Unravelling the Properties of Single α-Helical Domains in Myosin and other Proteins

Abstract

In most proteins, α-helices are stabilised by interactions with neighbouring secondary structure elements (e.g. coiled-coils). However, we recently showed that many proteins contain single α-helical (SAH) domains, which are stable in isolation and commonly inserted between two different functional domains. SAH domains are rich in arginine, lysine and glutamic acid residues. Their stability arises from the many potential (i, i ± 4) and (i, i ± 3) intrahelical interactions between either R and E, or K and E.To date, the best-studied SAH domains are those from myosins 6 and 10, and the Dictyostelium myosin myoM, where they are likely to form part of the functional lever. A SAH domain is also predicted for myosin 7a. We have shown that the SAH domain can functionally substitute for the canonical lever in myosin 5a in vitro. In cells, myosin 10 missing its SAH domain still moves to the tips of filopodia but with a reduced velocity.We have now determined that the SAH domains unfold at forces of less than ∼50 pN using single molecule force microscopy. Moreover, SAH domains unfold non-co-operatively during thermal melting, and have a high propensity to refold completely when the temperature is returned back to 10 °C, measured by circular dichroism. Molecular simulations agree with all the experimental data available, confirming the surprising stability of SAH domains and revealing unique properties that set the SAH domains apart. Among these is the ability to recover helical conformation rapidly after being extended through application of force.Thus SAH domains appear to function as ‘stretchable’ elements, unfolding and rapidly refolding at relatively low forces. In myosins, this may enable these motors to hold on to and traffic their cargoes in the crowded environment of the cell.