tailieunhanh - Báo cáo khoa học: Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state

Previous cross-linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002)Biochemistry41, 86–93] have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady-state ATPase parameters of actomyosin. | iFEBS Journal Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state Gyorgy Hegyi1 and Jozsef Belagyi2 1 Department of Biochemistry Eotvos University Budapest Hungary 2 Institute of Bioanalysis University of Pecs Hungary Keywords actin cross-linking actomyosin interactions EPR spectroscopy heavy meromyosin Correspondence G. Hegyi Department of Biochemistry Eotvos University Pazmany Peter setany 1. C H-1117 Budapest Hungary E-mail hegyi@ Received 3 January 2006 revised 20 February 2006 accepted 22 February 2006 doi Previous cross-linking studies Kim E Bobkova E Hegyi G Muhlrad A Reisler E 2002 Biochemistry 41 86-93 have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However it does not change the steady-state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains-1 and -2. In this study we use EPR spectroscopy to investigate the effects of cross-linking constituent monomers upon the molecular dynamics of the F-actin complex. We show that cross-linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin HMM to the cross-linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative even a 1 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5 Py-imido triphosphate AMPPNP a nonhydrolyzable