Comprehensive Analysis of Protein Dynamics Using HiBiT Tagging: Localization, Protein Interactions, and Degradation

Studying protein dynamics is crucial for understanding how cellular processes are regulated and contribute to biology. The HiBiT Protein Tagging System provides powerful tools for studying proteins in their native context. HiBiT is a small, 11-amino acid peptide that can be appended to endogenous proteins via CRISPR-mediated knock-in strategies. Tagging proteins endogenously with a small tag avoids overexpression artifacts and interference with protein function or localization. High affinity between HiBiT and LgBiT, the polypeptide component of the NanoBiT^® split luciferase reporter, enables exceptionally sensitive detection of HiBiT-tagged targets. Complementation of HiBiT and LgBiT yields a bright, bioluminescent signal that can be utilized for quantitative and functional analysis of proteins.  Beyond luminescence detection, HiBiT can function as an affinity handle for IP and immunofluorescence. Promega has built a suite of HiBiT assays that make the tag exceedingly versatile, allowing researchers to explore diverse areas of protein biology that include localization, degradation, post-translational modifications, and protein-protein interactions in both native cellular contexts and through pharmacologically driven mechanisms, such as targeted protein degradation.

In this article, we demonstrate utility of the HiBiT tag in study of SMARCA2, an essential chromatin remodeler protein¹. SMARCA2 is part of the SWI/SNF chromatin remodeling complex and is a key oncology target, with several degrader molecules advancing in clinical trials^2,3. Using CRISPR and clonal selection, a HeLa cell line was established with the HiBiT tag inserted on the 3’-end of endogenous SMARCA2 loci. By tagging the target endogenously and using HiBiT-based tools for analysis, a comprehensive overview of SMARCA2 biology is achieved. Here, we show native SMARCA2 localization, association with SWI/SNF complex components, and the kinetics of induced SMARCA2 degradation and mechanism of action by SMARCA2 degraders using a combination of HiBiT immunoassays and luminescent assays.

The HiBiT tagging system enables the cellular localization of endogenous proteins to be tracked. The Anti-HiBiT Monoclonal Antibody was used to confirm proper localization of SMARCA2-HiBiT in the nucleus via immunocytochemistry microscopy (Figure 1). These images demonstrate the HiBiT tag does not disrupt nuclear localization of SMARCA2 and its known role in chromatin remodeling. 

Figure 1. Immunocytochemistry of SMARCA2-HiBiT HeLa CRISPR cell line. Cells were fixed and treated with Anti-HiBiT Monoclonal Antibody, anti-mouse AlexaFluor® 488 secondary, and Hoechst stain. Nuclear localization of SMARCA2-HiBiT is observed, as expected.

Having established the proper localization of SMARCA2-HiBiT, we next sought to validate its interactions within proteins in the nucleus. Complexes containing SMARCA2-HiBiT were captured from cell lysate using Anti-HiBiT Magne^® Beads. Eluates from the beads were analyzed by mass spectrometry (MS) to identify native SMARCA2 interactors.  

Figure 2. IP-MS of SMARCA2-HiBiT complexes. A. Volcano Plot of Anti-HiBiT Magne Bead IP eluates comparing SMARCA2-HiBiT CRISPR knock-in cells with HeLa parental cell controls. B. Cartoon of SWI/SNF complex members. Proteins identified by MS analysis are shown in color.

Analysis of SMARCA2-HiBiT immunoprecipitation (IP) samples revealed enrichment of many known SWI/SNF complex members, including 10 out of 12 key proteins integral to the chromatin-remodeling function of this complex (Figure 2). The identification of established SWI/SNF components highlights the ability of HiBiT CRISPR cell lines to preserve native interaction profiles. Moreover, the sensitivity and specificity of the Anti-HiBiT Magne^® Beads allows robust capture of endogenously expressed HiBiT tagged proteins and relevant interactors. Interestingly, IP-MS of SMARCA2-HiBiT samples also identified accessory proteins outside of the SWI/SNF complex as potential interactors. Some of these have known roles in chromatin organization and gene regulation, This suggests an extended SMARCA2 interaction network that needs to be further explored.

