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.

Control of protein functional dynamics by peptide linkers

“…32 A range of stability occurs depending on the rigidity of the linker peptides. 33 Soft linkers confer flexibility, whereas more rigid peptides may act to keep domains apart.…”

“…32 Recombinant chimeric fusion proteins are routinely constructed to increase the expression of soluble proteins and to facilitate protein purification. 32,83 Other engineering approaches that link two proteins or protein domains by a peptide linker include immunoassays (e.g., using chimeras between antibody fragments and proteins 84,85 ), selection and production of antibodies, 86 and engineering of bifunctional enzymes.…”

“…32 Recombinant chimeric fusion proteins are routinely constructed to increase the expression of soluble proteins and to facilitate protein purification. 32,83 Other engineering approaches that link two proteins or protein domains by a peptide linker include immunoassays (e.g., using chimeras between antibody fragments and proteins 84,85 ), selection and production of antibodies, 86 and engineering of bifunctional enzymes. 87 In the respiratory chain, electron transfer protein domains of flavodoxin and cytochrome c553 from Desulfovibrio vulgaris and the heme domain of P450 BM3 from Bacillus megaterium have been used as molecular ''Lego''-type building blocks in different combinations to build artificial redox chains having variable redox potentials.…”

First Structural Glimpse of CCN3 and CCN5 Multifunctional Signaling Regulators Elucidated by Small Angle X-ray Scattering

“…Experiments done by Kubota et al (24) also demonstrated that certain biological effects were only seen when multiple domains were involved. Movement about linker regions has been seen to be important in other mosaic proteins (39), and the secondary structure, if any, of the linker regions can also have an effect on interdomain communication (40).…”

Solution Conformation and Thermodynamic Characteristics of RNA Binding by the Splicing Factor U2AF65

“…A short polymer of glycines, the residue with the fewest backbone restrictions, adopts a more extended conformation than an ␣-helical peptide (R g of ϳ1.5 Å and D max of ϳ5.7 Å per glycine residue (42) as compared with an average R g of ϳ0.4 Å per ␣-helical residue (43)). However, chimeric multidomain proteins with ␣-helical linkers are more elongated than counterparts containing flexible linkers, as reflected by increases in the R g and D max values derived from SAXS analysis (44). This difference is thought to arise from rearrangements of the flexible linker to accommodate molecular attraction between the protein domains, whereas sequences with ␣-helical propensity confer rigidity.…”