Probing Molecular Mechanisms of Signaling Proteins with Single-Molecule Techniques
报告题目：Probing Molecular Mechanisms of Signaling Proteins with Single-Molecule Techniques
Single-molecule fluorescent spectroscopy can provide unique perspectives which complement conventional bulk biochemical approaches. We use single-molecule techniques to illuminate the transmission of signals inside the cell. Brassinosteroids (BRs) are the plant hormones that involved in numerous plant development processes. 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.
Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. We study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred on a timescale around 200 µs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Combining kinetic rates of the dynamics process extracted from single molecule experiments, we can explain this functional conformational change from a quantitative point of view. 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.
报告人简介：谭砚文，女，1976年11月生于台湾台北。1994年九月进入国立台湾大学物理系。1998年六月自台湾大学物理系毕业后，于台湾中央研究院原子分子研究所范文祥实验室从事发光共轭高分子研究并于2000年六月取得台湾大学物理硕士学位。同年九月至美国哥伦比亚大学诺奖得主Horst Störmer实验室攻读博士学位。2006年底博士毕业，加入杨皓实验室，先后在加州伯克利大学（2007年二月至2009年六月）与普林斯顿化学系（2009年七月至2010年八月），以单分子荧光光谱学方法，研究蛋白质分子构象动力学相关的生物物理问题。2010年九月加入复旦大学物理系。迄今发表SCI论文十数篇，其中包含Nature一篇以及Phys. Rev. Lett. 5篇，JACS，Nature Communications各一篇。论文他引次数迄今超过6300次。