Targeting SMARCA2 has historically been challenging due to its high structural similarity with its paralog, SMARCA4, both integral components of the SWI/SNF chromatin remodeling complex. Traditional small-molecule inhibitors often lack the specificity to distinguish between these two proteins, leading to off-target effects and limited therapeutic efficacy. Targeted protein degraders, such as Proteolysis-Targeting Chimeras (PROTACs), can potentially address this issue by facilitating the selective degradation of SMARCA2. PROTACs are bifunctional molecules that recruit a target protein to an E3 ubiquitin ligase, resulting in their ubiquitination and subsequent degradation (Figure 3). Due to a catalytic mechanism of action, targeted protein degraders offer the potential to achieve greater therapeutic efficacy with lower concentrations and with more prolonged pharmacological effects. This is particularly valuable in diseases where SMARCA2 dysregulation contributes to pathogenesis. 

Figure 3. Key stages of PROTAC-mediated target degradation. PROTACs must first exhibit cell permeability and form a ternary complex with the target protein and E3 ligase. This leads to ubiquitination of the target, its recruitment to the proteasome, and subsequent degradation. Color-coded symbols represent the target protein (blue), E3 recruiter (light blue), and ubiquitin (pink).

Having characterized SMARCA2’s native interaction network, we set out to investigate the kinetics and mechanism of action of the ACBI1 proteolysis targeting chimera (PROTAC) that targets SMARCA2 for degradation. We leveraged NanoBRET^® technology to detect ternary complex formation and ubiquitination, key steps in the mechanism of action of PROTACs. The NanoBRET^® system, when combined with HiBiT tagging, provides a sensitive, real-time method to monitor these induced interactions directly in living cells.

The ternary complex forms when a PROTAC binds both a target protein and effector E3. NanoBRET^® reports on complex formation by energy transfer from complemented HiBiT-LgBiT as a luminescent energy donor to a HaloTag^® fluorescent acceptor when target protein and E3 are in proximity. To measure ternary complex formation, SMARCA2-HiBiT cells were co-expressed with LgBiT and HaloTag^®-VHL, representing the E3 ubiquitin ligase recruited by the ACBI1 PROTAC. HaloTag^® NanoBRET^® 618 Ligand  was added when cells were plated in assay plates. The following day cells were treated with either 1 µm ACBI1 or DMSO for 2 hours after which the NanoBRET^® Nano-Glo^® Substrate was added and the NanoBRET® ratio was measured on a GloMax^® Discover. ACBI1-treated cells had an increase in NanoBRET^® ratio compared to DMSO-treated cells, indicating the PROTAC facilitated formation of the SMARCA2-VHL complex (Figure 4), supporting the mechanism of VHL-mediated degradation of SMARCA2.

Figure 4. SMARCA2-HiBiT is recruited to HaloTag®-VHL, an E3 ligase, upon treatment with ACBI1 PROTAC.

In addition to ternary complex formation, we used NanoBRET^® to assess the ubiquitination of SMARCA2—the critical post-translational modification marking the protein for proteasomal degradation. By tagging ubiquitin with HaloTag^® to serve as the energy acceptor we were similarly able to use NanoBRET^® to detect ubiquitination of SMARCA2-HiBiT after ACBI1 treatment.  HaloTag^® NanoBRET^® 618 Ligand was added when cells were plated in assay plates. The following day cells were treated with either 1 µm ACBI1 or DMSO for 2 hours after which the NanoBRET^® Nano-Glo^® Substrate was added and the NanoBRET^® ratio was measured on a GloMax^® Discover. ACBI1 PROTAC treatment resulted in ubiquitination levels of SMARCA2-HiBiT approximately 3-times higher compared to DMSO (Figure 5). This approach provided a direct link between ternary complex formation and subsequent ubiquitination, illustrating the stepwise process that governs ACBI1’s mechanism of action in degrading SMARCA2.

