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, dansyl 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 shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.
Kim
E
E
Department of Chemistry and Biochemistry, UCLA 90095, USA.
Motoki
M
M
Seguro
K
K
Muhlrad
A
A
Reisler
E
E
D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D001704:Biopolymers / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D003619:Dansyl Compounds; D004735:Energy Transfer; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D005973:Glutamine / Q000737:chemistry; D066298:In Vitro Techniques; D008274:Magnesium / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry; D011487:Protein Conformation; D011817:Rabbits; D011503:Transglutaminases; D014364:Tryptophan / Q000737:chemistry
D000199:Actins; D001704:Biopolymers; D003619:Dansyl Compounds; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D005973:Glutamine; C032377:dansylethylenediamine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D011503:Transglutaminases; D008274:Magnesium; D002118:Calcium