Fluorescence studies of the carboxyl‐terminal domain of smooth muscle calponin

The fluorescence parameters of the environment‐sensitive acrylodan, selectively attached to Cys273 in the C‐terminal domain of smooth muscle calponin, were studied in the presence of F‐actin and using varying salt concentrations. The formation of the F‐actin acrylodan labeled calponin complex at 75 m m NaCl resulted in a 21‐nm blue shift of the maximum emission wavelength from 496 nm to 474 nm and a twofold increase of the fluorescent quantum yield at 460 nm. These spectral changes were observed at the low ionic strengths (< 110 m m ) where the calponin : F‐actin stoichiometry is 1 : 1 as well as at the high ionic strengths (> 110 m m ) where the binding stoichiometry is a 1 : 2 ratio of calponin : actin monomers. On the basis of previous three‐dimensional reconstruction and chemical crosslinking of the F‐actin–calponin complex, the actin effect is shown to derive from the low ionic strength interaction 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 but not quantitatively similar to that earlier reported for the complexes of calponin and Ca 2+ ‐calmodulin or Ca 2+ ‐caltropin. As the three calponin ligands 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 Å was measured by fluorescence 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 was allosteric reflecting a global conformational change in the C‐terminal domain of calponin.