Conformation of Xanthene Dyes in the Sulfhydryl 1 Binding Site of Myosin. 2

Katalin Ajtai, Thomas P. Burghardt

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33 Scopus citations

Abstract

The fluorescent dyes 5'-(iodoacetamido)tetramethylrhodamine (5'IATR) and 5'-(iodoacetamido)-fluorescein (5'IAF) bind covalently to the reactive sulfhydryl (SHI) of myosin subfragment 1 (SI), the 5'IATR as a dimer and the 5'IAF as a monomer. The conformation of the dimer and the dye-protein complex was investigated by comparison of several spectroscopic signals of the molecules before and after their association into a complex and interpretation of any changes using a coupled dipole oscillator model adapted for this problem [Burghardt & Ajtai (1995) Biophys. Chem. (submitted for publication)]. Absorption and fluorescence spectroscopies were performed on 5'IAF, 5'IATR, and rhodamine 6G (R6G) and rhodamine B (RB) as models of dimer conformation. Absorption, fluorescence, and circular dichroism (CD) spectroscopies were performed on 5'IATR-modified SI (5'R-S1) and 5'IAF-modified SI (5'F-S1). Combined spectroscopic and 2-D NMR data from rhodamines in solution determined the conformations of the dimers. Xanthene rings from dimers of identical dyes (homodimers) stacked in two structures having very different spectroscopic signatures. Xanthene rings from the heterodimer of R6G and RB stacked in one conformation. The two homodimer conformations of 5'IATR are equally likely to form in solution. The other rhodamine homodimers have one dominant, but not exclusive, structure. Both conformations of the 5'IATR dimer were coupled to a tryptophan as a model of the dye-protein interaction at SHI. The calculated CD from one dimer conformer (dimer A) coupled to tryptophan is negative for the lowest energy CD absorption band. The other dimer (dimer B) gives positive CD on the two lowest energy CD absorption bands. Both dimer structures of 5'IATR contributed to the early time-dependent CD signal from 5'IATR binding to SHI, but at equilibrium the CD signal indicated only dimer B, suggesting that the SHI binding pocket converts dimer A into dimer B. The time-dependent CD signal from 5'IAF changes amplitude but not shape during the reaction with SHI. The model calculation accounting for the spectroscopic signals of 5'R-S1 and 5'F-S1 indicates several likely conformations of the 5'IATR dimertryptophan and 5'IAF-tryptophan complexes embedded in SI. These structures fit to the a-carbon structure of the SHI binding pocket when the 5'IATR dimer and 5'IAF interact closely with Trp510 [Rayment et al. (1993) Science 261, 50-58], The additional constraints imposed by the known orientation of the rhodamine dimer relative to the principal hydrodynamic frame of SI reject all but one of the 5'IATR dimer-tryptophan and 5'IAF-tryptophan structures coordinated with Trp510.

Original languageEnglish (US)
Pages (from-to)15943-15952
Number of pages10
JournalBiochemistry
Volume34
Issue number49
DOIs
StatePublished - Dec 1995

ASJC Scopus subject areas

  • Biochemistry

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