Analysis of Conformation of the Skeletal Muscle Myosin Modified by F2DNB Using FRET

Abstract

Previously biochemical studies have demonstrated that the highly reactive cysteine residues SH1 and SH2 can be crosslinked by variety of bifunctional reagents with different spans (3-14 A) in the presence of nucleotides, suggesting that the region is highly flexible. The SH1-SH2 region is believed to play a key role in the conformational changes that occur in the myosin head during the force generation coupled to ATP hydrolysis. We have previously shown that the skeletal muscle heavy mero-myosin (HMM), which SH1-SH2 was crosslinked by 1,5-difluoro-2, 4-dinitrobenzene (F2DNB) in the presence of ADP, have a novel conformation using quick freeze deep etch electron microscopy (QFDE-EM). We have also demonstrated that conformational change of the myosin motor domain during ATP hydrolysis can be monitored by measuring the FRET using fluorescent ATP analogue NBD-ATP. In the present study, we analyzed the conformation of the myosin crosslinked by F2DNB using FRET between the ATP binding site and the essential light chain (ELC) A1. We prepared skeletal muscle myosin subfragment-1 (S1), which ELC was labeled by 6-bromoacetyl-2-dimethylaminonaphthalene (BD) at the Cys 177. And fluorescent ADP analogue NBD-ADP was trapped within the ATPase site of S1 labeled by BD. The FRET efficiency was estimated by measuring the change of fluorescence intensity of BD comparing with control BD-S1. The FRET efficiency of F2DNB-S1-NBD-ADP was lower than S1-NBD-ADP state. This suggests that the F2DNB-S1-ADP states form more kinked conformation than S1-ADP state.