Dual-color fluorescence cross-correlation spectroscopy for monitoring the kinetics of enzyme-catalyzed reactions

Dual-color fluorescence correlation spectroscopy is a biophysical technique that enables precise and sensitive analyzes of molecular interactions. It is unique in its ability to analyze reactions in real time at nanomolar substrate concentrations and below, especially when applied to the monitoring of enzyme-catalyzed reactions. Furthermore, it offers a wide range of accessible reactions, restricted only by the prerequisite that a chemical bond or a physical interaction between two spectrally distinguishable fluorophores is established or broken. Recently, the optical setup of dual-color fluorescence correlation spectroscopy has been extended toward two-photon excitation, resulting in several advantages compared with standard excitation, such as lower fluorescence background, an even larger spectrum of potential fluorescence dyes to be used, as well as a more stable and simplified optical setup. So far, the method has been successfully employed to analyze the kinetics of nucleic acid and peptide modifications catalyzed by nucleases, polymerases, and proteases.     

Practical guidelines for dual-color fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS on a commercially available setup. Although FCCS is closely related to two-color confocal microscopy, critical adjustments and test measurements are necessary to establish successful FCCS measurements, which are described in a step-by-step manner. Moreover, we discuss control experiments for a negative cross-correlation artifact, arising from a lack of detection volume overlap, and a positive artifact, arising from cross-talk. FCCS has been applied to follow molecular interactions in solutions, on membranes and in cells and to analyze dynamic colocalization during intracellular transport. It is a technique that is expected to see new applications in various fields of biochemical and cell biological research.     

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.     

Dual-color time-integrated fluorescence cumulant analysis

We introduce dual-color time-integrated fluorescence cumulant analysis (TIFCA) to analyze fluorescence fluctuation spectroscopy data. Dual-color TIFCA utilizes the bivariate cumulants of the integrated fluorescent intensity from two detection channels to extract the brightness in each channel, the occupation number, and the diffusion time of fluorophores simultaneously. Detecting the fluorescence in two detector channels introduces the possibility of differentiating fluorophores based on their fluorescence spectrum. We derive an analytical expression for the bivariate factorial cumulants of photon counts for arbitrary sampling times. The statistical accuracy of each cumulant is described by its variance, which we calculate by the moments-of-moments technique. A method that takes nonideal detector effects such as dead-time and afterpulsing into account is developed and experimentally verified. We perform dual-color TIFCA analysis on simple dye solutions and a mixture of dyes to characterize the performance and accuracy of our theory. We demonstrate the robustness of dual-color TIFCA by measuring fluorescent proteins over a wide concentration range inside cells. Finally we demonstrate the sensitivity of dual-color TIFCA by resolving EGFP/EYFP binary mixtures in living cells with a single measurement.     

Quantitative two-color fluorescence cross-correlation spectroscopy in the analysis of polymerase chain reaction

We present results of an approach in which low-density labeled DNA itself provides an amplification of the cross-correlated fluorescent signal in the two-color cross-correlation function. Tetramethylrhodamine-4-dUTP and Cy5-dCTP are incorporated by polymerase chain reaction at multiple positions of the same 217 bp target DNA. We call this novel approach the 'two-color FCS signal amplification'. The signal amplification is an example for interactions of two ligands with different colors at multiple positions of the same target.     

Protein–protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein–protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca²⁺-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein–protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.     

Cross-talk-free fluorescence cross-correlation spectroscopy by the switching method

Fluorescence cross-correlation spectroscopy (FCCS) is used as a powerful technique to analyze molecular interactions both in vitro and in vivo. This method basically requires two laser excitations for two target molecules labeled with fluorophores of different colors. Their coincidence in a microscopic detection volume is analyzed using two detectors. Any overlap of emission spectra of the two fluorophores, however, gives rise to false-positive data about their interaction. To overcome this problem, we have developed a new FCCS system, in which two excitation lasers are switched alternately by modulation using an acousto-optic tunable filter (AOTF). In this report, we demonstrate the feasibility of switching FCCS for enzymatic cleavage of proteins in living cells. A fusion protein of two fluorophores (EGFP and mRFP) with a cleavage site of caspase-3 inserted was expressed in HeLa cells, and proteolysis assay was performed during apoptotic cell death. Due to the absence of cross-talk signals, the FCCS measurement with the switching function gave a large change in relative cross-correlation amplitude after protein cleavage. Hence, switching FCCS enables more reliable measurement of molecular interactions than conventional FCCS.     

Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies

Two-photon dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC-FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC-FCCS for monitoring competitive displacement of the labeled antibody in antibody-antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the experimental points to a binding model. DC-FCCS offers the sensitivity and all other advantages of a solution phase fluorescence-based technique. For systems containing proteins of a similar size that interact without substantial changes in the fluorescence intensity, DC-FCCS serves as a preferred means of measuring solution phase binding constants.     

Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are powerful techniques to measure molecular interactions with high sensitivity in homogeneous solution and living cells. In this study, we developed methods for the detection of prion protein (PrP) using FCS and FCCS. A combination of a fluorescent-labeled Fab' fragment and another anti-PrP monoclonal antibody (mAb) enabled us to detect recombinant bovine PrP (rBoPrP) using FCS because there was a significant difference in the diffusion coefficients between the labeled Fab' fragment and the trimeric immune complex consisting of rBoPrP, labeled Fab' fragment, and another anti-PrP mAb. On the other hand, FCCS detected rBoPrP using two mAbs labeled with different fluorescence dyes. The detection limit for PrP in FCCS was approximately threefold higher than that in FCS. The sensitivity of FCCS in detection of abnormal isoform of PrP (PrP(Sc)) was comparable to that of enzyme-linked immunosorbent assay (ELISA). Because FCS and FCCS detect the PrP immune complex in homogeneous solution of only microliter samples with a single mixing step and without any washing steps, these features of measurement may facilitate automating bovine spongiform encephalopathy diagnosis.     

Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (K_ds) 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.     

Determining protease activity in vivo by fluorescence cross-correlation analysis

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions.     

Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Myosin II molecules assemble into filaments through their C-terminal rod region, and are responsible for several cellular motile activities. Three isoforms of nonmuscle myosin II (IIA, IIB and IIC) are expressed in mammalian cells. However, little is known regarding the isoform composition in filaments. To obtain new insight into the assembly properties of myosin II isoforms, especially regarding the isoform composition in filaments, we performed a combination analysis of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), which enables us to acquire information on both the interaction and the size of each molecule simultaneously. Using C-terminal rod fragments of IIA and IIB (ARF296 and BRF305) labelled with different fluorescent probes, we demonstrated that hetero-assemblies were formed from a mixture of ARF296 and BRF305, and that dynamic exchange of rod fragments occurred between preformed homo-assemblies of each isoform in an isoform-independent manner. We also showed that Mts1 (S100A4) specifically stripped ARF296 away from the hetero-assemblies, and consequently, homo-assemblies of BRF305 were formed. These results suggest that IIA and IIB can form hetero-filaments in an isoform-independent manner, and that a factor like Mts1 can remove one isoform from the hetero-filament, resulting in a formation of homo-filaments consisting of another isoform.     

One-step analysis of protein complexes in microliters of cell lysate using indirect immunolabeling & fluorescence cross-correlation spectroscopy

This protocol describes a 'mix-and-measure' procedure for the analysis of interactions of endogenous proteins in microliters of crude cell lysates. The proteins of interest are labeled by indirect immunofluorescence through simple addition of all primary and secondary antibodies to the lysate. Detection is based on fluorescence cross-correlation spectroscopy. Due to the minimal number of handling steps for sample preparation and the need of only microliters of sample, the approach enables the parallel and miniaturized analysis of protein-protein interactions. No heterologous expression of proteins with detection tags is required. For this reason, the cellular processes leading to protein-protein interactions are not skewed by overexpression of individual components. This makes the approach particularly suitable for the parallel monitoring of interactions in signaling networks. Additionally, the approach enables the screening and titration of compounds interfering with interactions, especially for those interactions based on signaling-dependent post-translational modifications. This protocol can be completed in approximately 22 h, including a 16-h incubation phase.     

