Use of surface plasmon resonance for real-time analysis of the interactionof ZO-1 and occludin

Surface plasmon resonance (SPR) spectroscopy was applied to study in real t ime, the interaction between the tight junction proteins ZO-1 and occludin. To imitate the morphology of tight junctions, a cytosolic tail of mouse oc cludin was immobilised at the sensor and guanylate kinase-like domain (Guk) was allowed to pass over the modified chip surface. The Guk domain of ZO-1 (residues 644-812) was found to bind to the cytoplasmic, carboxy-terminal region of occludin (residues 378-521). This interaction was systematically characterised with respect to the concentrations of both proteins and the b inding conditions. Under the given experimental conditions, association and dissociation showed saturation kinetics, with affinity in micromolar range : k(a) = 4.14 +/- 0.52 x 10(3) M-1 s(-1), k(d) = 3.04 +/- 0.38 x 10(-3) s(- 1), K-D = 639 +/- 51 nM. The results support the hypothesis that the Guk do main of ZO-1 is involved in the recruitment of the transmembrane protein oc cludin at tight junctions by interacting with the cytosolic carboxy-termina l sequence of occludin, located far from the cell membrane. We demonstrate the use of SPR spectroscopy as an effective approach for characterisation o f the interactions of junction proteins. (C) 2001 Academic Press.