The Interaction Between ILOV Protein And Ag Nanoparticle

Electron transfer and energy transfer play a significant role in numerous physical,chemical,biological processes.Compared to bulk materials,nanoparticles have been studied due to their unique physical,chemical properties and make them attractive for a wide range of applications.The interactions between metal nanostructure and proteins have attracted wide attentions in the potential applications and fundamental researches,including biosensor,gene therapy,biological imaging,molecular recognition,catalytic reaction.In this work,silver nanoparticles and iLOV protein system were chosen as model system to investigate their interactions and involved mechanisms by using steady-state fluorescence and time-resolved fluorescence,which are of great significance in understanding the light response behavior of LOV protein,mechanisms of energy transfer and electron transfer between Ag nanoparticles and iLOV proteins.This thesis is composed of four parts as follows:1.The preparation of Ag nanoparticles and iLOV proteins.Silver nanoparticles of different diameters were prepared through sol-gel methods by controlling the reaction temperature.The obtained silver nanoparticles with sizes of 67 nm,29 nm and 15 nm in diameter were characterized with absorption spectroscopy and dynamic light scattering.2.Protein purification.The iLOV protein and iLOV-W83 A protein were purified through a series of operations.The obtained target proteins of 13 kD were characterized by sequence analysis,polyacrylamide gel electrophoresis,absorption fluorescence and fluorescence lifetime in the present study.3.The observations of interaction between Ag nanoparticles and iLOV protein.Our experiments indicated that iLOV protein is a small protein and possesses outstanding fluorescence characteristics and good stability.With the addition of silver nanoparticles of 29 nm in diameter into the iLOV proteins,we observed a drastic quenching of fluorescence intensity of iLOV,and the quenching efficiency depends on the amount of silver nanoparticles in a given volume.Specifically,the fluorescence lifetime of iLOV demonstrated a gradual decrease of the fluorescence lifetime of iLOV with the increasing amount of Ag nanoparticles confirmed the interaction between iLOV protein and silver nanoparticles.In order to gain insight into the interaction between iLOV protein and Ag nanoparticles.The interactions between iLOV protein and silver nanoparticles of different particle sizes 67 nm,29 nm,15 nm in diameter were studied respectively.The fluorescence quenching efficiency and the decrease of fluorescence lifetime of iLOV proteins demonstrated a dependence on the Ag nanoparticle sizes.4.Mechanism of the interaction between Ag nanoparticles and iLOV protein.The observed fluorescence quenching of iLOV protein with the presence of Ag nanoparticles could arise from fluorescence resonance energy transfer(FRET),electron transfer or surface energy transfer(SET).Based on our experimental data and dynamical analysis,we excluded that the effective quenching was caused by fluorescence resonance energy transfer from iLOV to Ag nanoparticle.Thus,we concluded that electron transfer from iLOV protein to Ag nanoparticles would be responsible for the fluorescence quenching of iLOV protein with the presence of Ag nanoparticles.The electron transfer rates between different sized silver nanoparticles and iLOV proteins were observed and determined by kinetic analysis.5.Electron transfer pathway.According to the amino acid sequence of iLOV protein,there is a site residue tryptophan 83.Tryptophan usually plays important role in the electron / charge transfer process of biology systems.We successfully purified the iLOV-W83 A mutant protein and utilized it in the key control experiment for investigating the interaction between Ag nanoparticles and iLOV protein.The results clearly demonstrated that the fluorescence lifetime of iLOV-W83 A protein kept the same value both with and without the presence of silver nanoparticles,and showed independence on the size of silver nanoparticles.Therefore,we concluded that the electron transfer pathway from iLOV protein to Ag nanoparticle is mediated by the site residue tryptophan 83.In a word,the interactions between silver nanoparticles of different sizes and iLOV protein have been systematically investigated with steady-state spectroscopy and time-resolved fluorescent spectroscopy combined with time correlated single photon counting technique.The fluorescence quenching of iLOV protein in the interaction with Ag nanoparticles mainly originates from the electron transfer process,and the quenching rate is dependent on the silver particle sizes.From a series of control experiments and dynamical analysis,it can be concluded that the electron transfer pathway from iLOV protein to Ag nanoparticles is mediated by the tryptophan residue.This conclusion also indirectly confirms the photo-responsive mechanism of LOV protein by the photo-induced electron transfer via site residue tryptophan 83.The obtained results in this study are of significance in explaining the function of LOV protein and interaction mechanisms in biological molecular sensors,nano-photonics and molecular imaging fields..