All ETS proteins share a structurally conserved DNA binding domain (known as the ETS domain) that recognizes DNA sequences containing a central 5’-GGAA/T-3’ consensus ().
Aberrent expression of ETS-regulated genes are frequently implicated in human and veterinary cancers (–). ETS-family proteins regulate the expression of a functionally diverse array of genes throughout the Metazoan kingdom (–). PU.1 is a member of the ETS-family of proteins which comprise an evolutionarily conserved family of transcription factors (). To illustrate the utility of SPR in protein-DNA interactions, and methods to optimize experimental conditions for overcoming the limitations for some challenging systems, the interaction between the transcription factor PU.1 (Spi-1) with a specific DNA sequence will be used as an example in this protocol. Several approaches have been devised to mitigate these challenges, including the use of high flow rates, low immobilization densities, and the addition of DNA in the flow solution (–). These limitations have restricted use of these instruments with protein-DNA complex. These features give rise to several practical complications: i) mass transfer limits on kinetics, where the rates of transfer of components from the injected solution to the immobilized component is slower than the association reaction, ii) very slow dissociation rates with potential rebinding during the dissociation phase, and iii) limited time for the association reaction due to volume limitations in the injection syringe. Protein-DNA interactions present specific challenges for SPR characterization due to their generally high binding affinity and strong electrostatic nature. In addition, SPR generally requires only picomole to nanomole quantities of material which is minimal compared to other techniques generally used for evaluation of biomolecular interactions.ĭespite the widespread and long-standing use of SPR in characterizing protein-ligand and protein-protein interactions, evaluation of protein-DNA complexes with such instruments has been less extensive. The unique SPR detection mode has numerous advantages over conventional interaction analyses, such as optical methods for systems involving strong interactions and/or low fluorescence and absorbance. The real time responses allow the extent and rates of complex formation to be quantitatively determined. Upon complex formation on the sensor surface between these species, the refractive index changes are converted into SPR responses.
The other component(s) of the interaction is then injected over the sensor surface in a solution at the desired ionic strength and pH. In the SPR method one component of an interaction is immobilized on a sensor chip to create the biosensor interaction surface. From the initial development on protein-protein interactions, the applications of SPR have been significantly extended to diverse biomolecule complexes, including protein-nucleic acid, protein-small molecules, and nucleic acid-small molecules (–8). During the past twenty years, commercial biosensors using surface plasmon resonance (SPR) detection have been introduced to the scientific community and have emerged as a major and powerful approach for characterizing biomolecular interactions with high quality kinetic and thermodynamic information (–).
0 kommentar(er)
