email:
trybus@physiology.med.uvm.edu

Structure/Function Studies of Conventional 
and Unconventional Myosin Motor and Actin

A major aim of my research program is to understand how molecular motors produce force and motion, and how the motor’s activity is regulated. We are also focusing on the structure and function of actin.

Myosin V:

• A folded-to extended conformational transition that is controlled by calmodulin and calcium, and probably by cargo-binding itself, regulates myosin V’s ability to transport cargo in the cell (Krementsov, D.N., Krementsova, E.B., and Trybus, K.M. (2004) Myosin V: Regulation by calcium, calmodulin, and the tail domain. J. Cell Biol. 164, 877-886).
• Myosin V’s long lever arm, which binds 6 calmodulins, allowed us to use truncated constructs to show a correlation between unitary step-size and the number of IQ motifs, confirming that the myosin V neck acts a lever (Moore, J.R., Krementsova, E.B., Trybus, K.M., and Warshaw, D.M. (2004) Does the myosin V neck region act as a lever? J. Muscle Research and Cell Motil. 25, 29-35).
• A single-molecule processivity assay was used to infer that myosin V can travel down multiple pathways during an ATPase cycle, which allows it to maintain robust processivity (Baker, J.E., Krementsova, E.B., Kennedy, G.G., Armstrong, A., Trybus, K.M., and Warshaw, D.M. (2004) Myosin V processivity: Multiple kinetic pathways for head-to-head coordination. Proc. Natl. Acad. Sci. USA, 101, 5542-5546).

Actin:

• We have succeeded in expressing functional actin in the baculovirus/insect cell expression system. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization, by virtue of two point mutations in subdomain 4. The monomeric mutant actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation; this complex is being used for crystallization trials (Joel, P.B., Fagnant, P.M., and Trybus, K.M. (2004), Expression of a Non-polymerizable Actin Mutant in Sf9 Cells. Biochemistry 43:11554-59).
  
  
• We have crystallized the monomeric actin in order to examine nucleotide-dependent conformational changes, as well as structural effects of mutations involved in cardiac and skeletal muscle myopathies.

Smooth Muscle Myosin:

• Smooth muscle myosin is regulated by light chain phosphorylation The structure of the inhibited, dephosphorylated double-headed subfragment of smooth muscle myosin was obtained from 2-D crystals formed on lipid monolayers. An unexpected, asymmetric structure was observed that allowed us to propose a model by which the ATPase activity of both heads could be inhibited, albeit by different mechanisms (Wendt, T., Taylor, D., Trybus, K.M., and Taylor, K.A. (2001) 3-D image recons truction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and the placement of subfragment 2. Proc. Natl. Acad. Sci. USA, 98, 4361-4366).
• A smooth muscle myosin heterodimer was used to show that although maximal actin displacement requires two heads, only one head needs to be enzymatically functional. (Kad N.M., Rovner, A.S., Fagnant, P.M., Joel, P.B., Kennedy, G.G., Patlak, J.B., Warshaw, D.M., and Trybus, K.M. (2003) A mutant heterodimeric myosin with one inactive head generates maximal displacement. J. Cell Biol. 162, 481-488).