The cofactor-dependent folding energy landscape of a light-sensing protein revealed by single-molecule pulling experiments

Abstract

The functional link between cofactor binding and protein activity is well established, but how cofactor interactions with the polypeptide reshape the folding energy landscape of large and multidomain proteins is unknown. Here, we use optical tweezers in combination with a novel analytical framework that integrates clustering, bootstrapping and global fitting of kinetic and thermodynamic data to dissect the folding mechanism of the light-sensing Drosophila cryptochrome (dCRY), a 542-residue protein that binds FAD, one of the most common, complex cofactors. We show that FAD binds to multiple dCRY folding intermediates, some of which contain large amounts of unfolded polypeptide. Yet, binding occurs with association kinetics above the diffusion-limit and at sub-nanomolar affinity. Surprisingly, the first parts of dCRY to fold are independent of FAD, but later steps are FAD-driven as the remaining protein folds around the cofactor. Thus, dCRY coordinates cofactor-dependent and independent folding mechanisms to attain its native state. Furthermore, we find that not all the FAD chemical moieties are strictly required for folding: whereas the isoalloxazine ring linked to ribitol and one phosphate group (i.e., FMN) are sufficient to drive complete dCRY folding, the adenosine ring plus the phosphate groups (i.e., AMP and ADP) only allow partially folded structures. Lastly, by combining the results from optical tweezers experiments with structural data, we propose a model for the dCRY folding pathway wherein regions known to undergo conformational transitions during signal transduction are the last to fold. Altogether, our single-molecule experiments and data analysis illustrate the power and broad applicability of optical tweezers to dissect complex mechanisms that couple the folding of large proteins to cofactor binding.