The Study Of Protein Interaction Using Fluorescence Spectroscopy

Many involved biochemical reaction processes in signal transduction of cells, geneexpression, enzymatic metabolism, respiration, etc. in living cell are based on the protein-protein interactions. The further study of protein-protein interactions is of scientificsignificance and potential applications in understanding and revealing the functionalregulation mechanism, disease control and treatment in cells.Throughout evolutionary history, biological systems form self-regulating pathwaysin response to external stress caused by environmental changes. In the regulation of signaltransduction, the modification of kinase on substrate or the transformation of redox state forprotein is a dynamical process, which is difficult to be observed by using the traditionalmethods. In this thesis, we employed spectroscopic techniques to study the interactionbetween proteins as follows:(1) The glutathione peroxidase (GPX3) and thioredoxin (TRX) are selected as the researchsystem. By using protein-directed mutagenesis, intrinsic fluorescent probe (tryptophan)and artificial probes(TF3,TF5), fluorescence spectroscopy and SDS-polyacrylamide gel electrophoresis (PAGE), we studied the redox states conversion between the involvedproteins to und understand the protein interaction and the conversion pattern of electrontransfer in protein-protein interactions. The results indicate that the redox states ofglutathione peroxidase (GPX3) and thioredoxin (TRX) in different environmentsdemonstrating different static and dynamic features. Interestingly, our experimental datareveals that the transformation of electron between the two proteins is from GPX3toTRX, which is in contrast to the viewpoint of TRX as the hydrogen donor.(2) In order to get more insight of protein interaction, we selected glutathione peroxidase(AtGPX3) and thioredoxin (AtTRX9) from Arabidopsis as the model system to study theconnection between the switching of redox states and docking-undocking of interactionproteins. Using fluorescence labeling technique, multi-channel single molecule-totalinternal reflection fluorescence microscopy (TIRFM) system, fluorescence resonanceenergy transfer (FRET), the protein-protein interaction was studied systematically. Byobserving the change of intrinsic fluorescence spectra of tryptophan and the FRET effectbetween labeled AtGPX3and AtTRX9, the direct evidence of protein interaction wasobtained and the relevant scientific mechanism was discussed briefly. The experimentalresults show that the oxidized AtGPX3can be reduced by capturing an electron from reduced AtTRX9, which confirms the hypothesis of thioredoxin (TRX) acting as anelectron donor in this organism metabolism process. The ensemble FRET efficiencyclearly demonstrated concentration dependence of the interaction proteins, indicating thedocking process or complex formation. In the single molecule observation, the dynamicalfeature was monitored in real time on TIRFM, and the results presented a dynamicalprocess with the docking-undocking time of tens seconds. These results from ensembleand single molecule are consistent to support the existence of protein interaction betweenAtGPX3with AtTRX9, and imply that the protein conformational dynamics play animportant role in protein function regulation.The results obtained in this thesis are of significance in revealing the proteininteraction and understanding the signal transduction mechanisms in plant cell. Meanwhile,the techniques used in this work, e.g., single molecule TIRFM, enable us to study othermacromolecule systems, such as protein-DNA interaction, protein folding and unfolding, drugrecognition at single molecule level.