Structural changes in subdomain 2 of G-actin observed by fluorescence spectroscopy

Abstract

The influence of DNase I binding to Ca-ATP-G-actin and of Ca2+/Mg2+ and ATP/ADP exchange on the conformation of G-actin were investigated by measuring the fluorescence of dansyl cadaverine (DC) conjugated to Gln41 in subdomain 2 of the protein. Fluorescence resonance energy transfer (FRET) between this probe and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide (DDPM) attached to Cys374 in subdomain 1 was also measured. Contrary to an earlier report [dos Remedios, Kiessling and Hambly (1994) in Synchrotron Radiation in the Biosciences (Chance, B., Deisenhofer, J., Ebashi, S., Goodhead, D. T., Helliwell, J. R., Huxley, H. E., Iizuka, T., Kirz, J., Mitsui, T., Rubenstein, E. et al., eds.), pp. 418–425, Oxford University Press, Oxford], the distance between these probes did not change significantly when DNase I was bound to actin. A small but reproducible increase in the quantum yield and a blue shift of the DC fluorescence maximum were observed when bound Ca2+ was replaced by Mg2+. A large increase (about 70%) in the quantum yield and an approx. 12 nm blue shift of the emission spectrum occurred when ATP in Mg-G-actin was replaced by ADP. These changes were not accompanied by any significant change in the FRET distance between the dansyl donor 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-terminal segment in subdomain 1 of actin. The results are discussed in relation to recently published data obtained with another fluorescent probe and to earlier observations based on limited cleavage using proteolytic enzymes.