Analysis of Interaction between PHO4 and PHO2 Protein by Real Time BIA.

ABSTRACT

Applying real time BIA(biomolecular interaction analysis) the interaction between yeast PHO4 and PHO2 protein was analyzed. Recombinant PHO4 protein was coupled at the sensor chip via amino group. 5 &mgr;mol/L of recombinant PHO2 and PHO2 mutants which were fused with glutathione S-transferase were injected respectively. The mutant whose Ser 230 was changed into Asp (GST-2SD) showed high SPR (surface plasmon resonance) signal while wild type PHO2 and the mutant where Ser 230 was changed into Ala (GST-2SA) did not. This result indicated that there was interaction between PHO4 and GST-2SD proteins and the phosphorylation of the Ser 230 of PHO2 was essential. Five different concentration of GST-2SD (0.5 0.75 1.0 1.5 2.0&mgr;mol/L) were injected separately and the interaction kinetics was analyzed. The association rate constant is 2.4x10(4)(mol/L)(-1)s(-1) dissociation rate constant is 3.5x10(-5)s(-1) and association constant is 6.9x10(8) (mol/L)(-1).