A Comparative Biophysical Study of Conserved Ubiquitin-like Protein Folds

Ubiquitin and ubiquitin-like proteins (Ubls) constitute a conserved protein family that shares a common β-grasp structural fold despite substantial sequence and functional divergence. How this highly conserved fold is maintained while accommodating diverse biological functions remains an open question in protein structural biology. In this study, we provide atomistic insights into the structural features that determine the stability of these conserved ubiquitin-like folds. Using an integrated biophysical approach combining solution NMR spectroscopy, molecular dynamics simulations, and chemical denaturation experiments, we systematically compare the protein dynamics and thermodynamic stability of ubiquitin and representative Ubls, including SUMO1, SUMO2, NEDD8, and Hub1. Our results reveal that ubiquitin exhibits exceptional thermodynamic stability relative to other Ubls, whereas Ubls display a broad range of stability profiles without a uniform trend toward destabilization. Biophysical and molecular dynamics analyses show that the reduced stability of Ubls is not associated with the loss of the conserved fold but is accompanied by differences in local structural flexibility. Together, these findings indicate that the conserved ubiquitin fold has been maintained despite sequence divergence and variation in stability and dynamics, supporting functional diversity while preserving structural integrity.