Breeze through membrane protein stability with Uncle

Importance of the topic

The stability of membrane proteins is critical for drug discovery and biomarker research. These proteins are targets for therapies and vaccines, but their hydrophobic regions require detergent or alternative vehicles for solubilization. Characterizing their thermal and aggregation stability is challenging due to limited sample and heterogeneous formats, limiting throughput and insight.

Objectives and study overview

This application note evaluates the Uncle all-in-one stability platform to profile the thermal unfolding and aggregation of two membrane proteins, GPRC5D and CD20, in detergent micelles, nanodiscs, and virus-like particles (VLPs). The goal is to compare stability across formats using multiple biophysical readouts from minimal sample volumes.

Methodology and instrumentation

• Protein samples: detergent‐solubilized, MSP1D1 nanodiscs, and enveloped VLPs for GPRC5D and CD20 sourced from ACROBiosystems.
• Sample normalization: protein concentrations at ~0.15 mg/mL (GPRC5D) and ~0.25 mg/mL (CD20).
• Detection modes: intrinsic fluorescence (266 nm excitation), static light scattering (SLS), dynamic light scattering (DLS), and thiol-reactive dye CPM.
• Thermal gradient: 15–95 °C ramp at 0.6 °C/min with triplicate measurements in 9 µL volumes.

Used Instrumentation

• Uncle stability platform with full-spectrum fluorescence.
• Temperature control module (15–95 °C) and sealed 9 µL quartz cuvette array.
• 266 nm and 473 nm UV lasers for intrinsic and CPM fluorescence.
• Uncle Analysis software for Tm determination by barycentric mean and dye fluorescence increase.

Main results and discussion

GPRC5D in detergent showed a CPM-based melting temperature (Tm) of ~46.2 °C and an intrinsic fluorescence Tm of ~48.8 °C. Embedding in nanodiscs increased the CPM Tm by >10 °C, indicating a stabilizing lipid environment. CD20 in detergent exhibited two CPM Tms at ~43.2 °C and ~50.9 °C with no clear intrinsic unfolding signal; nanodisc incorporation raised each Tm by ~1 °C and yielded an intrinsic fluorescence Tm at ~82.4 °C. VLP-embedded proteins produced complex unfolding profiles with multiple intrinsic fluorescence transitions and size changes detected by DLS and SLS. VLPs decreased in hydrodynamic diameter after heating, and SLS intensity drops highlighted thermal disassembly events.

Benefits and practical applications of the method

The Uncle platform requires only 9 µL per sample, enabling high-throughput screening of scarce membrane proteins. Simultaneous multi-modal readouts deliver orthogonal data on unfolding and aggregation, facilitating formulation optimization, construct engineering, and vehicle selection.

Future trends and potential applications

Advances in membrane mimetics such as nanodiscs, liposomes, and VLPs will expand biophysical screening for drug targets and vaccine candidates. Integration with automated workflows and machine learning analytics may accelerate stability-guided design of membrane protein therapeutics.

Conclusion

The multi-parameter Uncle platform provides a comprehensive, low-volume solution for assessing membrane protein stability across detergent, nanodisc, and VLP formats. Combined fluorescence, SLS, and DLS readouts enable rapid comparison of conditions and vehicles, driving efficient development of membrane protein research and therapeutics.

References

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• S Gridley et al. Challenges and Approaches for Assay Development of Membrane and Membrane-Associated Proteins in Drug Discovery. Prog Mol Biol Transl Sci. 2010;91:209–239.
• P Rodriguez-Otero et al. GPRC5D as a novel target for the treatment of multiple myeloma: a narrative review. Blood Cancer J. 2024;14(1):24.
• W Wang et al. Antibody Structure, Instability, and Formulation. J Pharm Sci. 2007;96(1):1–26.
• The UniProt Consortium. UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res. 2023;51(D1):D523–D531.