Use of surface plasmon resonance for real-time analysis of the interaction of ZO-1 and occludin.
Surface plasmon resonance (SPR) spectroscopy was applied to study in real time, the interaction between the tight junction proteins ZO-1 and occludin. To imitate the morphology of tight junctions, a cytosolic tail of mouse occludin 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 binding 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 domain of ZO-1 is involved in the recruitment of the transmembrane protein occludin at tight junctions by interacting with the cytosolic carboxy-terminal sequence of occludin, located far from the cell membrane. We demonstrate the use of SPR spectroscopy as an effective approach for characterisation of the interactions of junction proteins.
Schmidt
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A
Institute of Molecular Pharmacology, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Utepbergenov
D I
DI
Krause
G
G
Blasig
I E
IE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D062793:Occludin; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance / Q000379:methods*; D019108:Tight Junctions / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein