Biomolecules under Pressure: Protein Phase Transitions from Folding to Misfolding, Aggregation, and Condensation

Abstract High hydrostatic pressure provides a unique and sensitive perturbation to protein structure, enabling direct measurement of volume changes associated with folding, misfolding, and assembly. Because pressure perturbs noncovalent forces while preserving covalent integrity, it reveals conformational intermediates that often remain hidden from thermal or chemical denaturation. Over the past decade, advances in high-pressure NMR, fluorescence spectroscopy, time-resolved crystallography, and single-molecule spectroscopy have transformed pressure into a high-resolution probe of folding landscapes. At the same time, pressure has become an incisive tool for studying the continuum from protein folding to misfolding, including amyloidogenesis, prion conversion, mutant p53 aggregation, and the physical chemistry of biomolecular condensates. This review summarizes conceptual developments in the last 20 years, highlighting how pressure reshapes our understanding of protein energy landscapes, cavity hydration, and the coupling between folding, phase separation, and disease.