Kinetic Pathways and Thermodynamic Stability in Protein Folding of Small Globular Proteins

Understanding the mechanisms of protein folding remains a central challenge in biophysics. This study focuses on the kinetic pathways and thermodynamic stability in the folding of small globular proteins. We aimed to elucidate the interplay between folding rates and stability by employing molecular dynamics simulations and experimental validation via nuclear magnetic resonance (NMR) spectroscopy. The simulations revealed that the transition state ensemble exhibits a significant diversity in its conformational space, with critical contacts forming at a rate-determining step. Our experimental results corroborated these findings, showing a folding half-time of 1.2 ± 0.3 ms for our model protein under physiological conditions. Additionally, we observed that mutations stabilizing native contacts can lead to a 25% increase in folding speed, indicating the delicate balance between stability and kinetic accessibility. The data suggest that the energy landscape of protein folding is more rugged than previously anticipated, with multiple pathways leading to the native state. These findings advance our understanding of the fundamental principles governing protein folding and highlight potential avenues for designing proteins with enhanced stability. In conclusion, our integrative approach offers new insights into the protein folding process, which may inform future research in protein engineering and therapeutic design.