Quantification of Cyclin-CDK dissociation constants in living cells using fluorescence cross-correlation spectroscopy with green and near-infrared fluorescent proteins.

The cell cycle is a highly coordinated process governed by cyclin-bound cyclin-dependent kinases (CDKs). While the interaction between cyclin and CDK are well-documented, the dissociation constants (Kd) between specific cyclin-CDK pairs within living cells remain poorly understood. Fluorescence cross-correlation spectroscopy (FCCS) enables the 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 bleed-through. This fluorescent protein pair enables us to measure the Kd values of the cyclin-dependent kinase Cdc2 and B-type cyclin Cdc13 in fission yeast cells. Moreover, we conducted a comprehensive analysis of the Kd values for 36 cyclin-CDK complexes, formed by 9 distinct cyclins and 4 CDKs, in mammalian cells, including unconventional cyclin-CDK pairs. These findings provide insights into the redundancy of cyclin-CDK binding in cell cycle progression, with potential implications for understanding cell cycle regulation in both fission yeast and higher eukaryotes.