Independently melting modules and highly structured intermodular junctionswithin complement receptor type 1

A Segment of complement receptor type 1 (CR1) corresponding to modules 15-1 7 was overexpressed as a functionally active recombinant protein with N-gly cosylation sites ablated by mutagenesis (referred to as CR1 similar to 15-1 7(-)). A protein consisting of modules 15 and 16 and another corresponding to module 16 were also overexpressed. Comparison of heteronuclear nuclear m agnetic resonance (NMR) spectra for the single, double, and triple module f ragments indicated that module 16 makes more extensive contacts with module 15 than with module 17. A combination of NMR, differential scanning calori metry, circular dichroism, and tryptophan-derived fluorescence indicated a complex unfolding pathway for CR1 similar to 15-17(-). As temperature or de naturant concentration was increased, the 16-17 junction appeared to melt f irst, followed by the 15-16 junction, and module 17 itself; finally, module s 15 and 16 became denatured. Modules 15 and 16 adopted an intermediate sta te prior to total denaturation. These results are compared with a previousl y published study [Clark, N. S., Dodd, I, Mossakowska, D. E., Smith, R. A, G., and Gore, M. G. (1996) Protein Eng. 9, 877-884] on a fragment consistin g of the N-terminal three CR1 modules which appeared to melt as a single un it.