The 28.6 kDa vaccinia virus complement control protein (VCP) is an inhibitor of the complement system and has therapeutic potential. It is composed of four domains or modules and is a homologue of complement receptor 1 (CR1) and other mammalian regulators of complement activation. A key aspect to structure-function relationships in these proteins is the extent of intramolecular module-module interactions, since these dictate the overall shape and flexibility of the molecules. A protein fragment (VCP similar to 2,3) encompassing modules 2 and 3 of VCP was over-expressed in Pichia pastoris. Ultracentrifugation showed that VCP similar to 2,3 is highly asymmetric with an axial ratio of 5.3: 1, which is consistent with an end-to-end arrangement of the two modules. NMR spectroscopy, differential scanning calorimetry, CD and intrinsic tryptophan fluorescence were used to monitor unfolding of VCP similar to 2,3. Experiments performed over a range of temperatures and concentrations of guanidinium chloride revealed that module 2 unfolds under milder conditions than, and independently of, module 3. Unfolding of module 2 is not associated with extensive changes in amide N-15 and H-1 chemical shifts of module 3, implying that the modules do not form an extensive intermodular interface. Results obtained in this work for VCP similar to 2,3 are compared with those obtained in a study of CRI modules 15-17 [Kirkitadze, Krych, Uhrin, Dryden, Smith, Cooper, Wang, Hauhart, Atkinson and Barlow (1999) Biochemistry 38, 7019-7031].
|Number of pages||9|
|Publication status||Published - 15 Nov 1999|