The thermal unfolding and domain structure of myosin subfragment 1 (SI) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 "C), while the binding of the nun-hydrolysable analogue of ATP, adenosine 5'-[/Y~-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (V,), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.loC, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADPVi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes Sl-AdoPP[NH]P and S1-ADP-V, is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis : one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-P,. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide @PDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-V, can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-P,.Muscle contraction and many other manifestations of biological motility are based on the cyclic interaction of myosin heads with actin, which is accompanied by ATP hydrolysis. During steady-state ATP hydrolysis the myosin head is subjected to conformational changes that can be detected by changcs in ultraviolet absorption Enzyme. Chymotrypsin (EC 3.4.21 .I).is used as a stable analogue of the S1-ATP complex.Complexes of S1 with ADP and orthovanadate (Vi) [S, 61 and S1 cross-linked by p-phenylene-N,N-dimaleimide (pPDM) in the presence of ADP [7] are often used as stable analogues of the S1-ADP-Pi complex. The use of stable analogues allows the structure and properties of S1 in the intermediates of the ATPase reaction to be studied. There are numerous data suggesting that the stable analogues of the S1-ATP and S1-ADP-Pi intermediates are similar in structure, but differ from nucleotide-free S1 and S1-ADP [8-201, It should be noted that all these data are in fact indirect since they are based on either studies of the actinbinding properties of S1 [8 -151 or studies of local conformational changes in S1 [16-201. In order to obtain direct proof of the assumption of two main structural states of S1, one must know the effects of nucleoti...

The thermal unfolding of the myosin subfragment 1 (Sl) in its stable complex with ADP and beryllium fluoride (Sl . ADP . BeF;) was studied by differential scanning calorimetry. It has been shown that the structure of the Sl molecule in the Sl ADP . BeF; complex is similar to that of Sl in its complex with ADP and orthovanadate (Sl ADP . Vi) but differs radically from that of nucleotide-free Sl and Sl in the Sl . ADP complex. It is concluded that the Sl . ADP . BeF; complex can be considered, like the Sl . ADP . Vi complex, a stable structural analogue of the myosin head ADP Pi transition state of the myosin-catalyzed ATP hydrolysis.

The structure of the myosin subfragment-l (SI) from rabbit skeletal muscle was studied using differential scanning microcalorimetry. Three independently melting regions (domains) were revealed in Sl. Selective denaturation of the middle 50 kDa segment of the SI heavy chain resulted in the disappearance of the heat sorption peak corresponding to the melting of the first, the most thermolabile domain without any effect on the thermally induced blue shift of the intrinsic tryptophan fluorescence spectrum which occurs within the temperature region of melting of the second domain. It is concluded that the most thermolabile domain seems to correspond to the N-terminal part of the 50 kDa segment devoid of tryptophan residues.

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