共 41 条
Site-selective photo-crosslinking for the characterisation of transient ubiquitin-like protein-protein interactions
被引:0
|作者:
Sandy, Zac
[1
]
Wang, Zijuan
[1
]
Behera, Deepak
[1
,2
]
Foster, Benjamin M.
[1
]
Martin, Finlay A.
[1
]
Bruninghoff, Kira
[3
]
Cain, Kathleen M.
[4
,5
]
Dorner, Wolfgang
[3
]
Cabello-Lobato, Maria Jose
[1
]
Forment, Josep V.
[6
]
Cliff, Matthew J.
[2
,4
]
Larrosa, Igor
[2
]
Barran, Perdita
[2
,4
,7
]
Smith, Duncan L.
[8
]
Mootz, Henning D.
[3
]
Schmidt, Christine K.
[1
]
机构:
[1] Univ Manchester, Fac Biol Med & Hlth, Manchester Canc Res Ctr, Sch Med Sci,Div Canc Sci, Manchester, England
[2] UNIV MANCHESTER, Sch Chem, MANCHESTER, England
[3] Univ Munster, Inst Biochem, Munster, Germany
[4] Univ Manchester, Manchester Inst Biotechnol MIB, Manchester, England
[5] Univ Manchester, Fac Sci & Engn, Mass Spectrometry & Separat Facil, Manchester, England
[6] AstraZeneca, Early Oncol R&D, Cambridge Biomed Campus, Cambridge, England
[7] Univ Manchester, Michael Barber Ctr Collaborat Mass Spectrometry, Manchester, England
[8] Univ Manchester, Manchester Inst, Canc Res UK, Manchester, England
来源:
基金:
英国生物技术与生命科学研究理事会;
英国工程与自然科学研究理事会;
关键词:
STRUCTURAL BASIS;
AMINO-ACID;
GENETIC-CODE;
ISG15;
RECOGNITION;
CYSTEINE;
D O I:
10.1371/journal.pone.0316321
中图分类号:
O [数理科学和化学];
P [天文学、地球科学];
Q [生物科学];
N [自然科学总论];
学科分类号:
07 ;
0710 ;
09 ;
摘要:
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.
引用
收藏
页数:12
相关论文