Processivity Determinants of Engineered Myosins

Abstract

Myosin superfamily proteins have diverse mechanical properties adapted for specific cellular tasks. Specialized dimeric myosins are capable of processive hand-over-hand motion along actin filaments. We have used protein engineering to explore structural requirements for processivity. Previous work has shown that processivity can be retained with artificial lever arms, without requiring detailed tuning of lever arm structure or mechanics[1-4]. However, we have found that myosins engineered for desired characteristics such as bidrectionality[4] are often less processive than natural motors. We asked whether processivity could be enhanced using two strategies: (1) multimerization to form three-headed and four-headed myosins, and (2) introduction of flexible spacers in lever arms. Models of uncoordinated stepping predict that trimeric and tetrameric myosins should be much more processive than their dimeric counterparts. However, coordinated stepping mechanisms may be disrupted by the presence of additional heads. Similarly, the introduction of flexible spacers may increase the accessibility of actin binding sites for suboptimal lever arm geometries, but is also expected to abrogate strain-mediated coordination between heads. After characterizing a panel of myosins with engineered lever arms and multimerization domains, we have found that (1) trimers and tetramers show large improvements in processivity over dimers, and (2) the addition of flexible regions greatly enhances the processivity of constructs with short lever arms. Our findings reinforce the the idea that gating is dispensable for processivity in high duty ratio myosins[3] and yield general strategies for increasing processivity in engineered myosins for use in synthetic biology or nanotechnology applications.[1] J.C. Liao et al. J. Mol. Biol. 2009.[2] M. Amrute-Nayak et al. Angew. Chem. Int. Ed. 2010.[3] M.W. Elting et al. Biophys. J. 2011.[4] L. Chen, M. Nakamura, T.D.S., and Z.B. Submitted.