2014.11.3 Probing Molecular Mechanisms of Signaling Proteins with Single-Molecule Techniques
题目： Probing Molecular Mechanisms of Signaling Proteins with Single-Molecule Techniques
Department of Physics, Fudan University
Single-molecule fluorescent spectroscopy can provide unique perspectives which complement conventional bulk biochemical approaches. We use single-molecule techniques to illuminate the details of the working mechanism of signaling proteins at different stages of a signaling transduction. Light-sensitive protein cryptochrome plays many different rolls in eukaryotic cells. It is a kind of signaling proteins that is known to retrain circadian clock and involves in the magnetic sensing capabilities of animals. Using smFRET (single-molecule Föster-type resonance energy transfer), we reveal how blue light would initiate in a conformational change in an algae cryptochrome under different redox states and this conformational change is believed to be functionally important. While conformational changes are usually speculated as the cause of activation of signaling function in various receptor proteins, smFRET can afford direct evidence to these hypotheses.
To illuminate the transmission of signals inside cells, we studied the signaling pathway of a plant hormone, Brassinosteroids (BRs). BRs are the plant hormones that involved in numerous plant development processes such as leaf expansion, shoot elongation and pollen tube formation. Once the signal transduction is initiated by the membrane receptor kinase, the downstream signaling pathway is realized by three proteins: BIN2 (brassinosteroid insensitive 2), BES1 (BRI1 ems suppressor1) and a kind of 14-3-3s protein. BRs signaling pathway have been extensively studied via genetics, proteomics, genomics and cell biology techniques. However, these bulk methods can’t follow the transduction process in situ or resolve molecular details at a rate matching the true signaling time-scale. Here we use a single molecular assay based on Total-Internally Reflected Fluorescence (TIRF) microscopy to observe the interaction of these three proteins. The result shows that BIN2 can phosphorylate BES1 on the order of seconds, and the dimeric 14-3-3s can only bind with BES1 in its phosphorylated form. In addition, we have, for the first time, found that the interaction between BIN2 and BES1 is oxygen dependent. This result may have implications on BRs signaling pathway’s involvement of stress acclimation in plants.