The conformation of xanthene dyes in the myosin sulfhydryl one binding site. Part I. Methods and model systems

Thomas P. Burghardt, Katalin Ajtai

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Derivatives of the fluorescent probes fluorescein and rhodamine specifically and covalently modify the highly reactive thiol (SH1) of myosin subfragment 1 (S1). Both probes develop circular dichroism (CD) upon modification of SH1 at the visible absorption band of the chromophore. A model system of chiral complexing agents (aromatic chiral amines) interacting with fluorescein in solvent develops a CD signal that mimics that produced by S1. The model system suggests that a specific interaction of the probe with an aromatic chiral residue in the SH1 binding pocket induces the CD signal. Several other spectroscopic signals, including absorption and fluorescence intensity and anisotropy, characterize the fluorescein or rhodamine binding to SH1. A coupled dipole method is adapted to interpret these spectroscopic signals in terms of the probe-S1 complex conformation. The computation of the orientation of the principal hydrodynamic frame (PHF) of S1 from its crystallographic α-carbon backbone structure permits the known orientation of the probe in the PHF of S1 to further constrain the conformation of the probe-S1 complex. The coupled dipole interpretation of spectroscopic data combined with constraints relating the probe dipole orientation to the PHF of S1 determines the conformation of the probe-S1 complex. The methods developed here are applied to the spectroscopic signals from fluorescein or rhodamine in the SH1 binding site of S1 to obtain an atomic resolution model of the probe-S1 conformation [Ajtai and Burghardt, Biochemistry, 34 (1995) 15943-15952].

Original languageEnglish (US)
Pages (from-to)119-133
Number of pages15
JournalBiophysical Chemistry
Volume60
Issue number3
DOIs
StatePublished - Jun 11 1996

Keywords

  • Chiral complexing agent
  • Circular dichroism
  • Coupled dipoles
  • Fluorescein
  • Myosin
  • Rhodamine dimer
  • Tryptophan

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Organic Chemistry

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