Automated Stability Screening of Protein Complexes Using ProteoPlex

Large macromolecular complexes play essential roles in biological processes, but their structural analysis is hindered by low stability and conformational heterogeneity upon purification. Conventional stabilization approaches are often manual, protein-specific, and require extensive biochemical knowledge. Traditional DSF methods fail to interpret overlapping unfolding transitions typically observed in multicomponent complexes. This limits their utility in structural biology, especially for challenging targets like ribosomes, spliceosomes, or ATPase assemblies. There is a critical need for a general, automated, and sensitive method to assess and enhance the stability of these biomolecular machines under varying chemical environments.

ProteoPlex automates stability screening through a novel extension of DSF tailored for protein complexes. The system uses a thermocycler, fluorescence detection, and liquid-handling robotics to perform thermal unfolding assays across a matrix of buffer and ligand conditions. During thermal denaturation, the dye Sypro Orange binds exposed hydrophobic regions, generating fluorescence profiles indicative of protein unfolding. A key innovation lies in ProteoPlex’s analytical software, which applies a thermodynamic unfolding model capable of distinguishing cooperative versus non-cooperative transitions. This model estimates enthalpy, entropy, and cooperativity parameters for each condition. The platform supports both manual and fully automated modes, including a Protein Concentration Determination (PCD) module, and can screen up to 88 buffer conditions or ligands in a single run. Resulting data enable identification of conditions that yield monodisperse, stable samples, verified through downstream structural methods such as electron microscopy.

• Automated high-throughput buffer and ligand screening for protein complexes without prior biochemical knowledge.
• Advanced deconvolution algorithm resolves overlapping thermal unfolding transitions unique to macromolecular assemblies.
• Minimal sample volume requirements, enabling screening of scarce or precious protein preparations.
• Enhanced structural homogeneity and stability, verified through direct correlation with EM or crystallization success.
• Fully integrated robotic system allows unattended operation from setup to analysis.

• Structural biology: stabilization of complexes for cryo-EM or X-ray crystallography.
• Drug discovery: screening ligand-induced stabilization for therapeutic target validation.
• Protein engineering: evaluating mutant stability in multimeric protein systems.
• Biochemistry: optimizing buffer conditions for functional assays or reconstitution studies.
• Academic research: characterization of novel or poorly understood macromolecular machines.

PCT application (WO2013034160A1; 06.09.2011), active in EP, US, JP, CN

Chari, A., Haselbach, D., Kirves, J. M., Ohmer, J., Paknia, E., Fischer, N., ... & Stark, H. (2015). ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space. Nature methods, 12(9), 859-865.