Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation Spectroscopy

Abstract

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (K(d)s) of biomolecules. The determination of a K(d) depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ~0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Förster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the K(d).

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

“…As reported previously, mEGFP exhibited sufficiently bright fluorescence, a small triplet fraction, and resistance to photobleaching. In contrast, substantial triplet fractions were detected in tdTomato and mCherry, as reported previously (38). Furthermore, Foo et al reported previously that the low maturation efficiency of mCherry affected FCCS analysis (38).…”

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

“…1 A); the value is the average from measurements on both membranes and at both temperatures, errors are given as the SE of the mean (mean 5 SE) unless stated otherwise. The apparent complex fraction <100% can be explained by incomplete maturation of mRFP and its prolonged residence in dark or dim states, as discussed in detail earlier (57,58). The apparent complex fraction of mRFP-EGFR-eGFP in COS-7 and HEK293 cells was 68 5 2% and 58 5 1%, respectively.…”

“…Only cells with expression levels of the two fluorescent proteins differing by a factor of <2 were selected for data processing and evaluation (57). Positive and negative controls were performed for each cell line and each temperature to determine the dynamic range of our method.…”

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

“…Quantitative analysis of protein-protein interactions in living cells has been performed by single-wavelength fluorescence cross-correlation spectroscopy [12,13,16], although twolaser-beam FCCS was also used to quantify biological interactions [15,[17][18][19]. In this work, we used two-laser-beam FCCS instead of single-beam FCCS because the two-laser FCCS has more flexible laser power tuning for each fluorescence probe, providing accurate measurement of fluorescence intensity.…”

“…The femtoliter confocal volume is well suited to resolve the different measurement positions even in live cells. FCCS has various applications to determine quantitative parameters, including determination of the dissociation constant in the living cell [12][13][14][15][16][17][18][19]. Indeed, single wavelength fluorescence cross-correlation spectroscopy has been employed for determination of dissociation constants [12,13,16].…”