期刊
JOURNAL OF BIOLOGICAL ENGINEERING
卷 13, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s13036-019-0157-4
关键词
beta-Lactam antibiotics; BlaR-CTD; Docking; Site-directed mutagenesis; Stability; Affinity
资金
- National Key R&D Program of China [2018YFD0500300, 2017YFC1600100]
- Natural Science Foundation of Hubei Province [2017CFB445]
- National Program for Risk Assessment of Quality and Safety of Livestock and Poultry Products [GJFP2019007, GJFP201800804]
Owing to the thermal instability and low affinity of BlaR-CTD to some -lactams, the receptor assay based on BlaR-CTD is limited in the detection of abundant variety of drugs and the result is often unstable. In this study, the three-dimensional structure of BlaR-CTD from Bacillus licheniformis ATCC14580 was constructed by homologous modeling based on the crystal structure of BlaR-CTD from B. licheniformis 749/I, and the binding sites of this protein to 40 -lactams were also obtained by molecular docking. To improve the stability and affinity of the protein, 23 mutant proteins were designed based on docking and homologous alignment results as well as by inserting disulfide bond and building the salt bridge. The mutation was rationality evaluated by SIFT and PloyPhen2 software. The heterologous expressed and purified mutant proteins were then subjected to the activity and stability assay. It was shown that among all mutant proteins, I188K/S19C/G24C, A138E/R50C/Q147C and S190Y/E183C/I188K respectively exhibited a higher affinity to 33, 22 and 21 -lactams than the wild-type protein, while I188K/S19C/G24C exhibited the best stability. This may due to that the conformation of the active site in mutant protein I188K/S19C/G24C changed, and the random coli in the surface of protein activity increased. Our study suggests a possible structure-function relationship on the stability and affinity of BlaR-CTD, which provides new insights into protein rational design study and lays a solid foundation for establishing the receptor-based screening assay for the detection of -lactam residues.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据