Fluorescence Resonance Energy Transfer (FRET) Induced by Conjugation of Metalloproteins to Nanoparticles

Pier P. Pompa1, R. Chiuri1, L. Manna1, T. Pellegrino1, Wolfgang J. Parak2, Roberto Cingolani1, and Ross Rinaldi1

1National Nanotechnology Laboratory of Istituto Nazionale di Fisica della Materia, Dipartimento di Ingegneria dell'Innovazione, Università di Lecce, Via per Arnesano, 73100 Lecce, Italy,
22Centre for Nanoscience, Ludwig Maximilians University, Amalienstraße 54, D-80799 Munich, Germany

e-mail: piero.pompa@unile.it

URL: http://www.nnl.it


In this study, we show the possibility of realizing a hybrid system composed of a semiconductor nanoparticle (NP) and a metalloprotein, in which the photophysical properties of the two species can be exploited to elicit Fluorescence Resonance Energy Transfer (FRET) mechanisms from the biomolecule to the NP. A specific conjugation process between CdSe/ZnS core/shell water soluble NPs, functionalized with surface exposed thiol groups (-SH), and the apo-form of the metalloprotein azurin (Az) has been achieved, via the sulphur atoms of Cys 3 and/or Cys 26 (which are the only solvent accessible cysteine residues in azurin), resulting in a fixed distance in the donor-acceptor pairs. The increase in the NP fluorescence intensity was found to be dependent on the Az to NP molar ratio, revealing that a single CdSe nanoparticle can bind, on the average, 10-15 azurin molecules. PLE experiments performed on the hybrid system confirmed the FRET process (Trp48->NP) as the mechanism responsible for the observed increase in the fluorescence. In addition, intrinsic fluorescence measurements performed on azurin indicate that the protein retains a native-like fold pattern even upon conjugation.