Detection of Synaptic Proteins with Fluorescent Molecular Rotor-labeled Peptides

荧光分子转盘标记多肽检测突触蛋白 摘要 我们的目标是建立用于即刻和特定用途的合成肽的常规开发方法 在固定的和活的神经元中检测内源性未修饰蛋白。我们会放映《一珠一号》 共同研究员(Co-I)Kit Lam发明的化合物(OBOC)组合肽库(自然354，82- 84)具有荧光分子旋转体(FMR)的多肽。FMR多肽只有在特定的情况下才会发出荧光 结合到他们的目标蛋白，但在溶液中不是游离的。最初的焦点将放在关键的突触后蛋白上 谷氨酸能突触(AMPAR(AMPA型谷氨酸受体)，NMDARs(NMDA型谷氨酸 受体)、PSD-95(在突触后部位锚定AMPAR和NMDAR))。信噪比(&gt；2300倍) 而且光稳定性(~10倍)优于目前的XFP标签。多肽将被制成膜 通过TAT序列或通过肉豆蔻酰化。这种变革性的方法将允许检测体内的蛋白质 在生命系统中的几分钟绕过技术复杂、耗时和昂贵的基因 蛋白质标签。它还将极大地加速(50倍)蛋白质在固定细胞中的分布，这是因为 超分辨率显微镜，因为它不需要使用一次和二次抗体，甚至 洗涤步骤。FMR多肽可以很容易地重新合成，消除抗体固有的可变性 探测器。多肽将被反复优化，以获得高亲和力和特异性。我的长期和 最主要的兴趣是确定支配突触后功能的分子机制(例如，科学 293，98；科学293,2205；科学信号10，eaaf9659；自然411,801；神经元74,1023；神经元78,483； 神经元81,249；神经元88,528；神经元97,1094；神经元98,783；EMBO J.26,4879；EMBO J.29,482；EMBO J.31,1203；EMBO J.33,1341；EMBO J.35,1330；EMBO J.36,1330；EMBO J.37,122)。PSD-95确定 谷氨酸受体的突触后定位。特异性荧光FMR多肽的研究进展 结合这些蛋白质将允许对它们的定位和突触的动态变化进行实时成像 随着时间的推移，这些蛋白质在静息和刺激的条件下在培养的神经元中经历，并最终在 活体中的大脑。最终，我设想开发针对&gt；100突触前和突触后蛋白的FMR多肽。其他 将把我们的技术应用于CNS内外的所有生物医学研究领域。 第1页

Detection of Synaptic Proteins with Fluorescent Molecular Rotor-labeled Peptides Abstract Our goal is to establish routine methodology for development of synthetic peptides for instant and specific detection of endogenous unmodified proteins in fixed and living neurons. We will screen One Bead One Compound (OBOC) combinatorial peptide libraries invented by co-investigator (Co-I) Kit Lam (Nature 354, 82- 84) for peptides with fluorescent molecular rotors (FMRs). FMR peptides will fluoresce only when specifically bound to their target protein but not when free in solution. Initial focus will be on the key postsynaptic proteins of glutamatergic synapses (AMPARs (AMPA-type glutamate receptors), NMDARs (NMDA-type glutamate receptors), PSD-95 (anchors AMPARs and NMDARs at postsynaptic sites)). Signal to noise ratio (>2300fold) and photostability (by ~ 10fold) is superior to current xFP tags. Peptides will be made membrane permeant with the tat sequence or by myristoylation. This transformative approach will allow detection of proteins within minutes in living systems circumventing technically complicated, time consuming, and expensive genetic protein tagging. It will also dramatically accelerate (by 50fold) protein distribution in fixed cells by high and super-resolution microscop as it will not require the use of primary and secondary antibodies, not even washing steps. FMR peptides can be easily re-synthesized eliminating the variability inherent to antibody probes. Peptides will be reiteratively optimized to achive high affinity and specificity. My long-standing and overarching interest is to determine the molecular mechanisms that govern postsynaptic function (e.g., Science 293,98; Science 293,2205; Science Signaling 10, eaaf9659; Nature 411,801; Neuron 74,1023; Neuron 78,483; Neuron 81,249; Neuron 88,528; Neuron 97, 1094; Neuron 98, 783; EMBO J. 26,4879; EMBO J. 29,482; EMBO J. 31,1203; EMBO J. 33,1341; EMBO J. 35,1330; EMBO J. 36,1330; EMBO J. 37, 122). PSD-95 determines postsynaptic localization of glutamate receptors. Development of FMR-peptides that fluoresce upon specific binding to these proteins will allow live imaging of their localization and of the dynamic changes synapses with those proteins undergo over time at resting and stimulated conditions in cultured neurons and ultimately in the brain in vivo. Ultimately, I envision to develop FMR-peptides for >100 pre- and postsynaptic proteins. Others will apply our technology inside and outside the CNS in all fields of biomedical research. Page 1