title: Deletional protein engineering based on stable fold.
abstract: Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191-196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.
authors.affiliation: Department of Chemical Engineering, Pusan National University, Busan, South Korea.
authors.forename: Govindan
authors.initials: G
authors.lastname: Raghunathan
authors.forename: Nagasundarapandian
authors.initials: N
authors.lastname: Soundrarajan
authors.forename: Sriram
authors.initials: S
authors.lastname: Sokalingam
authors.forename: Hyungdon
authors.initials: H
authors.lastname: Yun
authors.forename: Sun-Gu
authors.initials: SG
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001665:Binding Sites; D055592:Biophysical Phenomena; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion
chemical_list: D000596:Amino Acids; D049452:Green Fluorescent Proteins