Determining Stable Single Alpha Helical (SAH) Domain Properties by Circular Dichroism and Atomic Force Microscopy
Stable, single α-helical (SAH) domains exist in a number of unconventional myosin isoforms, as well as other proteins. These domains are formed from sequences rich in charged residues (Arg, Lys, and Glu), they can be hundreds of residues long, and in isolation they can tolerate significant changes in pH and salt concentration without loss in helicity. Here we describe methods for the preparation and purification of SAH domains and SAH domain-containing constructs, using the myosin 10 SAH domain as an example. We go on to describe the use of circular dichroism spectroscopy and force spectroscopy with the atomic force microscope for the elucidation of structural and mechanical properties of these unusual helical species.
Key wordsStable single alpha helical domains Circular dichroism Atomic force microscopy Myosin
This work was supported by Biotechnology and Biological Sciences Research Council grants BB/I007423/1 and BB/M009114/1.
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