Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions.
Non-covalent protein-protein interactions are one of the most fundamental building blocks in cellular signalling pathways. Despite this, they have been historically hard to identify using conventional methods due to their often weak and transient nature. Using genetic code expansion and incorporatio...
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Public Library of Science (PLoS)
2025-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0316321 |
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| author | Zac Sandy Zijuan Wang Deepak Behera Benjamin M Foster Finlay A Martin Kira Brüninghoff Kathleen M Cain Wolfgang Dörner Maria Jose Cabello-Lobato Josep V Forment Matthew J Cliff Igor Larrosa Perdita Barran Duncan L Smith Henning D Mootz Christine K Schmidt |
| author_facet | Zac Sandy Zijuan Wang Deepak Behera Benjamin M Foster Finlay A Martin Kira Brüninghoff Kathleen M Cain Wolfgang Dörner Maria Jose Cabello-Lobato Josep V Forment Matthew J Cliff Igor Larrosa Perdita Barran Duncan L Smith Henning D Mootz Christine K Schmidt |
| author_sort | Zac Sandy |
| collection | DOAJ |
| description | Non-covalent protein-protein interactions are one of the most fundamental building blocks in cellular signalling pathways. Despite this, they have been historically hard to identify using conventional methods due to their often weak and transient nature. Using genetic code expansion and incorporation of commercially available unnatural amino acids, we have developed a highly accessible method whereby interactions between biotinylated ubiquitin-like protein (UBL) probes and their binding partners can be stabilised using ultraviolet (UV) light-induced crosslinks. The stabilised protein complexes can be purified using affinity purification and identified by mass spectrometry. The resultant covalent bonds can withstand even the harshest washing conditions, allowing for the removal of indirect binders whilst retaining and capturing weak and transient interactors that are commonly lost during wash steps. This technique is widely applicable and highly effective for identifying site-selective non-covalent interactors. Members of our team have previously demonstrated the benefit of this method using the small ubiquitin-like modifier (SUMO). Here, we provide further proof-of-principle validation of the method and highlight its generality by applying an optimised workflow to a lesser studied UBL, interferon stimulated gene 15 (ISG15). We show that this method is able to capture known ISG15 interactors from a complex protein mixture in a site-selective manner, only capturing proteins that specifically interact with the region of ISG15 where the unnatural amino acid was incorporated. This exquisite degree of sensitivity and specificity greatly improves upon previous screens aimed at identifying downstream non-covalent binders, or readers, of ISG15. Taken together, the approach opens the possibility of characterising previously undetected protein-protein interactions, with the potential of elucidating molecular mechanisms behind the most complex and poorly understood processes in the cell. |
| format | Article |
| id | doaj-art-c651b5c7c0414bd9bcd0dbadcdaa335f |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-c651b5c7c0414bd9bcd0dbadcdaa335f2025-02-05T05:32:02ZengPublic Library of Science (PLoS)PLoS ONE1932-62032025-01-01201e031632110.1371/journal.pone.0316321Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions.Zac SandyZijuan WangDeepak BeheraBenjamin M FosterFinlay A MartinKira BrüninghoffKathleen M CainWolfgang DörnerMaria Jose Cabello-LobatoJosep V FormentMatthew J CliffIgor LarrosaPerdita BarranDuncan L SmithHenning D MootzChristine K SchmidtNon-covalent protein-protein interactions are one of the most fundamental building blocks in cellular signalling pathways. Despite this, they have been historically hard to identify using conventional methods due to their often weak and transient nature. Using genetic code expansion and incorporation of commercially available unnatural amino acids, we have developed a highly accessible method whereby interactions between biotinylated ubiquitin-like protein (UBL) probes and their binding partners can be stabilised using ultraviolet (UV) light-induced crosslinks. The stabilised protein complexes can be purified using affinity purification and identified by mass spectrometry. The resultant covalent bonds can withstand even the harshest washing conditions, allowing for the removal of indirect binders whilst retaining and capturing weak and transient interactors that are commonly lost during wash steps. This technique is widely applicable and highly effective for identifying site-selective non-covalent interactors. Members of our team have previously demonstrated the benefit of this method using the small ubiquitin-like modifier (SUMO). Here, we provide further proof-of-principle validation of the method and highlight its generality by applying an optimised workflow to a lesser studied UBL, interferon stimulated gene 15 (ISG15). We show that this method is able to capture known ISG15 interactors from a complex protein mixture in a site-selective manner, only capturing proteins that specifically interact with the region of ISG15 where the unnatural amino acid was incorporated. This exquisite degree of sensitivity and specificity greatly improves upon previous screens aimed at identifying downstream non-covalent binders, or readers, of ISG15. Taken together, the approach opens the possibility of characterising previously undetected protein-protein interactions, with the potential of elucidating molecular mechanisms behind the most complex and poorly understood processes in the cell.https://doi.org/10.1371/journal.pone.0316321 |
| spellingShingle | Zac Sandy Zijuan Wang Deepak Behera Benjamin M Foster Finlay A Martin Kira Brüninghoff Kathleen M Cain Wolfgang Dörner Maria Jose Cabello-Lobato Josep V Forment Matthew J Cliff Igor Larrosa Perdita Barran Duncan L Smith Henning D Mootz Christine K Schmidt Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions. PLoS ONE |
| title | Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions. |
| title_full | Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions. |
| title_fullStr | Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions. |
| title_full_unstemmed | Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions. |
| title_short | Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions. |
| title_sort | site selective photo crosslinking for the characterisation of transient ubiquitin like protein protein interactions |
| url | https://doi.org/10.1371/journal.pone.0316321 |
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