Structure and dynamics of lipoplex formation examined using two-photon fluorescence cross-correlation spectroscopy

The conditions required to form transfectable lipoplexes have been extensively studied [Zuhorn, I. S., and Hoekstra, D. (2002) J. Membr. Biol. 189, 167-179]. However, to date, experiments have not addressed either the order of events of lipoplex formation in solution or the maximum number of DNA molecules per vesicle in stable single-vesicle lipoplexes. In this study, we have employed two-photon excitation fluorescence correlation spectroscopy (TPE-FCS) and two-photon fluorescence cross-correlation spectroscopy (TPE-XCS) to examine both fluorescence-labeled DNA and cationic vesicle structure and dynamics simultaneously. The dependence of large aggregated lipoplex formation on DNA-to-cationic lipid charge ratio was determined, as was the maximum number of 40 bp double-stranded DNA oligonucleotides able to bind to a single vesicle.     

An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy

Site-specific exchange of genetic information is mediated by DNA recombinases, such as FLP or Cre, and has become a valuable tool in modern molecular biology. The so far low number of suitable recombinating enzymes has driven current research activities towards alteration of catalytic properties, such as thermostability or recognition sequences. However, identification and analysis of new mutants requires sensitive in vitro activity assays, which traditionally are based on gel electrophoresis. Here, we describe the development of a new sensitive DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy (DC-FCCS), which works in homogenous solution and does not require any separation step such as electrophoresis. The assay was validated with unlabeled FLP recombinase and different fluorescently labeled DNA substrates containing the FLP recognition target (FRT). This strategy fulfills all requirements for possible application in high throughput screening and engineering of new site-specific DNA recombinases starting from the FLP-FRT system, and is easily adjustable to other systems like Cre/loxP.     

Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates

Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.     

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.     

A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is becoming increasingly popular as a technique that aims at complementing live cell images with biophysical information. This article provides both a short overview over recent intracellular FCS applications and a practical guide for investigators, who are seeking to integrate FCS into live cell imaging to obtain information on particle mobility, local concentrations, and molecular interactions. A brief introduction to the principles of FCS is provided, particularly emphasizing practical aspects such as the choice of appropriate dyes and positioning of the measurement volume in the sample. Possibilities and limitations in extracting parameters from autocorrelation curves are discussed, and attention is drawn to potential artifacts, such as photobleaching and probe aggregation. The principle of dual-color cross-correlation is reviewed along with considerations for proper setup and adjustment. Practical implications of nonideal conditions including incomplete focus overlap and spectral cross-talk are considered. Recent examples of both auto- and cross-correlation applications demonstrate the potential of FCS for cell biology.     

Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy

We introduce a new analysis technique for fluorescence fluctuation data. Time-integrated fluorescence cumulant analysis (TIFCA) extracts information from the cumulants of the integrated fluorescence intensity. TIFCA builds on our earlier FCA theory, but in contrast to FCA or photon counting histogram (PCH) analysis is valid for arbitrary sampling times. The motivation for long sampling times lies in the improvement of the signal/noise ratio of the data. Because FCA and PCH theory are not valid in this regime, we first derive a theoretical model of cumulant functions for arbitrary sampling times. TIFCA is the first exact theory that describes the effects of sampling time on fluorescence fluctuation experiments. We calculate factorial cumulants of the photon counts for various sampling times by rebinning of the original data. Fits of the data to models determine the brightness, the occupation number, and the diffusion time of each species. To provide the tools for a rigorous error analysis of TIFCA, expressions for the variance of cumulants are developed and tested. We demonstrate that over a limited range rebinning reduces the relative error of higher order cumulants, and therefore improves the signal/noise ratio. The first four cumulant functions are explicitly calculated and are applied to simple dye systems to test the validity of TIFCA and demonstrate its ability to resolve species.