Divalent cation-, nucleotide-, and polymerization-dependent changes in the conformation of subdomain 2 of actin.
Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln41. Examination of changes in the fluorescence emission spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cation-dependent difference in the environment of Gln41. No significant difference between Ca- and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38-52 may be related to time-dependent changes in the conformation of subdomain 2 in DED- or DC-labeled G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln41 accompanying actin polymerization result in part from the release of Pi after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.
Moraczewska
J
J
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, PL-02-093 Warsaw, Poland.
Wawro
B
B
Seguro
K
K
Strzelecka-Golaszewska
H
H
D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives; D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000494:pharmacology; D003619:Dansyl Compounds; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D005973:Glutamine / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D018482:Muscle, Skeletal / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D002413:Cations, Divalent; D003619:Dansyl Compounds; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D009711:Nucleotides; D005973:Glutamine; C032377:dansylethylenediamine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium; C008542:monodansylcadaverine; D002103:Cadaverine; D002118:Calcium