Measuring Endogenous Protein Interactions in Live Cells

Protein-protein interactions (PPIs) are fundamental to nearly all cellular processes. However, when disrupted, they can lead to diseases including cancer and neurodegeneration. This clinical relevance makes them valuable therapeutic targets. Researchers are investigating PPIs to illuminate the complexities of cellular biology and develop new therapeutic strategies.^1,2 This requires tools for studying PPIs within an endogenous context. Live-cell assays provide valuable data on PPIs, generating insights into how these interactions occur in their native environment.

The significance of PPIs in cell biology

PPIs are the backbone of cellular signaling networks, controlling how cells sense and respond to internal and external stimuli. These highly orchestrated interactions control processes like cell cycle progression, apoptosis, differentiation, and development. Many PPIs are dynamic and transient, allowing for fine-tuned, reversible responses rather than permanent changes.

The downstream effects of a PPI depend on several key variables. Subcellular localization can affect downstream signaling. The relative abundance of interacting proteins influences whether an interaction occurs. Interactions can also be modulated through regulatory feedback or shifted by competitive interaction partners.

Because PPIs are highly regulated, even subtle molecular changes, such as single amino acid substitutions, can disrupt or enhance PPIs, rewiring entire pathways. Alternatively, post-translational modifications like phosphorylation or ubiquitination can alter a protein’s interaction capabilities.

Because of their roles in complex signaling networks, mapping and quantifying PPIs offers a system-level understanding of cell behavior. This research is critical for understanding biology at the molecular level and for defining emergent properties of whole-cell and tissue systems.

Applications of PPIs in drug discovery

Traditional drug discovery has focused on enzymes and receptors with active sites that could be inhibited. However, many disease-critical proteins act through interactions, not catalysis. Dysfunctional PPIs drive diseases, including cancers, neurodegenerative disorders and viral infections. Investigating PPIs allows drug discovery to move beyond static targets and into dynamic networks.

Strategies for targeting PPIs may depend on how the interaction is affecting the cell. Some PPIs drive disease progression by activating signaling cascades abnormally. Small molecules or biologics can disrupt these interactions to suppress abnormal signaling. Other PPIs are beneficial and need stabilization. Molecular glues promote or stabilize desirable interactions, leading to activation of beneficial processes like tumor suppression.

Finally, PPI profiling may help predict and overcome drug resistance by revealing how mutations alter interaction profiles and affect drug sensitivity. This helps them understand resistance mechanisms and design drugs for specific mutational contexts.

Methods for studying PPIs

Despite their disease relevance, many interactions are notoriously difficult to target due to absent binding pockets or context-specific behavior that is hard to replicate outside the native environment.

Biochemical assays play a key role in PPI studies, enabling robust and quantitative detection in lysates or with purified proteins. While these methods are well-suited for high-throughput screening, they require cell lysis and lack the ability to capture dynamic, localization-dependent interactions that occur in intact cells. To overcome these limitations, researchers use live-cell assays that provide real-time, quantitative measurements of PPIs under native conditions. These assays evaluate interactions in the native cellular context, capturing the effects of localization, feedback, and post-translational modifications. They are adaptable to diverse formats, ranging from single-cell imaging to high-throughput screening, and from monolayers to 3D spheroids. Importantly, they enable real-time analysis of PPI timing and dynamics under physiological conditions.

Live-cell protein complementation assays

Protein complementation assays detect PPIs by reconstituting an active reporter enzyme from two inactive fragments fused to target proteins. Interaction of these target proteins drives reporter assembly and signal production. For example, biomolecular fluorescence complementation (BiFC) uses split fluorescent proteins like GFP.³ While insightful, its slow kinetics and irreversibility limit accurate quantitation of PPI dynamics.

Luminescence-based complementation uses two subunits of a split luciferase enzyme.⁴ Luciferase complementation is reversible and generates light instantly upon interaction and substrate addition, allowing for real-time monitoring of both association and dissociation. Luciferase assays produce a proportional, luminescent signal that can be measured over time to assess interaction strength and kinetics.

Live-cell energy transfer assays

Energy transfer-based assays detect PPIs by measuring non-radiative energy transfer between two labels in close proximity. Fluorescence resonance energy transfer (FRET) is a widely used approach that employs fluorescent protein pairs genetically fused to proteins of interest.⁵ FRET enables real-time imaging of protein interactions and localization but is limited by high background fluorescence, photobleaching, and the need for external illumination, which can cause phototoxicity and complicate quantitation.

Bioluminescence resonance energy transfer (BRET) addresses these limitations by using a luciferase as the energy donor to activate the acceptor fluorophore.⁶ This eliminates the need for external light excitation, reduces background noise, and avoids phototoxicity. The reversibility of luciferase activation allows for continuous monitoring of association and dissociation kinetics, dose responses or drug-induced changes. Data is presented as a ratio between acceptor and donor signals, which normalizes for differences in expression levels or cell number.

While valuable, these techniques rely on transient overexpression from plasmids, which can introduce artifacts such as mislocalization or altered complex formation due to artificial promoters driving non-physiological expression. CRISPR/Cas9 genome editing overcomes this challenge by incorporating fluorescent or luminescent tags directly into endogenous gene loci and expressing fusion proteins under native regulatory control.⁷ This preserves physiological stoichiometry, minimizing artifacts ,and allowing more accurate analysis of weak or transient interactions that are highly sensitive to protein concentration. When paired with low-background, real-time detection methods, CRISPR knock-ins offer a robust strategy for mapping protein interaction networks in live cells with high fidelity.

Conclusion

The convergence of genome editing with complementation- and resonance energy transfer-based detection technologies marks a transformative step in the study of protein interactions. These tools allow researchers to move beyond overexpression models and investigate signaling complexes in their native cellular environments, governed by endogenous transcription, localization, and dynamic feedback.

As CRISPR-based knock-ins become more efficient and scalable, their adoption for physiologically relevant PPI analysis will continue to grow. Complementary reporter technologies extend this capability, enabling live-cell monitoring of interaction dynamics with high sensitivity and temporal resolution. Together, these approaches establish a versatile and physiologically grounded platform for drug discovery, particularly in identifying and characterizing modulators of protein interactions overlooked in conventional screening systems.

References

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7. Niles AL, Dibble MRC, Machleidt T, et al. Bioluminescence-based assays for quantifying endogenous protein interactions in live cells. J Biol Chem. 2025;301(8):110454. doi:10.1016/j.jbc.2025.110454