FisMat2017 - Submission - View

Abstract's title: Pyrene-labelled ferritin as an excimer fluorescence based probe to study protein conformational changes
Submitting author: Irene Benni
Affiliation: Università di Roma, La Sapienza
Affiliation Address: Piazzale Aldo Moro, 5, 00185 Roma
Country: Italy
Oral presentation/Poster (Author's request): Poster
Other authors and affiliations: Authors: Irene Benni1, Matilde C. Trabuco1, Simone De Panfilis2, Valeria de Turris2, Francesco Malatesta1, Alberto Boffi1,2, Paola Baiocco2 Author’s Affiliations: 1 Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza University of Rome, Rome, Italy 2 Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy

Pyrene is a fluorescent spatially sensitive probe, with the unique ability to form excimers (excited-state dimers) by stacking interaction of two close molecules. Excimers have a characteristic emission spectrum, very different from the pyrene-monomer spectrum, ideal for conformational changes studies on proteins. A new engineered ferritin from archaea bacteria (HumAfFt) has been recently demonstrated to maintain the unique oligomerization properties catalysed by divalent cations of Archaeoglobus fulgidus ferritin at neutral pH. The association/dissociation equilibrium of HumAfFt from dimer to a cage-like 24-mer has been investigated exploiting pyrene fluorescence properties. A cysteine residue per ferritin monomer has been specifically introduced by mutagenesis inside the protein cage and the thiol moieties have been selectively labelled with N-(1-Pyrenyl)maleimide. Dramatic changes in the fluorescence occurred in the pyrene-labelled protein as the ferritin associate in its 24-mer state. Interestingly, the amount of excimer decreased with the assembly of the 24-mer. The kinetic of the process has been studied by stopped-flow measurements, highlighting a very fast association rate and a slightly slower dissociation rate, within the range of micro seconds. Pyrene excimer emission has also been measured by two-photon fluorescence microscopy by excitation in the near IR region, a low-energy excitation wavelength which can significantly reduce photodamage and photobleach in vitro experiments. These features, together with pyrene high stability and long fluorescence lifetime highlighted the potential of these new pyrene labelled probes.