title: Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering.
abstract: We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3 approximately 4), and helical linkers, (EAAAK)n (n = 2 approximately 5). The conformations of the chimeric proteins with the various linkers were evaluated using small-angle X-ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2 approximately 3) causes multimerization, while the longer linkers (n = 4 approximately 5) solvate monomeric chimeric proteins. With the moderate-length linkers (n = 4), the observed radius of gyration (R(g)) and maximum dimension (D(max)) were 38.8 A and 120 A with the flexible linker, and 40.2 A and 130 A with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), R(g) and D(max) increased to 43.2 A and 140 A, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side-by-side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Arai
authors.forename: Willy
authors.initials: W
authors.lastname: Wriggers
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Nishikawa
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Fujisawa
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D017356:Synchrotrons / Q000295:instrumentation*; D014965:X-Rays
chemical_list: D011993:Recombinant Fusion Proteins