PH dependent unfolding characteristics of DLC8 dimer: Residue level details from NMR

Abstract

Environment dependence of folding and unfolding of a protein is central to its function. In the same vein, knowledge of pH dependence of stability and folding/unfolding is crucial for many biophysical equilibrium and kinetic studies designed to understand protein folding mechanisms. In the present study we investigated the guanidine induced unfolding transition of dynein light chain protein (DLC8), a cargo adaptor of the dynein complex in the pH range 7–10. It is observed that while the protein remains a dimer in the entire pH range, its stability is somewhat reduced at alkaline pH. Global unfolding features monitored using fluorescence spectroscopy revealed that the unfolding transition of DLC8 at pH 7 is best described by a three-state model, whereas, that at pH 10 is best described by a two-state model. Chemical shift perturbations due to pH change provided insights into the corresponding residue level structural perturbations in the DLC8 dimer. Likewise, backbone 15N relaxation measurements threw light on the corresponding motional changes in the dimeric protein. These observations have been rationalized on the basis of expected changes with increasing pH in the protonation states of the titratable residues on the structure of the protein. These, in turn provide an explanation for the change from three-state to two-state guanidine induced unfolding transition as the pH is increased from 7 to 10. All these results exemplify and highlight the role of environment vis-à-vis the sequence and structure of a given protein in dictating its folding/unfolding characteristics.