Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis

Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfr agment 1 (SI) introduces a chiral probe (TNP) into an interface of the cata lytic and lever arm domains of S1 [Muhlrad (1977) Biochim, Biophys. Acta 49 3, 154-166]. Characteristics of the TNP absorption and circular dichroism ( CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenyl ation rate in native S1, indicate a one-to-one correspondence between ATPas e transients and trapped phosphate analogues. Phosphate analogue-induced st ructures of TNP-Lys84-S1 were modeled using the crystallographic coordinate s of SI [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 a nd Gly710 to approximate conformational changes during ATPase. The CD and a bsorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, fi rst tested on a trinitrophenylated hexapeptide with known structure, were a pplied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitt ing just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhib ition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced ch anges in S1 Mg2+-and K+-EDTA ATPase are from collision of the lever arm wit h TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a ch iral reporter group for the effect of Lys84 modification on dynamic S1 stru cture. As such, TNP-Lys84-S1 is equivalent to a genetically engineered muta nt with intrinsic sensitivity to structure local to the modified residue.