title: Altering the orientation of a fused protein to the RNA-binding ribosomal protein L7Ae and its derivatives through circular permutation.
abstract: RNA-protein complexes (RNPs) are useful for constructing functional nano-objects because a variety of functional proteins can be displayed on a designed RNA scaffold. Here, we report circular permutations of an RNA-binding protein L7Ae based on the three-dimensional structure information to alter the orientation of the displayed proteins on the RNA scaffold. An electrophoretic mobility shift assay and atomic force microscopy (AFM) analysis revealed that most of the designed circular permutants formed an RNP nano-object. Moreover, the alteration of the enhanced green fluorescent protein (EGFP) orientation was confirmed with AFM by employing EGFP on the L7Ae permutant on the RNA. The results demonstrate that targeted fine-tuning of the stereo-specific fixation of a protein on a protein-binding RNA is feasible by using the circular permutation technique.
authors.affiliation: Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Shoji J
authors.initials: SJ
authors.lastname: Ohuchi
authors.affiliation: Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Sagawa
authors.affiliation: Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba, 275-0016, Japan.
authors.forename: Taiichi
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan. Electronic address: tan@kuchem.kyoto-u.ac.jp.
authors.forename: Tan
authors.initials: T
authors.lastname: Inoue
mesh_terms: D000495:Allosteric Site; D019843:Archaeal Proteins / Q000737:chemistry; D019611:Archaeoglobus fulgidus / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008024:Ligands; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009154:Mutation; D053758:Nanoparticles / Q000737:chemistry; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D019642:RNA, Archaeal / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000737:chemistry*; D015398:Signal Transduction
chemical_list: D019843:Archaeal Proteins; D008024:Ligands; D019642:RNA, Archaeal; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D049452:Green Fluorescent Proteins; D012313:RNA
keywords: Circular permutation; RNA-protein complex; RNP nano-object