Rational design-guided improvement of protein thermostability typically requires identification of residues or regions contributing to instability and introduction of mutations into these residues or regions. One popular method, B-FIT, utilizes B-factors to identify unstable residues or regions and combines them with other strategies, such as directed evolution. Here, we performed structure-based engineering to improve the thermostability of the subtilisin E-S7 (SES7) peptidase. The B-value of each residue was redefined in a normalized B-factor calculation, which was implemented with a refined bioinformatics analysis strategy to identify the critical area (loop 158-162) related to flexibility and to screen for suitable thermostable motif sequences in the Protein DataBank that can act as transplant loops. In total,weanalyzed 445 structures and identified 29 thermostable motifs as candidates. Using these motifs as a starting point, we performed iterative homologous modeling to obtain a desirable chimera loop and introduced five different mutations into this loop to construct thermostable SES7 proteins. Differential scanning fluorimetry revealed increases of 7.3 °C in the melting temperature ofanSES7variant designatedM5 compared with the WT. The X-ray crystallographic structure of this variant was resolved at 1.96Å resolution. The crystal structure disclosed that M5 forms more hydrogen bonds than the WT protein, consistent with design and molecular dynamics simulation results. In summary, the modified B-FIT strategy reported here has yielded a subtilisin variant with improved thermostability and promising industrial applications, supporting the notion that this modified method is a powerful tool for protein engineering.
Bibliographical noteFunding Information:
This work was supported by National Key Research and Development Pro-gram of China 2017YFB0308401 (to J. Z.), Program for Changjiang Scholars and Innovative Research Team in University Grant IRT_15R26, National First-class Discipline Program of Light Industry Technology and Engineering Grant LITE2018-08, National Institutes of Health Grant R35 GM118047 (to H. A.), and the China Scholarship Council fund. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Insti-tutes of Health.
Acknowledgments—This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by National Institutes of Health (NIH) Grant P30 GM124165. The Pilatus 6M detector on the 24-ID-C beamline is funded by NIH Office of Research Infrastructure Program (ORIP) High-End Instrumentation (HEI) Grant S10 RR029205. This research used resources of the Advanced Photon Source, a United States Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. We thank Editage for English language editing.
© 2019 Tang et al.