期刊
CELLULAR AND MOLECULAR LIFE SCIENCES
卷 73, 期 3, 页码 637-648出版社
SPRINGER BASEL AG
DOI: 10.1007/s00018-015-2015-y
关键词
Protein-protein interactions; Ultra-short UV-laser pulses; UV cross-linking; Living cells irradiation
资金
- University of Naples Federico II
- Compagnia di San Paolo
- Istituto Banco di Napoli-Fondazione
- STRAIN PROJECT (POR Campania FSE) [CUP B25B0900000000]
A hallmark to decipher bioprocesses is to characterize protein-protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.
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