Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)(n) (n = 3similar to4), and helical linkers, (EAAAK)(n) (n = 2similar to5). 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 = 2similar to3) causes multimerization, while the longer linkers (n = 4similar to5) 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 Angstrom and 120 Angstrom with the flexible linker, and 40.2 Angstrom and 130 Angstrom 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 Angstrom and 140 Angstrom, 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. (C) 2004 Wiley-Liss, Inc.