Quantification of cyclin–CDK dissociation constants using FCCS with green and near-infrared fluorescent proteins

The cell cycle is a highly coordinated process governed by cyclin-bound cyclin-dependent kinases (CDKs). Although the interactions between cyclins and CDKs are well documented, the dissociation constants (Kd) for specific cyclin-CDK pairs within living cells remain poorly understood. Fluorescence cross-correlation spectroscopy (FCCS) enables quantification of the Kd, but challenges remain in selecting an optimal pair of fluorescent molecules for FCCS in a living cell. In this study, we demonstrate that mNeonGreen and phycocyanobilin-bound miRFP670 represent a suitable pair for FCCS in living cells from the viewpoint of high photostability and low bleedthrough. This fluorescent protein pair enables us to measure the Kd values of the CDK Cdc2 and B-type cyclin Cdc13 in fission yeast cells. Moreover, we roughly estimated the Kd values for 36 cyclin-CDK complexes formed by nine distinct cyclins and four CDKs in mammalian cells, including unconventional cyclin-CDK pairs. These measurements suggest potential versatility of cyclin-CDK binding in cell cycle progression, with implications for understanding cell cycle regulation in both fission yeast and higher eukaryotes.