Nanoluc oligoproteins as a model system for protein misfolding and refolding studies

Protein misfolding can lead to protein malfunction, which may compromise cell viability. Chaperones, including the HSP70 system, are proteins that have evolved to restore the native structure of misfolded proteins. Although most chaperones, including DnaK (bacterial HSP70), were first described over 30 years ago, important questions related to their mechanisms remain unanswered. Only a small number of model proteins are used in the literature for misfolding and refolding studies. Previously, we described several NanoLuc (Nluc) luciferase-based constructs as models for DnaK-assisted chaperone refolding: Nluc₂, Nluc₃, and others, where the Nluc module was combined with the titin I91 domain. Here, we expanded this family of tandem multimodular proteins with Nluc₇, which allowed us to better analyze how interactions between modules affect Nluc_n activity, denaturation, and DnaK-assisted refolding. We found that interactions between internal modules of Nluc_n attenuate the module's activity differently than interactions between terminal and neighboring modules. Also, among the Nluc_n variants, Nluc₇ is the most resistant to precipitation during thermal denaturation, enabling the production of soluble misfolded proteins at elevated concentrations. After denaturation, Nluc₇ shows greater ability of DnaK-assisted refolding with elevated half-time compared with Nluc₂ and Nluc₃. The relatively large size of Nluc₇ allowed us to characterize both its native and denatured states using transmission electron microscopy, which showed no aggregation but indicated particle compactization after 10 min of denaturation and the formation of small soluble aggregates after 30 min of denaturation.