Moraczewska J, Strzelecka-Gołaszewska H, Moens P D, dos Remedios C G
Department of Anatomy and Histology, University of Sydney, Australia.
Biochem J. 1996 Jul 15;317 ( Pt 2)(Pt 2):605-11. doi: 10.1042/bj3170605.
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.
通过测量与蛋白质第2亚结构域中Gln41偶联的丹磺酰尸胺（DC）的荧光，研究了DNase I与Ca-ATP-G-肌动蛋白的结合以及Ca2+/Mg2+和ATP/ADP交换对G-肌动蛋白构象的影响。还测量了该探针与连接在第1亚结构域中Cys374上的N-[4-(二甲基氨基)-3,5-二硝基苯基]马来酰亚胺（DDPM）之间的荧光共振能量转移（FRET）。与早期报告[dos Remedios、Kiessling和Hambly（1994年），载于《生物科学中的同步辐射》（Chance、B.、Deisenhofer、J.、Ebashi、S.、Goodhead、D.T.、Helliwell、J.R.、Huxley、H.E.、Iizuka、T.、Kirz、J.、Mitsui、T.、Rubenstein、E.等人编），第418 - 425页，牛津大学出版社，牛津]相反，当DNase I与肌动蛋白结合时，这些探针之间的距离没有显著变化。当结合的Ca2+被Mg2+取代时，观察到量子产率有一个小但可重复的增加以及DC荧光最大值的蓝移。当Mg-G-肌动蛋白中的ATP被ADP取代时，量子产率大幅增加（约70%），发射光谱发生约12 nm的蓝移。这些变化并未伴随着丹磺酰供体和DDPM受体探针之间FRET距离的任何显著变化。在蛋白水解去除肌动蛋白的最后三个残基后，观察到DC-肌动蛋白的荧光有实质性变化，这与早期证据一致，即肌动蛋白第2亚结构域与第1亚结构域的C末端片段之间存在构象偶联。结合最近用另一种荧光探针获得的数据以及基于蛋白水解酶有限切割的早期观察结果对这些结果进行了讨论。
