binding process is likely. The assembly states of the protein then need to be characterized. 3. 3. Alternatively, multiple titrations should be performed at several different protein and/or DNA concentrations in order to detect any protein assembly-linked process as a shift in the isotherm. Determine whether each isotherm can be fit to a simple 1:1 Langmuir isotherm using the same equilibrium constant. 4. 4. In all titrations performed at constant DNA concentrations, one must be cautious under conditions such that PT ⪢ DT, because under these conditions assembly-linked DNA binding is not readily detected as a shift in the binding isotherm. 5. 5. Once the equilibrium binding scheme has been determined, performed multiple titrations at a series of DNA and protein concentrations and perform a simultaneous global fitting of these isotherms to resolve estimates of the equilibrium interaction constants and their associated uncertainties.47