Structural and biochemical investigation into stable FGF2 mutants with novel mutation sites and hydrophobic replacements for surface-exposed cysteines.
| Title: | Structural and biochemical investigation into stable FGF2 mutants with novel mutation sites and hydrophobic replacements for surface-exposed cysteines. |
|---|---|
| Authors: | An, Young Jun1 (AUTHOR), Jung, Ye-Eun2 (AUTHOR), Lee, Kyeong Won1 (AUTHOR), Kaushal, Prashant3 (AUTHOR), Ko, In Young4 (AUTHOR), Shin, Seung Min1 (AUTHOR), Ji, Sangho5 (AUTHOR), Yu, Wookyung5 (AUTHOR), Lee, Cheolju3 (AUTHOR), Lee, Won-Kyu4 (AUTHOR), Cha, Kiweon4 (AUTHOR), Lee, Jung-Hyun1 (AUTHOR) jlee@kiost.ac.kr, Cha, Sun-Shin2 (AUTHOR) chajung@ewha.ac.kr, Yim, Hyung-Soon1 (AUTHOR) yimh@kiost.ac.kr |
| Source: | PLoS ONE. 9/5/2024, Vol. 19 Issue 9, p1-22. 22p. |
| Subject Terms: | *FIBROBLAST growth factor 2, *PROTEIN stability, *ATOMIC structure, *STRUCTURAL engineering, *THERMAL stability |
| Abstract: | Fibroblast growth factor 2 (FGF2) is an attractive biomaterial for pharmaceuticals and functional cosmetics. To improve the thermo-stability of FGF2, we designed two mutants harboring four-point mutations: FGF2-M1 (D28E/C78L/C96I/S137P) and FGF2-M2 (D28E/C78I/C96I/S137P) through bioinformatics, molecular thermodynamics, and molecular modeling. The D28E mutation reduced fragmentation of the FGF2 wild type during preparation, and the substitution of a whale-specific amino acid, S137P, enhanced the thermal stability of FGF2. Surface-exposed cysteines that participate in oligomerization through intermolecular disulfide bond formation were substituted with hydrophobic residues (C78L/C78I and C96I) using the in silico method. High-resolution crystal structures revealed at the atomic level that the introduction of mutations stabilizes each local region by forming more favorable interactions with neighboring residues. In particular, P137 forms CH-π interactions with the side chain indole ring of W123, which seems to stabilize a β-hairpin structure, containing a heparin-binding site of FGF2. Compared to the wild type, both FGF2-M1 and FGF2-M2 maintained greater solubility after a week at 45 °C, with their Tm values rising by ~ 5 °C. Furthermore, the duration for FGF2-M1 and FGF2-M2 to reach 50% residual activity at 45 °C extended to 8.8- and 8.2-fold longer, respectively, than that of the wild type. Interestingly, the hydrophobic substitution of surface-exposed cysteine in both FGF2 mutants makes them more resistant to proteolytic cleavage by trypsin, subtilisin, proteinase K, and actinase than the wild type and the Cys → Ser substitution. The hydrophobic replacements can influence protease resistance as well as oligomerization and thermal stability. It is notable that hydrophobic substitutions of surface-exposed cysteines, as well as D28E and S137P of the FGF2 mutants, were designed through various approaches with structural implications. Therefore, the engineering strategies and structural insights adopted in this study could be applied to improve the stability of other proteins. [ABSTRACT FROM AUTHOR] |
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| Items | – Name: Title Label: Title Group: Ti Data: Structural and biochemical investigation into stable FGF2 mutants with novel mutation sites and hydrophobic replacements for surface-exposed cysteines. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22An%2C+Young+Jun%22">An, Young Jun</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Jung%2C+Ye-Eun%22">Jung, Ye-Eun</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Lee%2C+Kyeong+Won%22">Lee, Kyeong Won</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Kaushal%2C+Prashant%22">Kaushal, Prashant</searchLink><relatesTo>3</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Ko%2C+In+Young%22">Ko, In Young</searchLink><relatesTo>4</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Shin%2C+Seung+Min%22">Shin, Seung Min</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Ji%2C+Sangho%22">Ji, Sangho</searchLink><relatesTo>5</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Yu%2C+Wookyung%22">Yu, Wookyung</searchLink><relatesTo>5</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Lee%2C+Cheolju%22">Lee, Cheolju</searchLink><relatesTo>3</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Lee%2C+Won-Kyu%22">Lee, Won-Kyu</searchLink><relatesTo>4</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Cha%2C+Kiweon%22">Cha, Kiweon</searchLink><relatesTo>4</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Lee%2C+Jung-Hyun%22">Lee, Jung-Hyun</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> jlee@kiost.ac.kr</i><br /><searchLink fieldCode="AR" term="%22Cha%2C+Sun-Shin%22">Cha, Sun-Shin</searchLink><relatesTo>2</relatesTo> (AUTHOR)<i> chajung@ewha.ac.kr</i><br /><searchLink fieldCode="AR" term="%22Yim%2C+Hyung-Soon%22">Yim, Hyung-Soon</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> yimh@kiost.ac.kr</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22PLoS+ONE%22">PLoS ONE</searchLink>. 