CONFORMATION OF XANTHENE DYES IN THE SULFHYDRYL-1 BINDING-SITE OF MYOSIN .2.

The fluorescent dyes 5'-(iodoacetamido)tetramethylrhodamine (5'IATR) a nd 5'-(iodoacetamido)fluorescein (5'IAF) bind covalently to the reacti ve sulfhydryl (SH1) of myosin subfragment 1 (S1), the 5'IATR as a dime r and the 5'IAF as a monomer. The conformation of the dimer and the dy e-protein complex was investigated by comparison of several spectrosco pic signals of the molecules before and after their association into a complex and interpretation of any changes using a coupled dipole osci llator model adapted for this problem [Burghardt & Ajtai (1995) Biophy s. Chern. (submitted for publication)]. Absorption and fluorescence sp ectroscopies were performed on 5'IAF, 5'IATR, and rhodamine 6G (R6G) a nd rhodamine B (RB) as models of dimer conformation. Absorption, fluor escence, and circular dichroism (CD) spectroscopies were performed on 5'IATR-modified S1 (5'R-S1) and 5'IAF-modified S1 (5'F-S1). Combined s pectroscopic and 2-D NMR data from rhodamines in solution determined t he conformations of the dimers. Xanthene rings from dimers of identica l dyes (homodimers) stacked in two structures having very different sp ectroscopic 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 homod imers have one dominant, but not exclusive, structure. Both conformati ons of the 5'IATR dimer were coupled to a tryptophan as a model of the dye-protein interaction at SH1. The calculated CD from one dimer conf ormer (dimer A) coupled to tryptophan is negative for the lowest energ y CD absorption band. The other dimer (dimer B) gives positive CD on t he two lowest energy CD absorption bands. Both dimer structures of 5'I ATR contributed to the early time-dependent CD signal from 5'IATR bind ing to SH1, but at equilibrium the CD signal indicated only dimer B, s uggesting that the SH1 binding pocket converts dimer A into dimer B. T he time-dependent CD signal from 5'IAF changes amplitude but not shape during the reaction with SH1. The model calculation accounting for th e spectroscopic signals of 5'R-S1 and 5'F-S1 indicates several likely conformations of the 5'IATR dimer-tryptophan and 5'IAF-tryptophan comp lexes embedded in S1. These structures fit to the a-carbon structure o f the SH1 binding pocket when the 5'IATR dimer and 5'IAF interact clos ely with Trp510 [Rayment et al. (1993) Science 261, 50-58]. The additi onal constraints imposed by the known orientation of the rhodamine dim er relative to the principal hydrodynamic frame of S1 reject all but o ne of the 5'IATR dimer-tryptophan and 5'IAF-tryptophan structures coor dinated with Trp510.