Abstract
Circular permutation (CP) is a protein-engineering technique to improve the characteristics of protein. In contrast with traditional mutagenesis, CP requires a linker to connect the native termini and creates new termini to result in the rearrangement of protein sequence. Vigna radiata plant defensin 1 (VrD1) is a cysteine-rich protein containing four disulfide bonds, and has a disulfide bond (C3-C46) bridging the native termini. We applied CP to VrD1 for exploring the influence of linker length and the effect of CP on cysteine-rich protein. The characteristics of structure and function in CP-VrD1 were analyzed through circular dichroism, intrinsic fluorescence, and α-amylase inhibition assay. We found that linkers with length less than the distance between original termini resulted in structural change and losing functions. Therefore, we speculated the poor folding of CP-VrD1 with shorter linker was due to the simultaneous existence of shorter linker and the terminal disulfide bond (C3-C46). Site-directed mutagenesis was used to remove terminal disulfide-bond in CP-VrD1 with linkers of different length, and to further elucidate the influence of linker length and disulfide-bond. Our results showed that terminal disulfide-bond removed CP-VrD1 still has poor structural folding and less thermal stability, but the function was not changed. We concluded that poor folding of CP-VrD1 was caused by the constraint of linker with shorter length rather than the existence of terminal disulfide bond (C3-C46).