title: Design of the linkers which effectively separate domains of a bifunctional fusion protein.
abstract: With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting beta-can domains was compared with that of flexible linkers or of a three alpha-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2-5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an alpha-helix and the alpha-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This 'linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku,Tokyo 113-8656, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Arai
authors.forename: H
authors.initials: H
authors.lastname: Ueda
authors.forename: A
authors.initials: A
authors.lastname: Kitayama
authors.forename: N
authors.initials: N
authors.lastname: Kamiya
authors.forename: T
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D015195:Drug Design; D004735:Energy Transfer; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
chemical_list: D003432:Cross-Linking Reagents; D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins