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The Coiled-Coil Neck Domain of Human Pulmonary Surfactant Protein D Drives Trimerization and Stabilization of Thioredoxin, a Heterologous Non-Collagenous Protein

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

Document Type: Research Article

DOI: https://doi.org/10.2174/092986609787601769

Publication date: 2009-03-01

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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