Clustering intramolecular interactions of proteins significantly enhances cellular thermal stability

Thermophilic microorganisms, thriving in extreme temperatures, offer valuable insights into the molecular mechanisms of protein thermal stability, with significant implications for biotechnology. Herein, we employed multiscale molecular dynamics (MD) simulations to investigate the proteome thermal adaptation of Escherichia coli (E. coli), Zhurongbacter thermophilus 3DAC (3DAC), and Thermococcus eurythermalis A501 (A501), representing mesophilic, thermophilic, and hyperthermophilic microorganisms, respectively. Comparisons of cellular crowding effect, amino acid composition, secondary structures, global and surface net charges, salt bridges, hydrogen bonds, and hydrophobic interactions revealed no substantial differences among these microorganisms. It is noteworthy that significant differences were observed in the spatial distribution of intramolecular interactions. Thermophilic proteins exhibited a higher density of clustered salt bridges, hydrogen bonds, and hydrophobic interactions, particularly beyond active sites, which enhanced proteome rigidity and thermal stability without compromising protein functionality. These findings suggest that clustered intramolecular interactions, rather than specific amino acid compositions or structures, are a key strategy for thermophilic protein stability. Our study provides new perspectives on thermal adaptation mechanisms in early-diverging life forms and offers a framework for designing thermostable proteins for biotechnological applications.