THE SIGNAL TRANSDUCER GP130 - BACTERIAL EXPRESSION, REFOLDING AND PROPERTIES OF THE CARBOXY-TERMINAL DOMAIN OF THE CYTOKINE-BINDING MODULE

Gp130 is the signal transducing receptor subunit of the so-called inte rleukin-6-type cytokines. This transmembrane protein is a member of th e cytokine-receptor superfamily predicted to consist of six fibronecti n-type-III-like domains in its extracellular part. The second and the third domain constitute the so-called cytokine-binding module. Domain 2 is characterized by a set of four conserved Cys residues, domain 3 b y a conserved WSXWS motif. As a first approach to a more detailed char acterization of the cytokine-binding domains of human gp130, we have e xpressed in Escherichia coli two forms of domain 3 differing in length . Both proteins were purified and refolded in a single step applying s ize-exclusion chromatography. According to the rotational correlation times deduced from fluorescence anisotropy decay, they do not form agg regates. CD and fluorescence spectroscopy were used to study thermal u nfolding and denaturation by guanidinium hydrochloride. It was shown t hat N- and C-terminal extension by residues of the adjacent hinge regi ons substantially increase the thermal stability of the domain, which is conceivable from a molecular model. These results are the basis for further structural investigation by NMR spectroscopy.