title: Construction of a linker library with widely controllable flexibility for fusion protein design.
abstract: Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions.
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China.|Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China.|Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China.
authors.forename: Ziliang
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China. chongzhang@tsinghua.edu.cn.|Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China. chongzhang@tsinghua.edu.cn.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China.|Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China.
authors.forename: Bo-Jun
authors.initials: BJ
authors.lastname: Dong
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China.|Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China.
authors.forename: Ruo-Hai
authors.initials: RH
authors.lastname: Guo
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China.|Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China.
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Yue
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China.|Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China.
authors.forename: Li-Tang
authors.initials: LT
authors.lastname: Yan
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China. xhxing@tsinghua.edu.cn.|Key Laboratory of Industrial Biocatalysis, Ministry of Education, Beijing, China. xhxing@tsinghua.edu.cn.
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Xing
mesh_terms: D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins
keywords: DPD simulation; FRET; Flexibility/rigidity; Fusion protein; Linker; Molecular dynamics