Molecular engineering of myosin

Abstract

Protein engineering and design provide excellent tools to investigate the principles by which particular structural features relate to the mechanisms that underlie the biological function of a protein. In addition to studies aimed at dissecting the communication pathways within enzymes, recent advances in protein engineering approaches make it possible to generate enzymes with increased catalytic efficiency and specifically altered or newly introduced functions. Here, two approaches using state-of-the-art protein design and engineering are described in detail to demonstrate how key features of the myosin motor can be changed in a specific and predictable manner. First, it is shown how replacement of an actin-binding surface loop with synthetic sequences, whose flexibility and charge density is varied, can be employed to manipulate the actin affinity, the catalytic activity and the efficiency of coupling between actin-and nucleotide-binding sites of myosin motor constructs. Then the use of pre-existing molecular building blocks, which are derived from unrelated proteins, is described for manipulating the velocity and even the direction of movement of recombinant myosins.

The motor mechanism of myosin V: insights for muscle contraction

“…Further experimental support for the swinging lever arm hypothesis comes from studies that show that the unitary displacement and/or velocity of myosin II are related to lever arm length (Uyeda et al 1996;Warshaw et al 2000;Manstein 2004). In particular, three reports demonstrated a direct correlation between lever arm length and stroke size (the displacement of a single-headed interaction with actin) of myosin V (Purcell et al 2002;Sakamoto et al 2003;Moore et al 2004).…”

Controllable molecular motors engineered from myosin and RNA

“…Previously, synthetic nucleic acid motors 3–5 and modified natural protein motors 6–10 have been developed in separate complementary strategies for achieving tunable and controllable motor function. Integrating protein and nucleic acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities.…”

“…Protein engineering has previously been used to replace the myosin lever arm with alternative structures that can reproduce or modify wild-type behavior 6,7,9,19 , including modules that function as gearshifts that respond to optical or chemical signals 7,9 . Since nucleic acid engineering offers a complementary approach for precise design of programmable molecular structures and devices 20,21 , we asked whether a functional myosin lever arm could also be constructed from RNA.…”

“…M6-RB (for Myosin VI - RNA-Binding) was generated by fusing myosin VI to the L7Ae kink-turn binding domain 22 . Following a design principle established in previous work on engineered myosin lever arms 6,7,9,19 , we made use of a helix-sharing junction in which the C-terminal helix of the truncated myosin VI lever arm was fused to the N-terminal helix of the L7Ae domain. Guided by crystal structures of L7Ae-RNA 22 and of myosin VI 23,24 , we aligned the terminal helices and optimized the phasing of the junction to orient a bound kink-turn motif as a structural foundation from which to build extended RNA lever arms.…”

“…Changing the directionality of myosins by altering the structure of the lever arm has provided rigorous tests of the swinging crossbridge model and represents an additional demonstration that an attached structure functions as a lever 6,7,9,19 . Following a similar strategy used in protein engineering studies of myosins and kinesin-14 9,31 , we aimed to reverse the direction of the motor by designing an RNA lever arm that inverted the projection of the power stroke, yielding plus-end directed motility.…”