Abstract
Nuclear magnetic resonance (NMR) is used to determine protein structure in solution. The potential application is usually restricted by the presence of severe line broadening and resonance overlapping in a macromolecule. In the past decade, scientists widely used the strategy of segmental isotope labeling to overcome the problem. By employing split INTEIN to mediate protein trans-splicing (PTS), we can solve the problem by labeling one domain and leaving the other domain unlabeled. There is still no available split INTEIN structure in Protein Data Bank. Therefore, we reported the first solution structure of split INTEIN here. We developed a streamlined method to simply and quickly prepare Npu DnaE split INEIN and used NMR method to determine the solution structure. The demonstrated structure revealed differences to native INTEIN. It helps us to evaluate the interaction within the enzymatic site including the critical residues and the N- and C-terminus. Meanwhile, it might provide structural basis in understanding structural difference before and after PTS reaction. We also setup CS-ROSETTA system to allow us quickly predict the correct fold of native INTEIN based on very limited NOEs constraints. The strategy can dramatically curtail the time of structure determination and meanwhile, compromise the usage between conventional NMR structure determination and computer simulation.