Abstract
Single α-helix (SAH) domains are rich in charged residues (Arg, Lys, and Glu) and stable in solution over a wide range of pH and salt concentrations. They are found in many different proteins where they bridge two functional domains. To test the idea that their high stability might enable these proteins to resist unfolding along their length, the properties and unfolding behavior of the predicted SAH domain from myosin-10 were characterized. The expressed and purified SAH domain was highly helical, melted non-cooperatively, and was monomeric as shown by circular dichroism and mass spectrometry as expected for a SAH domain. Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (<30 pN) without a characteristic unfolding peak. Molecular dynamics simulations showed that the SAH domain unfolds progressively as the length is increased and refolds progressively as the length is reduced. This enables the SAH domain to act as a constant force spring in the mechanically dynamic environment of the cell.
Highlights
Single ␣-helix (SAH) domains bridge two functional domains in proteins
Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (
When SAH domains are inserted between I27 domains, their high thermal stability allows them to keep the I275 domains separate in the I275SAH2 construct, enabling them to refold. These results suggest that insertion of a SAH domain between two functional domains in any protein would be able to effectively separate the two domains even when they unfold and that this behavior can promote the refolding of these two domains
Summary
Single ␣-helix (SAH) domains bridge two functional domains in proteins. Their force response is poorly understood. Single ␣-helix (SAH) domains are rich in charged residues (Arg, Lys, and Glu) and stable in solution over a wide range of pH and salt concentrations They are found in many different proteins where they bridge two functional domains. It is important to determine how well the SAH domains maintain their highly helical structure over a long length of sequence Do they act as a stiff spacer or as a weak extensible element that unfolds at low forces before other regions of the protein unfold? We used single molecule atomic force spectroscopy to determine its unfolding properties, and we used molecular simulation to understand the physical properties that underlie its unique structural features These results provide novel insight into the properties of the SAH domain and suggest new ideas about its biological role
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