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Deciphering the liquid-liquid phase separation induced modulation in the structure, dynamics, and enzymatic activity of an ordered protein β-lactoglobulin.

Saurabh RaiSrikrishna PramanikSaptarshi Mukherjee
Published in: Chemical science (2024)
Owing to the significant role in the subcellular organization of biomolecules, physiology, and the realm of biomimetic materials, studies related to biomolecular condensates formed through liquid-liquid phase separation (LLPS) have emerged as a growing area of research. Despite valuable contributions of prior research, there is untapped potential in exploring the influence of phase separation on the conformational dynamics and enzymatic activities of native proteins. Herein, we investigate the LLPS of β-lactoglobulin (β-LG), a non-intrinsically disordered protein, under crowded conditions. In-depth characterization through spectroscopic and microscopic techniques revealed the formation of dynamic liquid-like droplets, distinct from protein aggregates, driven by hydrophobic interactions. Our analyses revealed that phase separation can alter structural flexibility and photophysical properties. Importantly, the phase-separated β-LG exhibited efficient enzymatic activity as an esterase; a characteristic seemingly exclusive to β-LG droplets. The droplets acted as robust catalytic crucibles, providing an ideal environment for efficient ester hydrolysis. Further investigation into the catalytic mechanism suggested the involvement of specific amino acid residues, rather than general acid or base catalysis. Also, the alteration in conformational distribution caused by phase separation unveils the latent functionality. Our study delineates the understanding of protein phase separation and insights into the diverse catalytic strategies employed by proteins. It opens exciting possibilities for designing functional artificial compartments based on phase-separated biomolecules.
Keyphrases
  • amino acid
  • protein protein
  • hydrogen peroxide
  • binding protein
  • molecular dynamics
  • ionic liquid
  • single cell
  • risk assessment
  • oxidative stress
  • stress induced