Rare Example of a Protein where an Isolated Domain is More Stable than the Full-Length

Abstract

Studying the contribution of individual domains to protein structure and function is of considerable interest over the years. Domains are in general less stable than the corresponding full-length proteins. Here, we report an exceptional case of utrophin tandem calponin-homology (CH) domain. Isolated C-terminal CH domain (CH2) is both thermodynamically and kinetically more stable than the full-length tandem CH domain. Reversible, equilibrium denaturant melts using both circular dichroism and protein fluorescence signals show that the CH2 is thermodynamically more stable by 4.0 kcal/mol when compared with the full-length tandem CH domain. Thermal melts indicate that CH2 unfolds at a higher temperature (15°C) than the full-length protein. Stopped-flow kinetics indicate that the CH2 unfolds slower (by 3 times) and folds faster (by 7 times) than the full-length protein, suggesting the higher kinetic stability of CH2. Analytical ultracentrifugation, size-exclusion chromatography, and dynamic light scattering show that both CH2 and the full-length protein are monomers in solution, confirming that the higher stability of CH2 is not due to formation of oligomers. Thus, the utrophin tandem CH domain is a rare example in which an isolated domain is more stable than the corresponding full-length protein.