Activating an Enzyme by an Engineered Coiled Coil Switch

摘要

We designed a de novo pro-tein based on a circular permutant of RNaseTl,in which the enzymatic ac-tivity can be manipulated by engi-neered peptide binding.The circular permutant of RNaseTl was obtained by tethering the original C-and N-ter-mini with a GPAG linker and cleaving the molecule between Glu~(82) and Asn~(83).This mutant lacked enzymatic activity,due to the destabilization of entire pro-tein structure.We previously reported the construction of ABC-type hetero-trimeric coiled coil peptides,in which the A-and B-type peptides cannot form the folded trimeric structure with-out the C-type peptide.The introduc-tion of the A-and B-type coiled coil peptides to the C-and N-termini of the circular permutant of RNaseTl,respec-tively,and the subsequent addition of the C-type coiled coil peptide enabled the RNaseTl domain to refold proper-ly,thus,restoring the enzymatic activi-ty.The formation of the trimeric coiled coil structure should bring the cleaved sites of RNaseTl close enough to refold the RNaseTl domain spontane-ously.