Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin - Effects of F-actin and salts

The fluorescence parameters of the environment-sensitive acrylodan, selecti vely attached to Cys273 in the C-terminal domain of smooth muscle calponin, were studied in the presence of F-actin and using varying salt concentrati ons. The formation of the F-actin acrylodan labeled calponin complex at 75 mM NaCl resulted in a 21-nm blue shift of the maximum emission wavelength f rom 496 nm to 474 nm and a twofold increase of the fluorescent quantum yiel d at 460 nm. These spectral changes were observed at the low ionic strength s (< 110 mM) where the calponin:F-actin stoichiometry is 1 : 1 as well as a t the high ionic strengths (> 110 mM) where the binding stoichiometry is a 1:2 ratio of calponin:actin monomers. On the basis of previous three-dimens ional reconstruction and chemical crosslinking of the F-actin-calponin comp lex, the actin effect is shown to derive from the low ionic strength intera ction of calponin with the bottom of subdomain-1 of an upper actin monomer in F-actin and not from its further association with the subdomain-1 of the adjacent lower monomer which occurs at the high ionic strength. Remarkably , the F-actin-dependent fluorescence change of acrylodan is qualitatively b ut not quantitatively similar to that earlier reported for the complexes of calponin and Ca2+-calmodulin or Ca2+-caltropin. As the three calponin liga nds bind to the same segment of the protein, encompassing residues 145-182, the acrylodan can be considered as a sensitive probe of the functioning of this critical region. A distance of 29 Angstrom was measured by fluorescen ce resonance energy transfer between Cys273 of calponin and Cys374 of actin in the 1 : 1 F-actin-calponin complex suggesting that the F-actin effect w as allosteric reflecting a global conformational change in the C-terminal d omain of calponin.