Novel Steroid-Sensing Model and Characterization of Protein Interactions Based on Fluorescence Anisotropy Decay

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• 作者：Ko-Shing Chang ; Liyang Luo ; Chih-Wei Chang ; Yen-Chieh Huang ; Chih-Yu Cheng ; Chen-Shiung Hung ; Eric Wei-Guang Diau ; Yaw-Kuen Li
• 刊名：Journal of Physical Chemistry B
• 出版年：2010
• 出版时间：April 1, 2010
• 年：2010
• 卷：114
• 期：12
• 页码：4327-4334
• 全文大小：304K
• 年卷期：v.114,no.12(April 1, 2010)
• ISSN：1520-5207

文摘

Intramolecular binding of a ligand with an alkyl link, (−CH₂)₃, covalently bound to a residue near the active site of the protein forms a novel steroid-sensing model. A genetically engineered ⁵-3-ketosteroid isomerase (KSI) was designed to conjugate uniquely with this ligand at its Cys-86 through the formation of a disulfide bond. The steady-state protein−ligand binding, mediated by hydrophobic interactions, was confirmed with fluorescence spectra, and the fluorophore-labeled peptide sequence was identified with tandem mass spectra. A comparison of steady-state fluorescence spectra of various fluorophore-labeled KSI mutants reveals that the emission characteristics vary with environmental factors. An evaluation of the decay of the fluorescence anisotropy of the fluorophore indicates the existence of an intramolecular protein−ligand binding interaction. The measurement of time-resolved fluorescence anisotropy of various protein−ligand complexes yielded values of anisotropy decay representing the degrees of freedom of the fluorophore related to its location, inside or outside the steroid-binding domain. When 19-norandrostenedione (19-NA) was added to this protein−ligand system, competitive binding between the ligand and the steroid was observed; this finding confirms the feasibility of the design of steroid detection with engineered KSI. On integration of this protein−ligand system with a silicon-based nanodevice (a p-type field-effect transistor with an ultrathin body), a noncharged steroid, 19-NA, became detectable at a micromolar level ( Biosens. Bioelectron. 2008, 23, 1883).