Conformational changes in actin-myosin isoforms probed by Ni(II).Gly-Gly-His reactivity.

Crucial information concerning conformational changes that occur during the mechanochemical cycle of actin-myosin complexes is lacking due to the difficulties encountered in obtaining their three-dimensional structures. To obtain such information, we employed a solution-based approach through the reaction of Ni(II).tripeptide chelates which are able to induce protein cleavage and cross-linking reactions. Three different myosin motor domain isoforms in the presence of actin and nucleotides were treated with a library of Ni(II).tripeptide chelates and two reactivities were observed: (1) muscle motor domains were cross-linked to actin, as also observed for the skeletal muscle isoform, while (2) the Dictyostelium discoideum motor domain was cleaved at a single locus. All Ni(II).tripeptide chelates tested generated identical reaction products, with Ni(II).Gly-Gly-His, containing a C-terminal carboxylate, exhibiting the highest reactivity. Mass spectrometric analysis showed that protein cleavage occurred within segment 242-265 of the Dictyostelium discoideum myosin heavy chain sequence, while the skeletal myosin cross-linking site was as localized previously within segment 506-561. Using a fusion protein consisting of the yellow and cyan variants of green fluorescent protein linked by Dictyostelium discoideum myosin segment 242-265, we demonstrated that the primary sequence of this segment alone is not a sufficient substrate for Ni(II).Gly-Gly-His-induced cleavage. Importantly, the cross-linking and cleavage reactions both exhibited specific structural sensitivities to the nature of the nucleotide bound to the active site, validating the conformational changes suggested from crystallographic data of the actin-free myosin motor domain.