Quantitative Analysis Of Protein Interactions Research Articles

Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells

FRET (Fluorescence Resonance Energy Transfer) measurements are commonly applied to proof protein-protein interactions. However, standard methods of live cell FRET microscopy and signal normalization only allow a principle assessment of mutual binding and are unable to deduce quantitative information of the interaction. We present an evaluation and normalization procedure for 3-filter FRET measurements, which reflects the process of complex formation by plotting FRET-saturation curves. The advantage of this approach relative to traditional signal normalizations is demonstrated by mathematical simulations. Thereby, we also identify the contribution of critical parameters such as the total amount of donor and acceptor molecules and their molar ratio. When combined with a fitting procedure, this normalization facilitates the extraction of key properties of protein complexes such as the interaction stoichiometry or the apparent affinity of the binding partners. Finally, the feasibility of our method is verified by investigating three exemplary protein complexes. Altogether, our approach offers a novel method for a quantitative analysis of protein interactions by 3-filter FRET microscopy, as well as flow cytometry. To facilitate the application of this method, we created macros and routines for the programs ImageJ, R and MS-Excel, which we make publicly available.

SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks.

The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental defects, ageing disorders, blood disorders such as Fanconi Anemia, increased inflammation and cancer. Therefore, the development of tools to study proteins associated with error-free DNA replication processes is of paramount importance. So far, methods to study proteins associated with nascent replication forks relied on conventional immunofluorescence and immunoprecipitation assays of 5'-ethylene-2'-deoxyuridine (EdU) labeled DNA (iPOND). While greatly informative and important, these methods lack specificities for nascent fork interactions (e.g., IF) or assay an average change of millions of cells without single-cell resolution (e.g., iPOND). The assay system described here combines proximity ligation assay (PLA) with EdU coupled click-iT chemistry, which we termed "in situ protein interaction with nascent DNA replication forks (SIRF)". This method enables sensitive and quantitative analysis of protein interactions with nascent DNA replication forks with single-cell resolution, and can further be paired with conventional immunofluorescence marker analysis for added multi-parameter analysis.

SIRFing the replication fork: Assessing protein interactions with nascent DNA.

Roy et al. (2018. J. Cell. Biol. https://doi.org/10.1083/jcb.201709121) describe an ingenious single-cell assay system, in situ analysis of protein interactions at DNA replication forks (SIRF), for the quantitative analysis of protein interactions with nascent DNA at active and stalled replication forks. The sensitive and accurate SIRF methodology is suitable for multiparameter measurements in cell populations.

SIRF: Quantitative in situ analysis of protein interactions at DNA replication forks.

DNA replication reactions are central to diverse cellular processes including development, cancer etiology, drug treatment, and resistance. Many proteins and pathways exist to ensure DNA replication fidelity and protection of stalled or damaged replication forks. Consistently, mutations in proteins involved in DNA replication are implicated in diverse diseases that include defects during embryonic development and immunity, accelerated aging, increased inflammation, blood disease, and cancer. Thus, tools for efficient quantitative analysis of protein interactions at active and stalled replication forks are key for advanced and accurate biological understanding. Here we describe a sensitive single-cell-level assay system for the quantitative analysis of protein interactions with nascent DNA. Specifically, we achieve robust in situ analysis of protein interactions at DNA replication forks (SIRF) using proximity ligation coupled with 5'-ethylene-2'-deoxyuridine click chemistry suitable for multiparameter analysis in heterogeneous cell populations. We provide validation data for sensitivity, accuracy, proximity, and quantitation. Using SIRF, we obtained new insight on the regulation of pathway choice by 53BP1 at transiently stalled replication forks.

Flow Cytometric Single-Cell Analysis for Quantitative in Vivo Detection of Protein-Protein Interactions via Relative Reporter Protein Expression Measurement.

Cell-based two-hybrid assays have been key players in identifying pairwise interactions, yet quantitative measurement of protein-protein interactions in vivo remains challenging. Here, we show that by using relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, quantitative analysis of protein interactions in a bacterial adenylate cyclase two-hybrid (BACTH) system can be achieved. A multicolor flow cytometer was used to measure simultaneously the expression levels of one of the two putative interacting proteins and the β-galactosidase (β-gal) reporter protein upon dual immunofluorescence staining. Single-cell analysis revealed that there exists bistability in the BACTH system and the RRPE is an intrinsic characteristic associated with the binding strength between the two interacting proteins. The RRPE-BACTH method provides an efficient tool to confirm interacting pairs of proteins, investigate determinant residues in protein-protein interaction, and compare interaction strength of different pairs.

Image‐based systems biology

Image‐based systems biology

Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells.

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein–protein interaction.

Proximity or no proximity: That is the question—But the answer is more complex

THE significance of molecular interactions has been recognized since the dawn of molecular biology, and the real importance of the vast amount of information hidden in the human genome can only be understood if the interactome is also defined. Conventionally, molecular interactions are investigated by chemical crosslinking and coprecipitation. Although these approaches are widely used, they do not provide quantitative information, suffer from the lack of cell-by-cell resolution, and potential artifacts arising from high protein expression and membrane solubilization in the case of membrane proteins. Cytometry offers several alternative methods to assess protein–protein interactions. Fluorescence resonance energy transfer (FRET) has made its way to become the golden standard in quantitative analysis of protein interactions (1). In FRET, a fluorescent donor dye residing on a protein of interest transfers its excitation energy to a nearby acceptor molecule which labels another protein. The rate of this radiationless interaction decreases with the sixth power of the separation distance making FRET a sensitive tool for molecular interactions. In conventional FRET, the pairwise interaction between two spectroscopically distinct molecules is analyzed, and the principle has recently been applied for the simultaneous investigation of two pairwise interactions (2) and for the investigation of the three-way interaction among three molecules (3,4). Although FRET usually takes place between spectroscopically distinct molecules, the same process can also occur between molecules of the same type. This flavor of the interaction is called homo-FRET, and it has been used for the detection of molecular trimers (5) and even larger clusters (6,7). The combination of total internal reflection microscopy with FRET allowed the high-throughput screening of molecular interactions (8). A multitude of correlation techniques are available for the analysis of protein clustering. The mother of all such approaches is fluorescence correlation spectroscopy (FCS) in which the autocorrelation of fluorescence signals is measured in a femtoliter volume to determine the diffusion constant (9,10). FCS has been applied in confocal microscopy for the accurate and quantitative measurement of association states and concentrations in the intracellular space (11,12) and for the monitoring of dynamic properties of membrane proteins (13,14). Fluorescence cross-correlation spectroscopy (FCCS) has been used for the detection of the association of two fluorescently labeled proteins. Dynamic and transient interactions are more conveniently detected by FCCS than by FRET (15). However useful the aforementioned correlation techniques are, they require dedicated instrumentation. The realization that a temporal confocal image series taken by a conventional confocal microscope contains all the necessary information to calculate the diffusion constant and the association state of the fluorescently labeled substance has lead to the development of raster image correlation spectroscopy (RICS) (16) and number and brightness analysis (N&B) (17). Both of these techniques

21] Small zone, high-speed gel filtration chromatography to detect protein aggregation associated with light chain pathologies

Small zone gel filtration chromatography can be used for qualitative and quantitative analysis of protein interactions and aggregation phenomena. The technique is fast, accessible to most laboratories, and can be combined with computer simulation to extract quantitative information from experimental data. The programs KRUNCH and SCIMZ will be furnished on written request to the authors.