Protein Expression and Purification, Vol.68, No.2, 201-207, 2009
Structural differences between the SH3-HOOK-GuK domains of SAP90/PSD-95 and SAP97
The SH3-HOOK-GUK domains of the postsynaptic scaffolding proteins SAP90/PSD-95 and SAP97 are established targets of synaptic plasticity processes in the brain. A crucial molecular mechanism involved is the transition of this domain to different conformational states. We purified the SH3-HOOK-GUK domain of both proteins to examine variations in protein conformation and stability. As monitored by circular dichroism and differential scanning calorimetry, SAP97 (T-m = 64 degrees C) is significantly more thermal stable than SAP90/PSD-95 (T-m = 52 degrees C) and follows a bimodal phase transition. GdmCl-induced equilibrium unfolding of both proteins follows the two-state transitions and thus does not involve the accumulation of stable intermediate state(s). Equilibrium unfolding of SAP97 is highly cooperative from a native state to an unfolded state. In contrast, SAP90/PSD-95 follows a non-cooperative transition from native to unfolded states. A highly cooperative unfolding reaction in case of SAP97 indicates that the protein existed initially as a compact, well-folded structure, while the gradual, non-cooperative melting reaction in case of SAP90/PSD-95 indicates that the protein is in comparison more flexible. (C) 2009 Elsevier Inc. All rights reserved.
Keywords:MAGUK;Post synaptic density;Thermal unfolding;Equilibrium unfolding;Hydrophobic interaction chromatography;GdmCl;guanidine hydrochloride;DSC;differential scanning calorimetry