Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (S1) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977) Biochim. Biophys. Acta 493, 154-166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of S1 [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+- and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.

The interaction of myosin with actin, coupled with hydrolysis of ATP, is the molecular basis of muscle contraction. The head segment of myosin, called S1, contains the distinct binding sites for ATP and actin and is responsible for the ATPase activity. The myosin-catalyzed ATP hydrolysis consists of several intermediate steps and each step is accompanied by conformational changes in the S1 segment. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1 x ADP x Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate, beryllium fluoride (BeFx) or aluminum fluoride (AlF4-), yields stable complexes which mimic the intermediates of the ATP hydrolysis. In this work, tertiary structure changes in S1 in the vicinity of aromatic residues was studied by comparing near-UV circular dichroism (CD) spectra from S1-nucleotide-phosphate analog complexes in the presence of Mg2+ and other cations. A significant difference between the MgATP and MgADP spectra indicated notable tertiary structural changes accompanying the M**ADP x Pi --> M*ADP transition. The spectra of the S1 x MgADP x BeFx and S1 x MgADP x AlF4- complexes resemble to those obtained upon addition of MgATPgammaS and MgATP to S1, and correspond to the M* x ATP and M** x ADP x Pi intermediates, respectively. We have found recently that the presence of divalent metal cations (Me2+) is essential for the formation of stable S1 x MeADP x PA complexes. Moreover, the nature of the metal cations strongly influences the stability of these complexes [Peyser, Y. M., et al. (1996) Biochemistry 35, 4409-4416]. In the present work we studied the effect of Mg2+, Mn2+, Ca2+, Ni2+, Co2+, and Fe2+ on the near-UV CD spectrum of the ATP, ADP, ADP x BeFx, and ADP x AlF4- containing S complexes. The CD spectra obtained with ADP, ATP ADP x BeFx and ADP x AlF4- were essentially identical in the presence of Co2+ and rather similar in the case of Ca2+, while they were partially different in other cases. An interesting correlation was found between actin activation and ATP versus ADP difference spectra in the presence of various metal ions. The distribution of the fractional concentration of the intermediates of ATP hydrolysis was estimated in the presence of each cation from the CD spectra with phosphate analogs. In the presence of Mg2+ the predominant intermediate is the M** x ADP x Pi state, which is in accordance with the kinetic studies. On the other hand with non-native cations the predominant intermediate is the M* x ADP state and the release of ADP is the rate limiting step in the myosin-catalyzed ATP hydrolysis. According to the results, the near-UV CD spectrum originating from aromatic residues in S1 not only can distinguish identifiable states in the ATP hydrolysis cycle but can also pinpoint to changes in the tertiary structure caused by complex formation with nucleotide or nucleotide analog and various divalent metal cations. These findings, that are correlative with actin activation, and thus with the power...