PH Dependence of Oligomerization and Functional Activity of Alpha B Crystallin

Abstract

αB-crystallin, a small heat shock protein dispersed throughout the body, impacts cellular processes in ways that can help rescue the cell from various insults or traumas. Environmentally dependent oligomerization enables αB-crystallin to carry out its role as a chaperone protein. Stress conditions such as heat, ischemia and acidosis can induce phosphorylation of three serine residues of αB-crystallin at positions 19, 45, and 59 on the N-terminal domain. The oligomeric states, subunit exchange and efficiency of αB-crystallin are linked to different phosphorylation states. We have studied the pH-dependence of the hyper-phosphorylated αB-crystallin using a S19E, S45E, S59E triple mutant of αB-crystallin. Chaperone efficiency using insulin as an aggregation model was measured by observing changes in light scattering of insulin in the presence of the wild type and mutant αB-crystallin. Maximal chaperone function was observed at pH 7 for both mutant and wild type with the mutant exhibiting enhanced function. Efficiency for both declined rapidly at higher and lower pHs. Dynamic information can shed light on both subunit exchange and oligomerization, and when studied at different pHs for both the mutant and wild type can inform us about this important regulatory mechanism. These data are obtained using dual-color Fluorescence Cross-Correlation Spectroscopy and single molecule FRET. αB-crystallin labeled at lysine residues with Alexa Fluor 488 is used for FCCS and Alexa Fluor 555 are used for our FRET experiments. Both experiments use Alexa Fluor 647 as an acceptor.