ABSTRACTThe ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence based methods like two-color fluorescence cross-correlation spectroscopy can provide this information but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross-talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photo-stability, bleaching and fluorescence brightness can have an impact.Here we take advantage of lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross-talk. To establish sc-FLCCS we first engineered and tested multiple GFP-like fluorescent proteins for their suitability. This identified a novel GFP variant termed slmGFP (short lifetime monomeric GFP) with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and slmGFP were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color(dc)-FCCS measurements.STATEMENT OF SIGNIFICANCEThe quantification of protein concentrations and protein-protein interactions in vivo is a crucial information needed for the understanding of complex processes inside cells. Determination of such information is unfortunately still challenging. Fluorescence-based method like fluorescence cross-correlation spectroscopy (FCCS) is the only method which provides this information in vivo and almost in the real time, however it suffers from limitations caused by experimental setup and biological origin of fluorescent proteins. We present single-color fluorescence lifetime cross-correlation spectroscopy as an alternative to FCCS, which uses the information of fluorescence lifetime to overcome some of these limitations. We challenged the method and determined its advantages and limitations and demonstrated the applicability of the method on the proteins of yeast proteasome.
Cross-Talk-Free Dual-Color Fluorescence Cross-Correlation Spectroscopy for the Study of Enzyme Activity
