Detection of Synaptic Proteins with Fluorescent Molecular Rotor-labeled Peptides

使用荧光分子转子标记肽检测突触蛋白

• 批准号: 10063961
• 负责人: JOHANNES W HELL
• 金额: $ 17.74万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063961
• 关键词: Ache; Affinity; Antibodies; Benchmarking; Binding; Biomedical Research; Brain; Cell Adhesion Molecules; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Confocal Microscopy; Consumption; Detection; Development; Dyes; Future; Gene Expression; Genetic; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Image; Imaging Techniques; Imaging technology; Immunohistochemistry; In Vitro; Knock-out; Label; Libraries; Measures; Mediating; Membrane; Methodology; Methods; Microscopy; Molecular; Morphologic artifacts; N-Methylaspartate; Nature; Neurons; Noise; Oranges; Organism; Peptide Library; Peptide Synthesis; Peptides; Procedures; Process; Proteins; Reproducibility; Research Personnel; Resolution; Rest; Risk; Science; Side; Signal Transduction; Site; Specificity; Stains; Structure; Surface; Synapses; System; Technology; Time; Tissues; Work; alpha Actinin; combinatorial; detection method; extracellular; in vivo; interest; microscopic imaging; myristoylation; postsynaptic; presynaptic; processing speed; protein distribution; receptor; side effect; synthetic peptide

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

荧光分子转子标记肽检测突触蛋白 摘要 我们的目标是建立常规的方法学，用于开发合成肽， 检测固定和活神经元中的内源性未修饰蛋白质。我们将筛选一个珠子一个 由共同研究者（Co-I）Kit Lam发明的化合物（OBOC）组合肽文库（Nature 354，82- 1999）。 84)用于具有荧光分子转子（FMR）的肽。FMR肽仅在特异性地 与其靶蛋白结合，但在溶液中不结合。最初的重点将是关键的突触后蛋白质 AMPAR（AMPA型谷氨酸受体）、NMDAR（NMDA型谷氨酸受体 受体）、PSD-95（在突触后位点锚定AMPAR和NMDAR））。信噪比（> 2300倍） 并且光稳定性（约10倍）上级当前的xFP标签。将使肽具有膜渗透性 用达特序列或豆蔻酰化。这种变革性的方法将允许检测蛋白质， 几分钟的生命系统规避技术复杂，耗时，昂贵的遗传 蛋白质标记它还将显著加速（50倍）蛋白质在固定细胞中的分布， 超分辨率显微镜，因为它将不需要使用第一和第二抗体，甚至不 洗涤步骤FMR肽可以很容易地重新合成，消除了抗体固有的变异性， probes.肽将被粘附优化以实现高亲和力和特异性。我的长期和 首要的兴趣是确定控制突触后功能的分子机制（例如，科学 293，98; Science 293，2205; Science Signaling 10，eaaf 9659; Nature 411，801; Neuron 74，1023; Neuron 78，483; 神经元81，249;神经元88，528;神经元97，1094;神经元98，783;欧洲分子生物学组织杂志26，4879;欧洲分子生物学组织杂志29，482;欧洲分子生物学组织杂志 J. 31，1203; EMBO J. 33，1341; EMBO J. 35，1330; EMBO J. 36，1330; EMBO J. 37，122）。PSD-95测定 谷氨酸受体的突触后定位。开发在特异性免疫反应中发荧光的FMR-肽 与这些蛋白质的结合将允许对它们的定位和突触的动态变化进行实时成像， 这些蛋白质在培养的神经元中的静息和刺激条件下随着时间的推移经历，并最终在神经元中经历。 大脑在体内。最终，我设想开发用于>100种突触前和突触后蛋白的FMR肽。别人 将在CNS内外的所有生物医学研究领域应用我们的技术。 第1页