CONFORMATIONAL-CHANGES IN SUBDOMAIN-2 OF G-ACTIN - FLUORESCENCE PROBING BY DANSYL ETHYLENEDIAMINE ATTACHED TO GLN-41

Gln-41 on G-actin was specifically labeled with a fluorescent probe, d ansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2 + with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shift s in the wavelength of maximum emission and increases in DED fluoresce nce. Excitation of labeled actin at 295 nm revealed energy transfer fr om tryptophans to DED. Structure considerations and Cu2+ quenching exp eriments suggested that Trp-79 and/or Trp-86 serves as energy donors t o DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerizatio n of Mg-G-actin with MgCl2 resulted in much smaller changes in DED flu orescence than divalent cation substitution. This suggests that the co nformation of loop 38-52 on actin is primed for the polymerization rea ction by the substitution of Ca2+ with Mg2+ on G-actin.