Myosin motors with artificial lever arms.

Abstract

The myosin head consists of a globular catalytic domain and a light chain binding domain (LCBD). The coupling efficiency between ATP hydrolysis and myosin‐induced actin movement is known to decline as the LCBD is truncated or destabilized. However, it was not clear whether the observed alteration in the production of force and movement reflects only the mechanical changes to the length of the LCBD or whether these changes also affect the kinetic properties of the catalytic domain. Here we show that replacement of the LCBD with genetically engineered domains of similar rigidity and dimensions produces functional molecular motors with unchanged kinetic properties. The resulting single‐chain, single‐headed motors were produced in Dictyostelium discoideum and obtained after purification from a standard peptone‐based growth medium at levels of up to 12 mg/l. Their actin motility properties are similar or greater than those of native myosin. Rates of 2.5 and 3.3 microm/s were observed for motor domains fused to one or two of these domains, respectively. Their kinetic and functional similarity to the extensively studied myosin subfragment 1 (S1) and their accessibility to molecular genetic approaches makes these simple constructs ideal models for the investigation of chemo‐mechanical coupling in the myosin motor.

Folding of the Striated Muscle Myosin Motor Domain

“…We have achieved a sliding actin filament velocity (3 m/s) approaching that of the intact embryonic chicken myosin used as the base to construct the motor domain::GFP chimera (9). The level of activity we observed is similar to that reported for a Dictyostelium myosin motor domain constructed with a synthetic lever arm supplied by two ␣-actinin repeats (19,21). The best motor activity for that chimera was achieved when it was attached to the surface by a mAb to an epitope tag on the distal end of the ␣-actinin repeat.…”

“…This corresponds to Lys-785 of the embryonic skeletal muscle myosin isoform used here. Dictyostelium myosin truncated at this residue and fused to artificial lever arms including GFP produces active motor molecules with motility properties similar to single-headed myosin (19,20). Chymotrypsin digestion of skeletal muscle myosin after removal of both light chains also yields a functional motor domain (21).…”

A Molecular Model for Muscle Contraction

“…In a similar experiment, Manstein and his collaborators (Anson et al, 1996) have replaced the neck region with arti®cial lever arms consisting of one or more -actinin repeats (a triple -helix motive) and have used the engineered myosin in vitro motility assays. As above, the authors ®nd the speed of transport is proportional to the length of the lever arm.…”

Molecular engineering of myosin

“…This helps to prevent steric clashes with the motor and makes it possible to rotate the lever arm the full 180 . Spectrin-like repeats, in particular a-actinin repeats, the second type of building block, have been used as artificial lever arms in a wide range of constructs, where they always performed reliably (Anson et al 1996;Kurzawa et al 1997;Ruff et al 2001;Ito et al 2003). The third building block, the 698 residue Dictyostelium MyoE motor domain (E698), resembles the motor domain of conventional myosins (Kollmar et al 2002).…”