School of Applied and Engineering Physics, 212 Clark Hall, Cornell University, Ithaca, New York 14853, USA.
J Am Chem Soc. 2010 Jun 2;132(21):7244-5. doi: 10.1021/ja100500k.
Quenching of fluorophores by the same proteins that they covalently label is a phenomenon that is neither well-known nor well-characterized. It is often assumed that fluorophores are unperturbed by their target proteins. However, it has been observed that attached fluorophores can be quenched by contact with amino acids within the same protein, and this property has been exploited to report on changing conformational states or intramolecular dynamics of proteins. We show in this communication that fluorescence of Alexa dyes is, in fact, quenched by interactions with Trp, Tyr, Met, and His residues through a combination of static and dynamic quenching mechanisms. In light of this finding, the potential effect of intramolecular quenching should be considered in the interpretation of data that involves quantitative measurements of fluorescence intensity in proteins.
荧光团被它们共价标记的相同蛋白质猝灭是一种既不为人知也未被充分描述的现象。人们通常认为荧光团不受其靶蛋白的干扰。然而,已经观察到,与同一蛋白质中的氨基酸接触会使附着的荧光团猝灭,并且已经利用该性质来报告蛋白质构象状态或分子内动力学的变化。在本通讯中,我们表明实际上,通过静态和动态猝灭机制的组合,Alexa 染料的荧光被色氨酸、酪氨酸、甲硫氨酸和组氨酸残基的相互作用猝灭。鉴于这一发现,在涉及蛋白质荧光强度定量测量的数据解释中,应考虑分子内猝灭的潜在影响。