Optimization of the Nanocrystal-Fluorescent Protein System and Applications

• Grant number: 07/07310-4
• Support type: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): July 01, 2009
• Effective date (End): June 30, 2012
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal researcher: Mario José Politi
• Grantee: Vitor Renaux Hering
• Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Recently our group has observed and characterized the resonance energy transfer (FRET) among fluorescent proteins of the GFP family and CdSe/ZnS nanocrystals. Such donor-acceptor pairs put together the exceptional photochemical properties of the nanocrystals along with the capacity of genetically integrating the fluorescent proteins to cell systems. The occurrence of FRET reveals the approximation between compounds with nanometric precision, being a useful phenomenon for the exploration of intracellular events as well as for the development of sensors. In these work we will sought the optimization of nanocrystals-fluorescent protein pairs aiming the maximization of FRET. Fluorescent proteins other then GFP5 and HcRed1, which have already been characterized, will be tested and ZnSe nanocrystals will be synthesized in the lab aiming to replace the expansive nanocrystals purchased from third parties. Preliminary results demonstrate that green emitting CdSe/ZnS nanocrystals and the dTomato protein provides an exceptional FRET pair. Preliminary results also demonstrate the success in the synthesis of ZnSe with intense violet emission. Once achieving optimized nanocrystals-fluorescent protein pairs we will map the main elements of the interaction between these compounds. Beyond the already characterized electrostatic nature of the interaction, the amino acids involved in the interaction will be determined as well as the effect of the nanocrystals´ functionalization over the dissociation constant. Functionalizations that by one side enable FRET and by the other side facilitate the penetration of the nanocrystals inside E. coli cells will be selected, aiming to obtain FRET in vivo. In parallel in vitro tests will be taken to detect biologically relevant molecules that may be able to interfere in the energy transfer among nanocrystals and fluorescent proteins in the way to establish the molecular basis for sensors.