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, 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 mm 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 mm) where the calponin : F-actin stoichiometry is 1 : 1 as well as at 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-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 Ca2+-calmodulin or Ca2+-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 A 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.
Bartegi
A
A
Centre de Recherches de Biochimie Macromolèculaire du CNRS, Montpellier, France.
Roustan
C
C
Kassab
R
R
Fattoum
A
A
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D000199:Actins / Q000737:chemistry*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D004735:Energy Transfer; D008840:Microfilament Proteins; D009130:Muscle, Smooth / Q000737:chemistry*; D011817:Rabbits; D012492:Salts; D013050:Spectrometry, Fluorescence; D000096985:Calponins
D015081:2-Naphthylamine; D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D012492:Salts; D000096985:Calponins; C038518:acrylodan