title: Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain.
abstract: The effects of mutations in an actin-binding surface loop of myosin (loop 2) are described. Part of loop 2, the segment between myosin residues 618 and 622, was replaced with sequences enlarged by the introduction of positively charged GKK or neutral GNN motifs. Constructs with loops carrying up to 20 additional amino acids and charge variations from -1 to +12 were produced. Steady-state and transient kinetics were used to characterize the enzymatic behavior of the mutant motor domains. Binding of nucleotide was not affected by any of the alterations in loop 2. In regard to their interaction with actin, constructs with moderate charge changes (-1 to +2) displayed wild-type-like behavior. Introduction of more than one GKK motif led to stronger coupling between the actin- and nucleotide-binding sites of myosin and an up to 1000-fold increased affinity for actin in the absence of ATP and at zero ionic strength. In comparison to the wild-type construct M765, constructs with 4-12 extra charges displayed an increased dependence on ionic strength in their interaction with actin, a 2-3-fold increase in kcat, a more than 10-fold reduction in Kapp for actin, and a 34-70-fold increase in catalytic efficiency.
authors.affiliation: Max-Planck-Institut für Medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Furch
authors.forename: M A
authors.initials: MA
authors.lastname: Geeves
authors.forename: D J
authors.initials: DJ
authors.lastname: Manstein
mesh_terms: D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D004023:Dictyostelium; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration
chemical_list: D000199:Actins; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D009218:Myosins