STRUCTURAL-CHANGES IN SUBDOMAIN-2 OF G-ACTIN OBSERVED BY FLUORESCENCESPECTROSCOPY

The influence of DNase I binding to Ca-ATP-G-actin and of Ca2+/Mg2+ an d ATP/ADP exchange on the conformation of G-actin were investigated by measuring the fluorescence of dansyl cadaverine (DC) conjugated to Gl n(41) in subdomain 2 of the protein. Fluorescence resonance energy tra nsfer (FRET) between this probe and N-[4-(dimethylamino)-3,5-dinitroph enyl]maleimide (DDPM) attached to Cys(374) in subdomain 1 was also mea sured. Contrary to an earlier report [dos Remedies, Kiessling and Hamb ly (1994) in Synchrotron Radiation in the Biosciences (Chance, B., Dei senhofer, J., Ebashi, S., Goodhead, D.T., Helliwell, J. R., Huxley, H. E., Iizuka, T., Kirz, J., Mitsui, T., Rubenstein, E. et al., eds.), p p. 418-425, Oxford University Press, Oxford], the distance between the se probes did not change significantly when DNase I was bound to actin . A small but reproducible increase in the quantum yield and a blue sh ift of the DC fluorescence maximum were observed when bound Ca2+ was r eplaced by Mg2+. A large increase (about 70%) in the quantum yield and an approx. 12 nm blue shift of the emission spectrum occurred when AT P in Mg-G-actin was replaced by ADP. These changes were not accompanie d by any significant change in the FRET distance between the dansyl do nor and DDPM acceptor probes. A substantial change in the fluorescence of DC-actin was observed after proteolytic removal of the last three residues of actin, in accordance with earlier evidence suggesting that there is a conformational coupling between subdomain 2 and the C-term inal segment in subdomain 1 of actin. The results are discussed in rel ation to recently published data obtained with another fluorescent pro be and to earlier observations based on limited cleavage using proteol ytic enzymes.