An in-cell NMR strategy optimized for proteins with poor solubility and low thermal stability

Motivation: The in-cell NMR method allows direct observations of proteins in cultured human cells, to evaluate protein conformations and interactions in an intracellular environment. Currently, target proteins must be highly soluble and thermally stable. Goal(s): A technical in-cell NMR strategy is required for proteins with poor solubility and low thermal stability. Approach: Protein concentration-dependent NMR analyses revealed the self-association sites of proteins. Site-directed mutants gained higher solubility. Cell treatment conditions at lower temperature were established. Results: We successfully constructed an in-cell NMR protocol that improves protein solubility and cell treatment conditions at 25°C, the lowest temperature established so far. Impact: By performing experiments according to our strategy, in-cell NMR can be applicable to more types of proteins with poor solubility and low thermal stability. Furthermore, we developed an in-cell system for evaluating therapeutic candidate compounds against target proteins.