Abstract
Purpose: :
Hsp27 forms oligomeric complexes that can reversibly dissociate into binding competent dimers depending upon phosphorylation state. We are using spectroscopic tools to examine the role of the N-terminal region in the recognition and binding of substrate.
Methods: :
Residues along the N-terminal region were selected for EPR analysis on both wild-type and phosphorylation mimic backgrounds. Single cysteine mutations were constructed and reacted with a sulfhydryl specific spin label. Spin labeled mutants were analyzed for changes in EPR lineshape and solvent accessibility in the both the presence and absence of destabilized T4 lysozyme (T4L) mutants.
Results: :
EPR data in the absence of phosphorylation revealed a sterically packed and solvent-inaccessible environment for the N-terminal region. After phosphorylation EPR spectra report a transition to a more solvent-exposed environment suggesting exposure of the N-terminal region. Phosphorylation does not change the environment of the α-crystallin domain. Binding of Hsp27 to destabilized T4L mutants causes the N-terminal region to transition from a solvent-exposed to solvent-inaccessible environment; implying N-terminal region exposure results in contact with substrate and subsequent reassembly to a Hsp27/T4L complex. Our data revealed that mutation of several critical N-terminal residues modulated the Hsp27 dissociation equilibrium. These mutants either prevented Hsp27 oligomer dissociation on the phosphorylation mimic or destabilized the Hsp27 oligomer in the wild-type.
Conclusions: :
Previous work from our laboratory correlated the phosphorylation-induced dissociation of Hsp27 with increased levels of substrate binding. Our results suggest this enhancement for substrate binding is the result of exposure of the N-terminal region after phosphorylation. We propose that the interaction between the N-terminal region of Hsp27 and substrate is a critical event for its chaperone activity.
Keywords: protein structure/function • crystallins • chaperones