Single-molecule dissection of coupling mechanisms between cofactor binding and folding of a complex protein

Abstract

Dissecting protein folding pathways is challenging, especially for large or multidomain proteins. This challenge is further exacerbated when considering that many proteins harbor one or more cofactors in their structure, which can alter their fold and thermodynamic properties. Here, we use optical tweezers to mechanically unfold and refold drosophila cryptochrome (dCRY), a large, multidomain protein that harbors a FAD cofactor in its structure. By applying force and simultaneously varying the FAD concentration and the refolding and rebinding dwell time, we dissect the mechanisms that coupled dCRY folding and FAD binding.