Novel steroid-sensing model and characterization of protein interactions based on fluorescence anisotropy decay

Ko Shing Chang, Liyang Luo, Chih Wei Chang, Yen Chieh Huang, Chih Yu Cheng, Chen Shiung Hung, Eric Wei Guang Diau, Yaw Kuen Li

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Intramolecular binding of a ligand with an alkyl link, (-CH 2)3, covalently bound to a residue near the active site of the protein forms a novel steroid-sensing model. A genetically engineered Δ5-3-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).

Original languageEnglish
Pages (from-to)4327-4334
Number of pages8
JournalJournal of Physical Chemistry B
Volume114
Issue number12
DOIs
Publication statusPublished - 2010 Apr 1

Fingerprint

steroids
Anisotropy
Fluorescence
Steroids
Ligands
steroid delta-isomerase
proteins
Proteins
fluorescence
ligands
anisotropy
Fluorophores
decay
interactions
disulfides
Silicon
Field effect transistors
Disulfides
Peptides
mass spectra

All Science Journal Classification (ASJC) codes

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

Cite this

Chang, Ko Shing ; Luo, Liyang ; Chang, Chih Wei ; Huang, Yen Chieh ; Cheng, Chih Yu ; Hung, Chen Shiung ; Diau, Eric Wei Guang ; Li, Yaw Kuen. / Novel steroid-sensing model and characterization of protein interactions based on fluorescence anisotropy decay. In: Journal of Physical Chemistry B. 2010 ; Vol. 114, No. 12. pp. 4327-4334.
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Novel steroid-sensing model and characterization of protein interactions based on fluorescence anisotropy decay. / Chang, Ko Shing; Luo, Liyang; Chang, Chih Wei; Huang, Yen Chieh; Cheng, Chih Yu; Hung, Chen Shiung; Diau, Eric Wei Guang; Li, Yaw Kuen.

In: Journal of Physical Chemistry B, Vol. 114, No. 12, 01.04.2010, p. 4327-4334.

Research output: Contribution to journalArticle

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