G-Quadruplexes Catalyze Protein Folding: A Research Report

Academic Background

Protein folding is a complex and unsolved problem in biology. Many proteins fold very slowly in vitro, far exceeding the time ranges acceptable under physiological conditions. To address this challenge, ATP (adenosine triphosphate)-dependent chaperonins are thought to accelerate protein folding, enabling it to occur within physiological timescales. However, whether this capability is limited to ATP-dependent chaperones remains an open question. The core issue of this study is to explore whether other molecules can catalyze protein folding similarly to ATP-dependent chaperonins, thereby assisting cells in completing protein folding more rapidly.

G-quadruplexes (G4s) are four-stranded structures formed by guanine-rich nucleic acid sequences. In eukaryotes, G4s form under stress conditions and dissociate when stress dissipates. Recent studies suggest that G4s play a significant role in proteostasis, but their specific mechanism in protein folding remains unclear. This research aims to investigate whether G4s can catalyze protein folding and uncover their molecular mechanisms.

Paper Source

This paper was co-authored by Zijue Huang, Kingshuk Ghosh, Frederick Stull, and Scott Horowitz. The authors are affiliated with the Department of Chemistry and Biochemistry, Knoebel Institute for Healthy Aging, and Department of Physics at the University of Denver, as well as the Department of Chemistry at Western Michigan University. The paper was published on February 6, 2025, in the Proceedings of the National Academy of Sciences (PNAS), titled “G-quadruplexes catalyze protein folding by reshaping the energetic landscape.” The study was funded by the National Institutes of Health (NIH) and the National Science Foundation (NSF).

Research Process and Results

1. Catalytic Effect of G-Quadruplexes on Protein Folding

The core question of this study is whether G-quadruplexes can catalyze protein folding. The research team selected the fluorescent protein tagRFP675 as the subject, studying its folding and unfolding kinetics in the presence and absence of G4s through in vitro experiments.

• Investigation of Protein Folding Mechanism: Initially, the team observed the folding process of tagRFP675 without G4s using fluorescence spectroscopy. The experiment revealed that tagRFP675’s folding involves at least one fluorescent intermediate state (i1) and the final native state (n). Through kinetic modeling, the team found that tagRFP675’s folding also includes an off-pathway intermediate state (i2), indicating that some proteins get “trapped” in incorrect pathways, slowing down the folding process.

• Topology Dependence of G-Quadruplexes: To explore how the topology of G4s affects their catalytic activity, the team tested parallel, antiparallel, and 3+1 hybrid G4 structures on tagRFP675 folding. The results showed that the parallel G4 structure (seq576) had the strongest effect on promoting protein folding, while the antiparallel structure had no significant effect.

2. Mechanism of G-Quadruplex-Catalyzed Protein Folding

• Binding of G-Quadruplexes to Proteins: In the presence of G4s, the team’s kinetic modeling revealed that proteins form complexes with G4s during folding (ug4, i1g4, ng4). This “folding-while-bound” model significantly accelerated the folding process and effectively reduced the formation of off-pathway intermediates (i2).

• Thermodynamic Impact of G-Quadruplexes: By conducting experiments at different temperatures, the team used the Eyring equation to calculate thermodynamic parameters such as enthalpy change (ΔH), entropy change (ΔS), and free energy change (ΔG) during protein folding. They found that G4s alter the thermodynamic driving forces of protein folding, accelerating the process. Specifically, G4s significantly reduced the entropy barrier, facilitating the transition from intermediate states to the native state.

Research Conclusions

This study is the first to reveal that G-quadruplexes, as non-ATP-dependent molecules, can catalyze protein folding. The results show that G4s accelerate protein folding by reshaping the thermodynamic landscape and reducing the formation of off-pathway intermediates. This finding suggests that cellular protein folding acceleration is not solely dependent on ATP-dependent chaperones; other molecules like G4s may also play crucial roles.

Key Highlights

1. Discovery of a New Mechanism: This study provides the first evidence that G4s can catalyze protein folding, challenging the previous notion that only ATP-dependent chaperones have this capability.
2. Topology Dependence: The research shows that the topological structure of G4s significantly influences their catalytic activity, with parallel G4s exhibiting the strongest effect.
3. Thermodynamics-Driven Protein Folding: Through thermodynamic analysis, the study reveals how G4s modify the enthalpy and entropy changes in protein folding pathways, accelerating the folding process.

Application Value and Future Prospects

This research offers new insights into the complex mechanisms of protein folding within cells and may provide novel approaches for treating diseases related to protein misfolding. For instance, neurodegenerative diseases like Alzheimer’s disease are closely associated with protein misfolding, and G4s could become potential targets for future therapies. Additionally, the study suggests that nucleic acids within cells may globally regulate protein folding times, providing important theoretical foundations for further research on cellular protein folding regulation networks.

The findings of this study not only expand our understanding of protein folding mechanisms but also open up possibilities for developing new tools to regulate protein folding.