9/5/2024, Vol. 19 Issue 9, p1-22. 22p. – Name: Subject Label: Subject Terms Group: Su Data: *<searchLink fieldCode="DE" term="%22FIBROBLAST+growth+factor+2%22">FIBROBLAST growth factor 2</searchLink><br />*<searchLink fieldCode="DE" term="%22PROTEIN+stability%22">PROTEIN stability</searchLink><br />*<searchLink fieldCode="DE" term="%22ATOMIC+structure%22">ATOMIC structure</searchLink><br />*<searchLink fieldCode="DE" term="%22STRUCTURAL+engineering%22">STRUCTURAL engineering</searchLink><br />*<searchLink fieldCode="DE" term="%22THERMAL+stability%22">THERMAL stability</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: Fibroblast growth factor 2 (FGF2) is an attractive biomaterial for pharmaceuticals and functional cosmetics. To improve the thermo-stability of FGF2, we designed two mutants harboring four-point mutations: FGF2-M1 (D28E/C78L/C96I/S137P) and FGF2-M2 (D28E/C78I/C96I/S137P) through bioinformatics, molecular thermodynamics, and molecular modeling. The D28E mutation reduced fragmentation of the FGF2 wild type during preparation, and the substitution of a whale-specific amino acid, S137P, enhanced the thermal stability of FGF2. Surface-exposed cysteines that participate in oligomerization through intermolecular disulfide bond formation were substituted with hydrophobic residues (C78L/C78I and C96I) using the in silico method. High-resolution crystal structures revealed at the atomic level that the introduction of mutations stabilizes each local region by forming more favorable interactions with neighboring residues. In particular, P137 forms CH-π interactions with the side chain indole ring of W123, which seems to stabilize a β-hairpin structure, containing a heparin-binding site of FGF2. Compared to the wild type, both FGF2-M1 and FGF2-M2 maintained greater solubility after a week at 45 °C, with their Tm values rising by ~ 5 °C. Furthermore, the duration for FGF2-M1 and FGF2-M2 to reach 50% residual activity at 45 °C extended to 8.8- and 8.2-fold longer, respectively, than that of the wild type. Interestingly, the hydrophobic substitution of surface-exposed cysteine in both FGF2 mutants makes them more resistant to proteolytic cleavage by trypsin, subtilisin, proteinase K, and actinase than the wild type and the Cys → Ser substitution. The hydrophobic replacements can influence protease resistance as well as oligomerization and thermal stability. It is notable that hydrophobic substitutions of surface-exposed cysteines, as well as D28E and S137P of the FGF2 mutants, were designed through various approaches with structural implications. Therefore, the engineering strategies and structural insights adopted in this study could be applied to improve the stability of other proteins. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of PLoS ONE is the property of Public Library of Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1371/journal.pone.0307499 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 22 StartPage: 1 Subjects: – SubjectFull: FIBROBLAST growth factor 2 Type: general – SubjectFull: PROTEIN stability Type: general – SubjectFull: ATOMIC structure Type: general – SubjectFull: STRUCTURAL engineering Type: general – SubjectFull: THERMAL stability Type: general Titles: – TitleFull: Structural and biochemical investigation into stable FGF2 mutants with novel mutation sites and hydrophobic replacements for surface-exposed cysteines. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: An, Young Jun – PersonEntity: Name: NameFull: Jung, Ye-Eun – PersonEntity: Name: NameFull: Lee, Kyeong Won – PersonEntity: Name: NameFull: Kaushal, Prashant – PersonEntity: Name: NameFull: Ko, In Young – PersonEntity: Name: NameFull: Shin, Seung Min – PersonEntity: Name: NameFull: Ji, Sangho – PersonEntity: Name: NameFull: Yu, Wookyung – PersonEntity: Name: NameFull: Lee, Cheolju – PersonEntity: Name: NameFull: Lee, Won-Kyu – PersonEntity: Name: NameFull: Cha, Kiweon – PersonEntity: Name: NameFull: Lee, Jung-Hyun – PersonEntity: Name: NameFull: Cha, Sun-Shin – PersonEntity: Name: NameFull: Yim, Hyung-Soon IsPartOfRelationships: – BibEntity: Dates: – D: 05 M: 09 Text: 9/5/2024 Type: published Y: 2024 Identifiers: – Type: issn-print Value: 19326203 Numbering: – Type: volume Value: 19 – Type: issue Value: 9 Titles: – TitleFull: PLoS ONE Type: main |
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