Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.

Many and diverse modifications of the myosin subfragment 1 (S-1) increase (modulate) its ATPase activity, including interaction of this particle with actin; a recent addition to these modifications is the extensive lysine modification of S-1 that seems prerequisite to crystallizing it for structure analysis. In this study we first established kinetically the ATPase modulations induced by various treatments of the myosin S-1 enzyme, and we also measured two properties of the S-1 active site--the affinity with which the site binds (a fluorescent analog of) the enzymatic nucleotide product and the access that a fluorescence quencher has to the bound ADP product--in an effort to get at the mechanism of modulation. Modulations achieved by substituting Ca2+ for the normal Mg2+ cocatalyst or by substituting Cl- for the normal carboxylate anion seem due to the product being held more loosely by the modulated enzyme.

On how a myosin tryptophan may be perturbed.

A well-known indication that a nucleotide has bound to myosin is the enhancement of the fluorescence of a specific tryptophan in the "subfragment 1" segment of the protein. Empirically the effect has been enormously useful in myosin enzymology. But beyond an early suggestion that it arises from a purine-tryptophan charge-transfer complex, the mechanism of the effect has not been considered.

The region in myosin S-1 that may be involved in energy transduction.

Newly-reported structural information about certain proximities between points on bound nucleotide and points on the heavy chain of myosin S-1 are incorporated into a previously-reported [Botts, J. Thomason, J.F.

The sequence location of the actin metal.

We have incorporated Fe2+ into the high-affinity metal-ion-binding site of actin. By supplying the system with oxygen from air and a reductant (dithiothreitol or ascorbate), we have induced free-radical generation, with the intent of causing peptide cleavage at the metal-ion-binding site. By analysis of the resulting fragments from actin in the F-form, we have deduced that cuts occurred at positions 159-160 and 301-302 (at the latter location we could not be sure if more than one cut occurred).

A search for protein structural changes accompanying the contractile interaction.

It appears that small movements (detected hitherto only by fluorescence resonance energy transfer measurements and crosslinking studies) in a region of the myosin S-1 particle may mediate chemomechanical energy transduction in the contractile system. Here we find under conditions of high precision at 10 degrees C and 20 degrees C that ATP binding to S-1 causes small (0.4%) changes in CD signal, delta epsilon 222, as do temperature changes in the regime below 16 degrees C.