Structural dynamics of actin during active interaction with myosin: different effects of weakly and strongly bound myosin heads.
We have used optical spectroscopy (transient phosphorescence anisotropy, TPA, and fluorescence resonance energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374 and (b) a FRET donor-acceptor pair, IAEDANS attached to Cys 374 and a nucleotide analogue (TNPADP) in the nucleotide-binding cleft. Strong interactions were detected in the absence of ATP, and weak interactions were detected in the presence of ATP or its slowly hydrolyzed analogue ATP-gamma-S, under conditions where a significant fraction of weakly bound acto-S1 complex was present and the rate of nucleotide hydrolysis was low enough to enable steady-state measurements. The results show that actin in the weakly bound complex with S1 assumes a new structural state in which (a) the actin filament has microsecond rotational dynamics intermediate between that of free actin and the strongly bound complex and (b) S1-induced changes are not propagated along the actin filament, in contrast to the highly cooperative changes due to the strongly bound complex. We propose that the transition on the acto-myosin interface from weak to strong binding is accompanied by transitions in the structural dynamics of actin parallel to transitions in the dynamics of interacting myosin heads.
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA. ewa@ddt.biochem.umn.edu
Ewa
E
Prochniewicz
Timothy F
TF
Walseth
David D
DD
Thomas
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D016880:Anisotropy; D004735:Energy Transfer; D006868:Hydrolysis; D008163:Luminescent Measurements; D015335:Molecular Probes; D009038:Motion; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012399:Rotation; D013050:Spectrometry, Fluorescence
D000199:Actins; D015335:Molecular Probes; D000255:Adenosine Triphosphate; D009218:Myosins