title: Redesigning the type II' β-turn in green fluorescent protein to type I': implications for folding kinetics and stability.
abstract: Both Type I' and Type II' β-turns have the same sense of the β-turn twist that is compatible with the β-sheet twist. They occur predominantly in two residue β-hairpins, but the occurrence of Type I' β-turns is two times higher than Type II' β-turns. This suggests that Type I' β-turns may be more stable than Type II' β-turns, and Type I' β-turn sequence and structure can be more favorable for protein folding than Type II' β-turns. Here, we redesigned the native Type II' β-turn in GFP to Type I' β-turn, and investigated its effect on protein folding and stability. The Type I' β-turns were designed based on the statistical analysis of residues in natural Type I' β-turns. The substitution of the native "GD" sequence of i+1 and i+2 residues with Type I' preferred "(N/D)G" sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β-turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β-turns in natural β-hairpins can be further optimized by converting the sequence to Type I'.
authors.affiliation: Department of Chemical Engineering, Pusan National University, Busan, 609-735, South Korea.
authors.forename: Bharat
authors.initials: B
authors.lastname: Madan
authors.forename: Sriram
authors.initials: S
authors.lastname: Sokalingam
authors.forename: Govindan
authors.initials: G
authors.lastname: Raghunathan
authors.forename: Sun-Gu
authors.initials: SG
authors.lastname: Lee
mesh_terms: D000465:Algorithms; D020816:Amino Acid Motifs; D000818:Animals; D003198:Computer Simulation; D030562:Databases, Protein; D005103:Expert Systems; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D037402:Hydrozoa / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D058849:Protein Refolding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins
keywords: GFP; protein folding; type I' β-turn; type II' β-turn; β-hairpins; β-turns