Deletional Protein Engineering Based on Stable Fold

Title
Deletional Protein Engineering Based on Stable Fold
Author(s)
윤형돈Govindan Raghunathan[Govindan Raghunathan]Nagasundarapandian Soundrarajan[Nagasundarapandian Soundrarajan]Sriram Sokalingam[Sriram Sokalingam]이선구[이선구]
Keywords
GREEN-FLUORESCENT PROTEIN; SECONDARY STRUCTURE ANALYSES; SULFOLOBUS-SOLFATARICUS; NATIVE-LIKE; CRYSTAL-STRUCTURE; INCLUSION-BODIES; SEQUENCE SPACE; STABILITY; EVOLUTION; ACYLPHOSPHATASE
Issue Date
201212
Publisher
PUBLIC LIBRARY SCIENCE
Citation
PLOS ONE, v.7, no.12
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.
URI
http://hdl.handle.net/YU.REPOSITORY/26680http://dx.doi.org/10.1371/journal.pone.0051510
ISSN
1932-6203
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