Single-molecule Analysis of the Role of Flavin Adenine Dinulcleotide in the Conformational Landscapes of Drosophila Cryptochrome
The circadian clock regulates biological and behavioral adaptations in response to day-night cycles. Cryptochromes (CRYs) are members of a family of photolyases involved in DNA repair that are responsible for the oscillation of the circadian clock. CRYs harbor a conserved flavin adenine dinulcleotide (FAD), which is reduced by light and induces conformational changes to activate the protein. The underlying mechanisms of the light-induced conformational changes of CRYs coupled to FAD reduction, however, are still not well understood. In this study, we use optical tweezers to study the conformational landscape of the dCRY in the presence and absence of FAD, and in light activated condition. In the presence of FAD, dCRY unfolds in several steps whose total extension correspond to the fully folded protein. In contrast, in the absence of FAD the protein fails to fold into its native tertiary structure. By titrating FAD and monitoring the fraction of folded protein, we constructed a single-molecule binding curve and determine a Kd for FAD of 0.1 nM. Studying the kinetics of cofactor binding to the protein folding revealed folding of the protein through intermediates that bind to FAD with a very high affinity and in a very short time scale and the presence of intermediates bound to FAD promotes formation of native structure of the protein. Interestingly, the flavin mononucleotide (FMN) moiety binds with a Kd similar to that of FAD and promotes complete folding, whereas the dinucleotide moiety (adenine and phosphate groups) contribute very little to the binding interactions of FAD and has minimal effects on promoting the folded structure. These results indicate that FMN is the dominant moiety responsible for the binding energy of FAD, and that FAD is strictly required for the global structural integrity and stability of the protein. Taken together, the study uncovered the photoreceptor activity of the protein dependent on the presence of FAD.
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