NMR Insights Into Stress-Induced Modulation of the Monomer–Dimer Equilibrium in a Small Heat Shock Protein

Abstract

Small heat shock proteins (sHSPs) are essential molecular chaperones that play a crucial role in maintaining protein homeostasis and protecting cells from stress-induced damage. HSPB8 (Small heat shock protein B8), in particular, plays a crucial role in protein folding and degradation pathways and has been associated with protein aggregation disorders. However, its structural and dynamic behavior under different environmental stress conditions remains poorly defined. In particular, the effect of pH, temperature, and concentration on its oligomeric state and structural integrity needs further investigation. In this study, we performed the biophysical characterization of full-length HSPB8 and its α-crystallin domain (ACD) using solution-state nuclear magnetic resonance (NMR) spectroscopy under different environmental perturbations. The effect on the monomer–dimer equilibrium of the ACD was characterized by monitoring changes in chemical shifts and linewidths in response to the perturbations, including protein concentration, pH, and temperature. It was observed that at low pH, reduced protein concentrations, and elevated temperatures, the ACD favored sharper resonances, reflecting a monomeric state. The relative contribution of disulfide bond and noncovalent interactions in stabilizing the dimer form of ACD was also established. These results provide NMR-based molecular insights into the dynamic monomer–dimer equilibrium, crucial for HSPB8 function in protein folding.