Femtosecond UV-laser pulses to unveil protein-protein interactions in living cells.
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.
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy.
Francesco
F
Itri
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy.|Istituto Nazionale di Biostrutture e Biosistemi (INBB), Rome, Italy.
Daria M
DM
Monti
Department of Physics, University of Naples Federico II, 80126, Naples, Italy.
Bartolomeo
B
Della Ventura
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy.
Roberto
R
Vinciguerra
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy.
Marco
M
Chino
Department of Physics, University of Naples Federico II, 80126, Naples, Italy.
Felice
F
Gesuele
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy.
Angelina
A
Lombardi
Department of Physics, University of Naples Federico II, 80126, Naples, Italy.|Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), UdR, Naples, Italy.
Raffaele
R
Velotta
Department of Physics, University of Naples Federico II, 80126, Naples, Italy. caltucci@unina.it.|Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), UdR, Naples, Italy. caltucci@unina.it.
Carlo
C
Altucci
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy. birolo@unina.it.
Leila
L
Birolo
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy.|Istituto Nazionale di Biostrutture e Biosistemi (INBB), Rome, Italy.
Renata
R
Piccoli
Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy. anarciel@unina.it.|Istituto Nazionale di Biostrutture e Biosistemi (INBB), Rome, Italy. anarciel@unina.it.
Angela
A
Arciello
D000818:Animals; D003198:Computer Simulation; D019281:Dimerization; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D008958:Models, Molecular; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014466:Ultraviolet Rays
D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
Living cells irradiation; Protein–protein interactions; UV cross-linking; Ultra-short UV-laser pulses