Too close for comfort: Self-crowding transforms protein structure and stability beyond volume exclusion

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Proteins function in crowded cellular environments, but their behavior is often studied under dilute conditions.
• Understanding protein behavior in vivo requires considering macromolecular crowding effects.

Purpose of the Study:

• To investigate the impact of protein self-crowding on protein structure and interactions at physiological concentrations.
• To determine if self-crowding induces significant conformational and oligomeric changes.

Main Methods:

• Circular dichroism (CD) spectroscopy to assess secondary structure changes.
• Small-angle X-ray scattering (SAXS) to probe protein assembly and interactions.
• Experiments conducted on lysozyme (LYS) and bovine serum albumin (BSA) at varying concentrations.

Main Results:

• Self-crowding induced reversible structural changes in LYS and BSA, shifting from alpha-helices to beta-sheets and turns.
• LYS exhibited attractive interprotein interactions, forming dense states, while BSA showed repulsive interactions, destabilizing its dimer.
• BSA acted as a self-hydrotrope at low concentrations, stabilizing monomers, but promoted dimer dissociation at higher concentrations.

Conclusions:

• Protein self-crowding significantly alters protein conformation and assembly, leading to distinct dense protein states.
• These findings challenge classical volume-exclusion models of macromolecular crowding.
• Self-crowding drives reversible restructuring, highlighting its importance in physiological protein behavior.