Figure 5. SMARCA2-HiBiT is ubiquitinated to a greater extent upon treatment with ACBI1 PROTAC.

After assessing ternary complex formation and ubiquitination, we were interested in examining the kinetics of SMARCA2 degradation within live cells. ACBI2 was chosen to induce degradation in live cells. ACBI2 is a PROTAC designed specifically to target SMARCA2, whereas ACBI1 targets both SMARCA2 and SMARCA4. Live-cell, kinetic quantification of degradation using the HiBiT tagging system offers significant advantages, including minimizing the risk of overlooking dynamic cellular responses to degrader that may be missed with end-point assays alone.

To quantify SMARCA2-HiBiT degradation, we first introduced LgBiT into live cells with the ViaScript™ LgBiT mRNA Delivery System. The next day Nano-Glo^® Endurazine Substrate was added to cells and given 2.5 hours to reach substrate equilibrium before the cells were treated with a dilution series of ACBI2. The luminescent signal from SMARCA2-HiBiT cells was measured over 24 hours (Figure 6). The real-time degradation profile of SMARCA2 in response to PROTAC treatment revealed rapid, potent, and near-complete degradation of SMARCA2 that persisted over time.

Figure 6. Live-cell degradation of SMARCA2-HiBiT levels upon ACBI2 treatment

The kinetic profile provides critical insight into the action of ACBI2, and the efficiency of all mechanistic steps following from ternary complex formation to proteasomal degradation. Such data are invaluable for optimizing PROTAC-based therapeutic strategies by quantifying the relationships between concentration, time, and degradation efficacy, facilitating more informed compound ranking as well as providing deeper insights into degrader mode of action.

HiBiT technology, with its high sensitivity and non-intrusive tagging, enables precise, live-cell monitoring of protein levels in response to targeted treatments. By combining assays that elucidate the kinetics and mechanistic steps of targeted protein degraders with findings on localization and native protein interactions, we gain a comprehensive understanding of induced SMARCA2 degradation within its native cellular context. This underscores the value of HiBiT technology for detailed investigations of protein dynamics under endogenous cellular regulation.

The HiBiT tagging system, with its small size, high sensitivity, and compatibility with multiple detection platforms, is a versatile tool for investigating protein dynamics in their endogenous cellular environment. In this study, we demonstrated HiBiT’s utility for characterizing several aspects of SMARCA2 biology:

• Maintenance of Native Properties: SMARCA2-HiBiT was shown to localize to the nucleus, as expected for a chromatin remodeler. Additionally, IP-MS analysis revealed that SMARCA2-HiBiT interacts with expected SWI/SNF complex core proteins. The ability of HiBiT to capture these interactions illustrates its sensitivity and specificity for mapping protein-protein interactions and further exemplifies its non-disruptive nature in faithfully reporting endogenous biology.
• Characterization of Targeted Protein Degradation Mechanisms: The formation of ternary complexes and subsequent ubiquitination, assessed with NanoBRET® technology, provided insight into the mechanisms by which SMARCA2 is recruited to VHL by PROTAC ACBI1. Live-cell monitoring of degradation kinetics with ACBI2 revealed a rapid dose-dependent degradation profile, highlighting the power of HiBiT in studying dynamic processes. 

We demonstrate the ability for HiBiT to capture dynamic events inSMARCA2 biology in both native and pharmacologically induced contexts, and in real time. Together, these findings highlight the versatility of HiBiT as a minimally invasive, powerful tagging system for both foundational and applied research. With its adaptability across different assay platforms, HiBiT opens new avenues for studying endogenous protein behavior, helping to unlock a deeper understanding of complex biological processes, and expanding the toolkit available for drug discovery and therapeutic development.