Deletional protein engineering based on stable fold.
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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3519881?pdf=render |
| _version_ | 1828740583088193536 |
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| author | Govindan Raghunathan Nagasundarapandian Soundrarajan Sriram Sokalingam Hyungdon Yun Sun-Gu Lee |
| author_facet | Govindan Raghunathan Nagasundarapandian Soundrarajan Sriram Sokalingam Hyungdon Yun Sun-Gu Lee |
| author_sort | Govindan Raghunathan |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T00:42:23Z |
| format | Article |
| id | doaj.art-abc03c980ff54994a8a7cc4938bcb915 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T00:42:23Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-abc03c980ff54994a8a7cc4938bcb9152022-12-22T03:10:06ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01712e5151010.1371/journal.pone.0051510Deletional protein engineering based on stable fold.Govindan RaghunathanNagasundarapandian SoundrarajanSriram SokalingamHyungdon YunSun-Gu LeeDiversification 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.http://europepmc.org/articles/PMC3519881?pdf=render |
| spellingShingle | Govindan Raghunathan Nagasundarapandian Soundrarajan Sriram Sokalingam Hyungdon Yun Sun-Gu Lee Deletional protein engineering based on stable fold. PLoS ONE |
| title | Deletional protein engineering based on stable fold. |
| title_full | Deletional protein engineering based on stable fold. |
| title_fullStr | Deletional protein engineering based on stable fold. |
| title_full_unstemmed | Deletional protein engineering based on stable fold. |
| title_short | Deletional protein engineering based on stable fold. |
| title_sort | deletional protein engineering based on stable fold |
| url | http://europepmc.org/articles/PMC3519881?pdf=render |
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