Abstract

The coiled-coil neck domain of pulmonary surfactant protein D (SP-D) is required for trimeric association and the subsequent assembly of functional dodecamers of SP-D. It is also necessary and sufficient for trimerization of a heterologous collagen sequence. To investigate whether it is capable of driving trimerization of heterologous noncollagenous proteins, we expressed and purified a fusion of a heterologous non-collagenous sequence (thioredoxin) to the coiled-coil neck domain of human SP-D here. While western blot analysis detected a small population of stable trimers of the fusion protein, chemical cross-linking and SEC-HPLC indicated that the fusion protein was predominantly a trimer. In contrast, purified thioredoxin without the fusion was found only as monomers and dimers. We also measured the thermal stabilities (with circular dichroism) and degradation rates of these two proteins. Our data showed that the fusion protein had a melting temperature that was 13 K higher than that of thioredoxin and a longer degradation half life than thioredoxin. Our findings indicate that the coiled-coil neck domain of SP-D enables the trimerization and stabilization of the heterologous non-collagenous thioredoxin. It may provide new clues for further study on the application of this human original coiled-coil domain in protein engineering to construst trimeric functional fusion proteins.

Keywords: pulmonary surfactant protein D, trimerization, stabilization, coiled-coil, thioredoxin, fusion proteins