Hasegawa Tetsuya, Hagihara Masaki, Fukuda Masatora, Nakano Shun, Fujieda Nobutaka, Morii Takashi
Institute of Advanced Energy, Institute of Sustainability Science, and Pioneering Research Unit for Next Generation, Kyoto University, Uji, Kyoto 611-0011, Japan.
J Am Chem Soc. 2008 Jul 9;130(27):8804-12. doi: 10.1021/ja801734f.
Tools for selective recognition and sensing of specific phosphorylated tyrosine residues on the protein surface are essential for understanding signal transduction cascades in the cell. A stable complex of RNA and peptide, a ribonucleopeptide (RNP), provides effective approaches to tailor RNP receptors and fluorescent RNP sensors for small molecules. In vitro selection of an RNA-derived pool of RNP afforded RNP receptors specific for a phosphotyrosine residue within a defined amino-acid sequence Gly-Tyr-Ser-Arg. The RNP receptor for the specific phosphotyrosine residue was successfully converted to a fluorescent RNP sensor for sequence-specific recognition of a phosphorylated tyrosine by screening a pool of fluorescent phosphotyrosine-binding RNPs generated by a combination of the RNA subunits of phosphotyrosine-binding RNPs and various fluorophore-modified peptide subunits. The phosphotyrosine-binding RNP receptor and fluorescent RNP sensor constructed from the RNP receptor not only discriminated phosphotyrosine against tyrosine, phosphoserine, or phosphothreonine, but also showed specific recognition of amino acid residues surrounding the phosphotyrosine residue. A fluorescent RNP sensor for one of the tyrosine phosphorylation sites of p100 coactivator showed a binding affinity to the target site ~95-fold higher than the other tyrosine phosphorylation site. The fluorescent RNP sensor has an ability to function as a specific fluorescent sensor for the phosphorylated tyrosine residue within a defined amino-acid sequence in HeLa cell extracts.
用于选择性识别和检测蛋白质表面特定磷酸化酪氨酸残基的工具对于理解细胞中的信号转导级联反应至关重要。RNA与肽的稳定复合物，即核糖核肽（RNP），为定制用于小分子的RNP受体和荧光RNP传感器提供了有效的方法。对RNA衍生的RNP文库进行体外筛选，得到了对特定氨基酸序列Gly-Tyr-Ser-Arg中的磷酸酪氨酸残基具有特异性的RNP受体。通过筛选由磷酸酪氨酸结合RNP的RNA亚基和各种荧光团修饰的肽亚基组合产生的荧光磷酸酪氨酸结合RNP文库，成功地将特定磷酸酪氨酸残基的RNP受体转化为用于序列特异性识别磷酸化酪氨酸的荧光RNP传感器。由RNP受体构建的磷酸酪氨酸结合RNP受体和荧光RNP传感器不仅能区分磷酸酪氨酸与酪氨酸、磷酸丝氨酸或磷酸苏氨酸，还能特异性识别磷酸酪氨酸残基周围的氨基酸残基。针对p100共激活因子的一个酪氨酸磷酸化位点的荧光RNP传感器对靶位点的结合亲和力比另一个酪氨酸磷酸化位点高约95倍。该荧光RNP传感器能够作为HeLa细胞提取物中特定氨基酸序列内磷酸化酪氨酸残基的特异性荧光传感器发挥作用。
