Amine landscaping to maximize protein-dye fluorescence and ultrastable protein-ligand interaction
Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate...
| Main Authors: | , , , |
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| Format: | Journal article |
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Cell Press
2017
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| _version_ | 1797067567243722752 |
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| author | Jacobsen, M Fairhead, M Fogelstrand, P Howarth, M |
| author_facet | Jacobsen, M Fairhead, M Fogelstrand, P Howarth, M |
| author_sort | Jacobsen, M |
| collection | OXFORD |
| description | Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central tool for biological research. Dye-NHS modification of streptavidin severely damaged ligand binding. We generated a streptavidin variant retaining ultrastable binding after dye-NHS. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit—“amine landscaping”. Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (Fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. We confirmed Flavidin’s imaging benefit, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing general insight into simultaneously optimizing behavior of both biomolecule and chemical probe. |
| first_indexed | 2024-03-06T21:58:10Z |
| format | Journal article |
| id | oxford-uuid:4dac912b-29f5-4db5-830b-f51bdbbbc1c9 |
| institution | University of Oxford |
| last_indexed | 2024-03-06T21:58:10Z |
| publishDate | 2017 |
| publisher | Cell Press |
| record_format | dspace |
| spelling | oxford-uuid:4dac912b-29f5-4db5-830b-f51bdbbbc1c92022-03-26T15:56:38ZAmine landscaping to maximize protein-dye fluorescence and ultrastable protein-ligand interactionJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:4dac912b-29f5-4db5-830b-f51bdbbbc1c9Symplectic Elements at OxfordCell Press2017Jacobsen, MFairhead, MFogelstrand, PHowarth, MChemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central tool for biological research. Dye-NHS modification of streptavidin severely damaged ligand binding. We generated a streptavidin variant retaining ultrastable binding after dye-NHS. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit—“amine landscaping”. Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (Fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. We confirmed Flavidin’s imaging benefit, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing general insight into simultaneously optimizing behavior of both biomolecule and chemical probe. |
| spellingShingle | Jacobsen, M Fairhead, M Fogelstrand, P Howarth, M Amine landscaping to maximize protein-dye fluorescence and ultrastable protein-ligand interaction |
| title | Amine landscaping to maximize protein-dye fluorescence and ultrastable protein-ligand interaction |
| title_full | Amine landscaping to maximize protein-dye fluorescence and ultrastable protein-ligand interaction |
| title_fullStr | Amine landscaping to maximize protein-dye fluorescence and ultrastable protein-ligand interaction |
| title_full_unstemmed | Amine landscaping to maximize protein-dye fluorescence and ultrastable protein-ligand interaction |
| title_short | Amine landscaping to maximize protein-dye fluorescence and ultrastable protein-ligand interaction |
| title_sort | amine landscaping to maximize protein dye fluorescence and ultrastable protein ligand interaction |
| work_keys_str_mv | AT jacobsenm aminelandscapingtomaximizeproteindyefluorescenceandultrastableproteinligandinteraction AT fairheadm aminelandscapingtomaximizeproteindyefluorescenceandultrastableproteinligandinteraction AT fogelstrandp aminelandscapingtomaximizeproteindyefluorescenceandultrastableproteinligandinteraction AT howarthm aminelandscapingtomaximizeproteindyefluorescenceandultrastableproteinligandinteraction |